Dissertations / Theses on the topic 'DNA supercoiling'

DNA supercoiling is a fundamental biological process occurring in all cells. We developed a theory of braiding (supercoiling) of a pair of DNA molecules that takes into account the contribution of the bending and the electrostatic energy. The electrostatic interaction was calculated within the framework of the Kornyshev-Leikin theory of DNA interactions, which takes into account realistic helical patterns of charge. Because of the chirality of the charge patterns, we predict that left-handed braiding of a pair of DNA molecules is more favourable than right-handed braiding. Applying our model to the case of closed loop DNA supercoiling and to single molecule DNA micromanipulations, we predict novel effects that have not yet been experimentally observed. We show that supercoiling may occur in topologically relaxed plasmids, as a consequence of attractive chiral forces. We speculate about the potential biological role of the predicted effects in the case of topoisomerase action, and the occurrence of positively supercoiled DNA in hyperthermophilic bacteria and archea. Our findings also suggest alternative an explanation of well-known experiments that proved that divalent ions overwind DNA. We also give an explanation for pairing of homologous DNA molecules in monovalent salt, and explain the occurrence of tight supercoiling observed in cryo-electron and atomic force microscopy. The analysis of existing experimental data shows that in most cases the chiral effects that we predict remain elusive. The theory therefore awaits final experimental verification.

DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. Deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that behaves like a conventional type II topoisomerase, suggesting that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNA-binding domains of the A subunits. However, these results do not unveil the detailed mechanism by which the transported DNA segment is captured and directed through the DNA gate. This mechanism was addressed by probing the topology of the bound DNA segment at distinct steps of the catalytic cycle. A model is proposed in which gyrase captures a contiguous DNA segment with high probability, irrespective of the superhelical density of the DNA, while the efficiency of strand passage depends on the superhelical free-energy. This mechanism is concerted, in that capture of the transported segment induces opening of the DNA gate, which in turn, stimulates ATP hydrolysis. Mutation of Glu42 to Ala in the B subunit of DNA gyrase abolishes ATP hydrolysis but not nucleotide binding. Gyrase complexes that contain one wild-type and one Ala42 mutant B protein were formed and the ability of such complexes to hydrolyse ATP was investigated. It was found that ATP hydrolysis was able to proceed only in the wild-type subunit, albeit at a lower rate. With only one ATP molecule hydrolysed at a time, gyrase could still perform supercoiling but the limit of this reaction was lower than that observed when both subunits can hydrolyse the nucleotide. Limited proteolysis was used to identify conformational changes in DNA gyrase and the proteolytic signatures observed were interpreted in terms of four complexes of gyrase, each representing a particular conformational state. Quinolone binding to the gyrase-DNA complex induces a conformational change that results in the blocking of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis. The kinetics of this reaction have been studied and found to differ from those of the reaction of the drug-free enzyme. By observing the conversion of the ATPase rate to the quinolone-characteristic rate, the formation and dissociation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that of DNA cleavage reveals that formation of the gyrase-DNA-quinolone complex does not correspond to the formation of cleaved DNA. Quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinolones on DNA gyrase.

A principle challenge of modern biology is to understand how the human genome is organised and regulated within a nucleus. The field of chromatin biology has made significant progress in characterising how protein and DNA modifications reflect transcription and replication state. Recently our lab has shown that the human genome is organised into large domains of altered DNA helical twist, called DNA supercoiling domains, similar to the regulatory domains observed in prokaryotes. In my PhD I have analysed how the maintenance and distribution of DNA supercoiling relates to biological function in human cells. DNA supercoiling domains are set up and maintained by the balanced activity of RNA transcription and topoisomerase enzymes. RNA polymerase twists the DNA, over-winding in front of the polymerase and under-winding behind. In contrast topoisomerases relieve supercoiling from the genome by introducing transient nicks (topoisomerase I) or double strand breaks (topoisomerase II) into the double helix. Topoisomerase activity is critical for cell viability, but the distribution of topoisomerase I, IIα and IIβ in the human genome is not known. Using a chromatin immunoprecipitation (ChIP) approach I have shown that topoisomerases are enriched in large chromosomal domains, with distinct topoisomerase I and topoisomerase II domains. Topoisomerase I is correlated with RNA polymerase II, genes and underwound DNA, whereas topoisomerase IIα and IIβ are associated with each other and over-wound DNA. This indicates that different topoisomerase proteins operate in distinct regions of the genome and can be independently regulated depending on the genomic environment. Transcriptional regulation by DNA supercoiling is believed to occur through changes in gene promoter structure. To investigate DNA supercoiling my lab has developed biotinylated trimethylpsoralen (bTMP) as a DNA structure probe, which preferentially intercalates into under-wound DNA. Using bTMP in conjunction with microarrays my lab identified a transcription and topoisomerase dependent peak of under-wound DNA in a meta-analysis of several hundred genes (Naughton et al. (2013)). In a similar analysis, Kouzine et al. (2013) identified an under-wound promoter structure and proposed a model of topoisomerase distribution for the regulation of promoter DNA supercoiling. To better understand the role of supercoiling and topoisomerases at gene promoters, a much larger-scale analysis of these factors was required. I have analysed the distribution of bTMP at promoters genome wide, confirming a transcription and expression dependent distribution of DNA supercoils. DNA supercoiling is distinct at CpG island and non-CpG island promoters, and I present a model in which over-wound DNA limits transcription from both CpG island promoters and repressed genes. In addition, I have mapped by ChIP topoisomerase I and IIβ at gene promoters on chromosome 11 and identified a different distribution to that proposed by Kouzine et al. (2013), with topoisomerase I maintaining DNA supercoiling at highly expressed genes. This study provides the first comprehensive analysis of DNA supercoiling at promoters and identifies the relationship between supercoiling, topoisomerase distribution and gene expression. In addition to regulating transcription, DNA supercoiling and topoisomerases are important for genome stability. Several studies have suggested a link between DNA supercoiling and instability at common fragile sites (CFSs), which are normal structures in the genome that frequently break under replication stress and cancer. bTMP was used to measure DNA supercoiling across FRA3B and FRA16D CFSs, identifying a transition to a more over-wound DNA structure under conditions that induce chromosome fragility at these regions. Furthermore, topoisomerase I, IIα and IIβ showed a pronounced depletion in the vicinity of the FRA3B and FRA16D CFSs. This provides the first experimental evidence of a role for DNA supercoiling in fragile site formation.

The self-association of guanine bases in a tetrameric square planar arrangement was first determined in the early 1960s. The tetramer, commonly termed the G-quartet, can stack upon other G-quartets to form four-stranded helices termed G-quadruplexes. Bioinformatics studies have revealed that guanine-rich sequences with the propensity to adopt these structures are found in telomeric DNA and throughout the human genome, particularly in gene promoter regions. It is thought that the location of these sequences is not a coincidence and that the folding potential of guanine-rich DNA in vivo may play an important role in biological events such as gene regulation. Repetitive guanine tracts of G-quadruplex-forming DNAs form highly polymorphic structures with parallel or antiparallel strand orientations, depending the on ionic condition and the length of the connecting loops, and can be assembled as inter- or intra-molecular complexes. While extensive research has demonstrated their formation in vitro, there is little direct evidence to support their formation in vivo. With the exception of the single-stranded telomeric DNA, all genomic guanine-rich sequences are always present in the duplex configuration. Therefore, these structures will need to compete with the duplex that is normally generated with the complementary cytosine-rich strand. In order for this to happen would first require the local dissociation of the strands. Negative supercoiling results from the unwinding of the DNA helix and is known to provide energy to facilitate the formation of a number of alternative DNA structures. The work described in this thesis therefore aims to investigate the formation of G-quadruplexes under negatively supercoiled conditions. This was examined by preparing plasmids that contained multiple copies of G-rich oligonucleotides, based on the sequences (G3T)n and (G3T4)n, cloned into the pUC19 vector. The formation of G-quadruplexes within these repeats has been assessed using the chemical probes dimethyl sulphate (DMS) and potassium permanganate, and the single-strand specific endonuclease S1. DMS probing revealed some evidence for G-quadruplex formation in (G3T)n sequences, though this was not affected by DNA supercoiling. However, probing with KMnO4 failed to detect exposed thymines in the loop regions, though there was some supercoil-dependent reactivity in the surrounding sequences, suggesting that this had been affected by the G-rich region. In contrast, the (G3T4)n sequences did not demonstrate protection from DMS, suggesting that G-quadruplex formation had not taken place. Surprisingly, the KMnO4 reactions identified structural alterations around, but not within, the inserted G-rich fragments. S1 nuclease digestions did not detect any structural perturbations in any of the sequences apart from a mutant plasmid containing an inverted quadruplex repeat at the 3’-end. Two-dimensional gel electrophoresis of DNA topoisomers was also conducted to detect any supercoil-dependent B-DNA to quadruplex transitions. Neither the (G3T)n nor (G3T4)n plasmids showed any such structural changes. However, the mutant plasmid did demonstrate some supercoil-dependent changes, though these may correspond to cruciform rather than G-quadruplex formation. These results do not support the suggestion that negative supercoiling can induce the formation of G-quadruplex structures.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.
Includes bibliographical references.
The procession of helix-tracking enzymes along a DNA molecule results in the formation of supercoils in the DNA, with positive supercoiling (overwinding) generated ahead of the enzyme, and negative supercoiling (underwinding) in its wake. While the structural and physiological consequences of negative supercoiling have been well studied, technical challenges have prevented extensive examination of positively supercoiled DNA. Studies suggest that at sufficiently high levels of overwinding, DNA relieves strain by adopting an elongated structure, where the bases are positioned extrahelically and the backbones occupy the center of the helix. This transition has only been identified, however, at a degree of supercoiling substantially higher than is generated physiologically. To examine the structural changes resulting from physiological levels of positive DNA supercoiling, I have developed a method for preparing highly purified positively supercoiled plasmid substrates. Based on a method previously developed in this laboratory, this allows for preparation of large quantities of very pure, highly positively supercoiled plasmid. It also expands on earlier methods by exploiting ionic strength to modulate the direction of supercoiling introduced, allowing preparation of either positively or negatively supercoiled substrates.
(cont.) A combination of approaches has been used to elucidate changes to DNA structure that result from physiological levels of positive supercoiling. Enzymatic probes for regions of single-stranded character are not reactive with positively supercoiled plasmid, indicating that stably unpaired regions are not present. Additionally, the effect of supercoiling on the activity of restriction enzymes has been examined. With the enzymes tested, no substantial differences in cleavage rates were observed with either positively or negatively supercoiled substrates. To examine structural changes at a wider range of superhelical densities, design and preparation was undertaken on 2-aminopurine-containing DNA substrates for use in fluorescence studies with a magnetic micromanipulator. Technical limitations rendered these experiments infeasible with current instrumentation, but important insights were gained for future fluorescence-based A destabilizing effect on the base pairs, however, can be seen using Raman difference spectroscopy, suggesting a subtle shift toward the more extreme extrahelical state.
(cont.) The Raman data suggest that structural adjustments due to positive supercoiling are small but significant, and in addition to the base-pairing effects, alterations are observed in phosphodiester torsion and the minor groove environment, as well as a slight shift in sugar pucker conformation to accommodate lengthening of the DNA backbone. These results point to subtle changes in DNA structure caused by biologically relevant levels of positive superhelical tension and positive supercoiling. All of the changes are consistent with the mechanical effects of helical overwinding and suggest a model in which base pair destabilization in overwound DNA could affect the search mechanisms used by DNA repair enzymes and the binding of other proteins to DNA.
by Marita Christine Barth.
Ph.D.

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells.

La structure spatiale de l'ADN est fortement affectée par le surenroulement. Lorsqu'il est surenroulé, l'ADN circulaire (ou linéaire et contraint topologiquement aux extrémités) se replie et forme des boucles appelées plectonèmes, rapprochant dans l'espace des régions éloignées du chromosome, perturbant ainsi de l réseau de régulation de la transcription de la cellule. Les chromosomes bactériens sont surenroulés négativement et ce surenroulement est connu pour jouer un rôle important dans la régulation de la transcription. Cependant, à cause de la nature globale de la contrainte topologique associée à l'ADN, les méthodes actuelles ne sont capables de simuler que de petites molécules (jusqu'à quelques milliers de paires de bases, KB). Cette thèse présente un nouvel algorithme utilisé pour réaliser des simulations de Monte-Carlo d'ADN surenroulé, basé sur une molécule. Cet algorithme a été utilisé pour réaliser des simulations de longues molécules (plusieurs dizaines de KB) et apporter un nouvel éclairage sur des questions encore débattues à propos de la structure de l'ADN surenroulé à cette échelle. Cette méthode permet d'étudier l'effet de la position des gènes le long de l'ADN sur leur co-localisation et leur co-régulation, et laisse entrevoir la possibilité de simuler le repliement d'un chromosome bactérien entier
Superhelicity strongly affects the 3D structure of DNA. When supercoiled, circular DNA (or linear DNA with topolocically constrained ends) folds and forms loops called plectonemes, bringing some distant parts of the chromosme close to one another in space, thus perturbing the transcription regulation network of the cell. Bacterial chromosomes are negatively supercoiled and superhelicity is known to play a important role in the regulation of the transcription. However, due to the global nature of the topological constraint imposed to the DNA, current methods have only been able to simulate small moelcules (up to a few kilobasepairs, KB). This thesis presents a novel algorithm used to performed Monte-Carlo simulations of supercoiled DNA, featuring a local approach to the topological constraint via the computation of the twist of the molecule. Using this efficient algorithm, stimulations of long molecules (tens of KB) were performed and shed a new light on debated questions about the structure of supercoiled DNA at this scale. This method allows to study the effect of the position of genes along the DNA on their co-localisation and co-regulation, and to envision the possibility of simulating the folding of a whole bacterial chromosome

Previous studies have shown that translocation of an actively transcribing RNA polymerase leads to local increases or decreases in DNA torsion (twin-supercoiled domain), which cannot be resolved in vivo due to interactions of the template DNA, nascent RNA and polymerase with the crowded cellular environment. Local changes in DNA supercoiling are biologically relevant as they have been shown to regulate transcription initiation at promoters located downstream. Current in vitro single-molecule approaches are not able to directly probe transcription-coupled DNA supercoiling due to an inability to simultaneously monitor changes in torsional stress and localise individual transcribing RNA polymerase(s) on the DNA. Described here is a novel optical microscope, which utilises a combination of magnetic tweezers, bright-field illumination and wide-field epifluorescence imaging to permit the visualisation of fluorescently tagged polymerases transcribing in real-time on a torsionally constrained DNA template. With this unique geometry, transcription as a function of applied torsion can be probed directly in vitro. Unlike standard magnetic tweezers configurations this system extends tethers horizontally relative to the microscope slide surface, which allows individual enzymes to be directly viewed via attached fluorophores. The magnetic tweezers allow both the relative extension and linking number of the DNA tether to be manipulated, thus enabling transcription to be studied under conditions of constant DNA extension and defined torsional stress.

Aufgrund einer komplexen Wechselwirkung mit Proteinen ist das Genom in einer Zelle ständig mechanischer Spannung und Torsion ausgesetzt. Daher ist es wichtig die Mechanik und die Dynamik von verdrillter DNA unter Spannung zu verstehen. Diese Situation wurde experimentell mittels einer sog. magnetischen Pinzette nachgestellt, indem sowohl Kraft als auch Drehmoment auf ein einzelnes DNA Molekül ausgeübt und gleichzeitig die mechanische Antwort des Polymers aufgezeichnet wurde. Als erstes Beispiel wurde der Übergang von linearer zu sog. plectonemischer DNA untersucht, d.h. die Absorption eines Teils der induzierten Verdrillung in einer superhelikalen Struktur. Eine abrupte Längenänderung am Anfang dieses Übergangs wurde bereits im Vorfeld publiziert. In der vorliegenden Arbeit wird gezeigt, dass diese abrupte DNA Verkürzung insbesondere von der Länge der DNA und der Ionenkonzentration der Lösung abhängt. Dieses Verhalten kann mittels eines Modells verstanden werden, in dem die Energie pro Verwringung der ersten Schlinge innerhalb der Superhelix größer ist als die aller nachfolgenden. Des Weiteren wurden DNA-DNA Wechselwirkungen in der Umgebung monovalenter Ionen durch die Analyse des Superspiralisierungsverhaltens einzelner DNA Moleküle bei konstanter Kraft charakterisiert. Solche Wechselwirkungen sind für die Kompaktierung des Genoms und die Regulation der Transkription wichtig. Oft wird DNA als gleichmäßig geladener Zylinder modelliert und ihre elektrostatischen Wechselwirkungen im Rahmen der Poisson-Boltzmann-Gleichung mit einem Ladungsanpassungsfaktor berechnet. Trotz erheblicher Anstrengung ist eine präzise Bestimmung dieses Parameters bisher nicht gelungen. Ein theoretisches Modell dieses Prozesses zeigte nun eine erstaunlich kleine effektive DNA Ladung von ~40% der nominalen Ladungsdichte. Abgesehen von Gleichgewichtsprozessen wurde auch die Dynamik eines Faltungsvorgangs von DNA untersucht. Spontane Branch Migration einer homologen Holliday-Struktur wurde genutzt, um die intramolekulare Reibung der DNA zu erforschen. Mittels einer magnetischen Pinzette wurde eine torsionslimitierte Holliday-Struktur gestreckt während die Längenfluktuationen der Zweige mit schneller Videomikroskopie bei ~3 kHz aufgezeichnet wurden. Einzelne diffusive Schritte der Basenpaare sollten auf einer sub-Millisekunden Zeitskala auftreten und viel kleiner als die Gesamtfluktuationen der DNA sein. Eine Analyse der spektralen Leistungsdichte der Längenfluktuationen ermöglicht eine eindeutige Beschreibung der Dynamik der Branch Migration. Die Holliday-Struktur wurde außerdem als nanomechanischer Linearversteller eingesetzt, um einen einzelnen fluoreszierenden Quantenpunkt durch ein exponentiell abfallendes evaneszentes Feld zu bewegen. Durch die Aufzeichnung der Emission des Quantenpunkts sowohl in dem evaneszenten Feld als auch unter gleichmäßiger Beleuchtung kann die Intensitätsverteilung des Anregungsfelds ohne weitere Dekonvolution bestimmt werden. Diese neue Technik ist von besonderem wissenschaftlichen Interesse, weil die Beschreibung dreidimensionaler inhomogener Beleuchtungsfelder eine große Herausforderung in der modernen Mikroskopie darstellt. Die Ergebnisse dieser Arbeit werden dem besseren Verständnis einer Vielzahl biologischer Prozesse, die in Verbindung mit DNA Superspiralisierung stehen, dienen und weitere technische Anwendungen des DNA-basierten Linearverstellers hervorbringen
The genome inside the cell is continuously subjected to tension and torsion primarily due to a complex interplay with a large variety of proteins. To gain insight into these processes it is crucial to understand the mechanics and dynamics of twisted DNA under tension. Here, this situation is mimicked experimentally by applying force and torque to a single DNA molecule with so called magnetic tweezers and measuring its mechanical response. As a first example a transition from a linear to a plectonemic DNA configuration is studied, i.e. the absorption of part of the applied twist in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. Here, it is found that this abrupt DNA shortening strongly depends on the length of the DNA molecule and the ionic strength of the solution. This behavior can be well understood in the framework of a model in which the energy per writhe for the initial plectonemic loop is larger than for subsequent turns of the superhelix. Furthermore DNA-DNA interactions in the presence of monovalent ions were comprehensively characterized by analyzing the supercoiling behavior of single DNA molecules held under constant tension. These interactions are important for genome compaction and transcription regulation. So far DNA is often modeled as a homogeneously charged cylinder and its electrostatic interactions are calculated within the framework of the Poisson-Boltzmann equation including a charge adaptation factor. Despite considerable efforts, until now a rigorous quantitative assessment of this parameter has been lacking. A theoretical model of this process revealed a surprisingly small effective DNA charge of ~40% of the nominal charge density. Besides describing equilibrium processes, also the dynamics during refolding of nucleic acids is investigated. Spontaneous branch migration of a homologous Holliday junction serves as an ideal system where the friction within the biomolecule can be studied. This is realized by stretching a torsionally constrained Holliday junction using magnetic tweezers and recording the length fluctuations of the arms with high-speed videomicroscopy at ~3 kHz. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Apart from studying intramolecular friction, the four-arm DNA junction was also used as a nanomechanical translation stage to move a single fluorescent quantum dot through an exponentially decaying evanescent field. Recording the emission of the quantum dot within the evanescent field as well as under homogeneous illumination allows to directly obtain the intensity distribution of the excitation field without additional deconvolution. This new technique is of particular scientific interest because the characterization of three-dimensional inhomogeneous illumination fields is a challenge in modern microscopy. The results presented in this work will help to better understand a large variety of biological processes related to DNA supercoiling and inspire further technical applications of the nanomechanical DNA gear

In this thesis, on the basis of the asymmetrical charge distribution of E. coli topoisomerase I, I developed a new rapid procedure to purify E. coli DNA topoismoerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-sepharose FF and Q-sepharose FF columns. E. coli topoisomerase I purified here is free of nuclease contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were determined as well. I also used isothermal titration calorimetry to investigate the energetics of DNA supercoiling by using the unwinding properties of DNA intercalators, ethidium and daunomycin. After comparing the enthalpy changes of these DNA intercalators binding to supercoiled and nicked or relaxed plasmid DNA pXXZ06, I determined the DNA supercoiling enthalpy is about 12 kcal/mol per turn of DNA supercoil, which is in good agreement with the previously published results.

In the last few decades, the constant development of novel microscopy techniques have created the basis for a new paradigm in the field of biophysics. Single-molecule techniques enabled to carry out experiments providing new information: the nanomanipulation of individual biomolecules revealed unknown insights into the elasticity and mechanics of molecules, improving the understanding of the fundamental relation between structural properties and biological functions. In particular, an AFM and mostly a MT setup were used during this thesis work, both located in biophysics laboratory of Prof. Francesco Mantegazza, at the University of Milano-Bicocca. Similar issues were encountered at the cellular level, because bulk experiments of conventional microscopy techniques provide information on average only, without taking into account the intrinsic biological heterogeneity. Recent developments in microfluidics enabled to follow individual cells over a long time and under controlled conditions. During the last part of this thesis project I used one of these microfluidic devices to perform time-lapse microscopy experiments at the single-cell level. These experiments were carried out during a visiting period of seven months in Prof. Pietro Cicuta’s laboratory, in Cavendish laboratory at University of Cambridge. In this thesis I dealt with three main research topics: • DNA structural polymorphism • nanomechanics of DNA-ligand interactions • the dual role of H-NS protein: DNA condensation and gene regulation The study of the conformational changes of DNA, namely the property of structural polymorphism, is addressed during two projects: one about the nanomechanics of a DNA analogue and another concerning the behavior of DNA at high supercoiling. The study of a DNA analogue enables to observe how a chemical modification of nucleotides can induce structural re- arrangements of the double-helix, biasing towards an A-like-form of DNA. The regimes of high supercoiling, both positive and negative supercoiling, show instead how an applied torsion at a certain forces can promote the formation of plectonemes or denaturation bubbles, which are conditions that favor particular structural transitions. The second major theme concerns the analysis of the nanomechanics of DNA-ligand complexes, particularly the interactions of DNA with anticancer drugs or with the H-NS protein and the crowding agent PEG. The project about the interactions between DNA and drugs clearly shows how the mechanical properties and the stability of DNA change due to the binding with compounds commonly used in clinics to treat tumors. On the other hand, the H-NS protein forms relatively stable DNA loops and influences the stability of the double helix, as well as the crowding agent. The protein binding mechanism has a preference for some DNA sequences and an unexpected concentration-dependent behavior. The analysis of the the DNA-H-NS interactions also enables, particularly in crowding conditions, to better understand the mechanism of DNA condensation inside the cell, one of the biological roles of H-NS. The second important function of this NAP is the gene regulation. To investigate the dual role of H-NS in great detail two complementary techniques have been combined. The nanoma- nipulation technique is employed to observe the structural role of H-NS and its combined activity with a crowding agent leading to a clear and abrupt compaction of DNA. Time-lapse fluorescence microscopy is instead used to study the regulatory role of the protein, more precisely the gene silencing mechanism, at the single-cell level. This activity has also a strong influence in the cell physiology, by significantly changing the growth rate of bacteria.
In the last few decades, the constant development of novel microscopy techniques have created the basis for a new paradigm in the field of biophysics. Single-molecule techniques enabled to carry out experiments providing new information: the nanomanipulation of individual biomolecules revealed unknown insights into the elasticity and mechanics of molecules, improving the understanding of the fundamental relation between structural properties and biological functions. In particular, an AFM and mostly a MT setup were used during this thesis work, both located in biophysics laboratory of Prof. Francesco Mantegazza, at the University of Milano-Bicocca. Similar issues were encountered at the cellular level, because bulk experiments of conventional microscopy techniques provide information on average only, without taking into account the intrinsic biological heterogeneity. Recent developments in microfluidics enabled to follow individual cells over a long time and under controlled conditions. During the last part of this thesis project I used one of these microfluidic devices to perform time-lapse microscopy experiments at the single-cell level. These experiments were carried out during a visiting period of seven months in Prof. Pietro Cicuta’s laboratory, in Cavendish laboratory at University of Cambridge. In this thesis I dealt with three main research topics: • DNA structural polymorphism • nanomechanics of DNA-ligand interactions • the dual role of H-NS protein: DNA condensation and gene regulation The study of the conformational changes of DNA, namely the property of structural polymorphism, is addressed during two projects: one about the nanomechanics of a DNA analogue and another concerning the behavior of DNA at high supercoiling. The study of a DNA analogue enables to observe how a chemical modification of nucleotides can induce structural re- arrangements of the double-helix, biasing towards an A-like-form of DNA. The regimes of high supercoiling, both positive and negative supercoiling, show instead how an applied torsion at a certain forces can promote the formation of plectonemes or denaturation bubbles, which are conditions that favor particular structural transitions. The second major theme concerns the analysis of the nanomechanics of DNA-ligand complexes, particularly the interactions of DNA with anticancer drugs or with the H-NS protein and the crowding agent PEG. The project about the interactions between DNA and drugs clearly shows how the mechanical properties and the stability of DNA change due to the binding with compounds commonly used in clinics to treat tumors. On the other hand, the H-NS protein forms relatively stable DNA loops and influences the stability of the double helix, as well as the crowding agent. The protein binding mechanism has a preference for some DNA sequences and an unexpected concentration-dependent behavior. The analysis of the the DNA-H-NS interactions also enables, particularly in crowding conditions, to better understand the mechanism of DNA condensation inside the cell, one of the biological roles of H-NS. The second important function of this NAP is the gene regulation. To investigate the dual role of H-NS in great detail two complementary techniques have been combined. The nanoma- nipulation technique is employed to observe the structural role of H-NS and its combined activity with a crowding agent leading to a clear and abrupt compaction of DNA. Time-lapse fluorescence microscopy is instead used to study the regulatory role of the protein, more precisely the gene silencing mechanism, at the single-cell level. This activity has also a strong influence in the cell physiology, by significantly changing the growth rate of bacteria.

Advances in optical imaging techniques have allowed quantitative studies of many biological systems. This dissertation elaborates on our efforts in both developing novel imaging modalities based on detection of optical absorption and applying high-sensitivity fluorescence microscopy to the study of biology.
Chemistry and Chemical Biology

Within every human cell, approximately two meters of DNA must be compacted into a nucleus with a diameter of around ten micrometers. Alongside this daunting storage problem, the 3D organisation of the genome also helps determine which genes are up- or down-regulated, which in turn effects the functionality of the cell itself. While the organisational structure of the genome can be revealed using experimental techniques such as chromosome conformation capture and its high-throughput variant Hi-C, the mechanisms driving this organisation are still unclear. The first two results chapters of this thesis use molecular dynamics simulations to investigate the effect of a potential organisational mechanisms for DNA known as the "bridging-induced attraction". This mechanism involves multivalent DNA-binding proteins bridging genomically distant regions of DNA, which in turn promotes further binding of proteins and compaction of the DNA. In chapter 2 (the first results chapter) we look at a model where proteins can bind non-specifically to DNA, leading to cluster formation for suitable protein-DNA interaction strengths. We also show the effects of protein concentration on the DNA, with a collapse from a swollen to a globular phase observed for suitably high protein concentrations. Chapter 3 develops this model further, using genomic data from the ENCODE project to simulate the "specific binding" of proteins to either active (euchromatin) or inactive (heterochromatin) regions. We were then able to compare contact maps for specific simulated chromosomes with the experimental Hi-C data, with our model reproducing well the topologically associated domains (TADs) seen in Hi-C contact maps. In chapter 4 of the thesis we use numerical methods to study a model for the coupling between DNA topology (in particular, supercoiling in DNA and chromatin) and transcription in a genome. We present details of this model, where supercoiling flux is induced by gene transcription, and can diffuse along the DNA. The probability of transcription is also related to supercoiling, as regions of DNA which are negatively supercoiled have a greater likelihood of being transcribed. By changing the magnitude of supercoiling flux, we see a transition between a regime where transcription is random and a regime where transcription is highly correlated. We also find that divergent gene pairs show increased transcriptional activity, along with transcriptional waves and bursts in the highly correlated regime { all these features are associated with genomes of living organisms.

Molecular biophysics is a rapidly evolving field aimed at the physics-based investigation of the biomolecular processes that enable life. In this thesis, we explore two such processes: the thermodynamics of DNA bending, and the mechanism of the RecQ DNA helicase. A computational approach using a coarse-grained model of DNA is employed for the former; an experimental approach relying heavily on single-molecule fluorescence for the latter. There is much interest in understanding the physics of DNA bending, due to both its biological role in genome regulation and its relevance to nanotechnology. Small DNA bending fluctuations are well described by existing models; however, there is less consensus on what happens at larger bending fluctuations. A coarse-grained simulation is used to fully characterize the thermodynamics and mechanics of duplex DNA bending. We then use this newfound insight to harmonize experimental results between four distinct experimental systems: a 'molecular vise', DNA cyclization, DNA minicircles and a 'strained duplex'. We find that a specific structural defect present at large bending fluctuations, a 'kink', is responsible for the deviation from existing theory at lengths below about 80 base pairs. The RecQ DNA helicase is also of much biological and clinical interest, owing to its essential role in genome integrity via replication, recombination and repair. In humans, heritable defects in the RecQ helicases manifest clinically as premature aging and a greatly elevated cancer risk, in disorders such as Werner and Bloom syndromes. Unfortunately, the mechanism by which the RecQ helicase processes DNA remains poorly understood. Although several models have been proposed to describe the mechanics of helicases based on biochemical and structural data, ensemble experiments have been unable to address some of the more nuanced questions of helicase function. We prepare novel substrates to probe the mechanism of the RecQ helicase via single-molecule fluorescence, exploring DNA binding, translocation and unwinding. Using this insight, we propose a model for RecQ helicase activity.

Computer simulations provide a potentially powerful complement to conventional experimental techniques in elucidating the structures, dynamics and interactions of macromolecules. In this thesis, I present three applications of computer simulations to investigate important biomolecules with sizes ranging from two-residue peptides, to proteins, and to whole chromosome structures. First, I describe the results of 441 independent explicit-solvent molecular dynamics (MD) simulations of all possible two-residue peptides that contain the 20 standard amino acids with neutral and protonated histidine. 3JHNHα coupling constants and δHα chemical shifts calculated from the MD simulations correlated quite well with recently published experimental measurements for a corresponding set of two-residue peptides. Neighboring residue effects (NREs) on the average 3JHNHα and δHα values of adjacent residues were also reasonably well reproduced. The intrinsic conformational preferences of each residue, and their NREs on the conformational preferences of adjacent residues, were analyzed. Finally, these NREs were compared with corresponding effects observed in a coil library and the average β-turn preferences of all residue types were determined. Second, I compare the abilities of three derivatives of the Amber ff99SB force field to reproduce a recent report of 3JHNHα scalar coupling constants for hundreds of two-residue peptides. All-atom MD simulations of 256 two-residue peptides were performed and the results showed that a recently-developed force field (RSFF2) produced a dramatic improvement in the agreement with experimental 3JHNHα coupling constants. I further show that RSFF2 also improved modestly agreement with experimental 3JHNHα coupling constants of five model proteins. However, an analysis of NREs on the 3JHNHα coupling constants of the two-residue peptides indicated little difference between the force fields’ abilities to reproduce experimental NREs. I speculate that this might indicate limitations in the force fields’ descriptions of nonbonded interactions between adjacent side chains or with terminal capping groups. Finally, coarse-grained (CG) models and multi-scale modeling methods are used to develop structural models of entire E. coli chromosomes confined within the experimentally-determined volume of the nucleoid. The final resolution of the chromosome structures built here was one-nucleotide-per-bead (1 NTB), which represents a significant increase in resolution relative to previously published CG chromosome models, in which one bead corresponds to hundreds or even thousands of basepairs. Based on the high-resolution final 1 NTB structures, important physical properties such as major and minor groove widths, distributions of local DNA bending angles, and topological parameters (Linking Number (Lk), Twist (Tw) and Writhe (Wr)) were accurately computed and compared with experimental measurements or predictions from a worm-like chain (WLC) model. All these analyses indicated that the chromosome models built in this study are reasonable at a microscopic level. This chromosome model provides a significant step toward the goal of building a whole-cell model of a bacterial cell.

Les ADN topoisomérases (Topos) sont des éléments essentiels de la vie cellulaire eucaryote et procaryote. Ces enzymes interviennent lors de la réplication, de la réparation et également lors de la transcription en modulant la topologie de l'ADN. L'ADN gyrase, une Topoisomérase IIA (TopoIIA) bactérienne particulière, est la seule topoisomérase capable de surenrouler l’ADN négativement en présence d’ATP, une activité indispensable au génome bactérien. Les différentes études structurales et fonctionnelles sur ces enzymes ont permis de proposer un mécanisme catalytique de surenroulement très sophistiqué mais la vision morcelée de ces complexes multi-­‐conformationnels laisse aujourd’hui de nombreuses questions mécanistiques en suspens. Ce travail de thèse a combiné une approche structurale et fonctionnelle pour essayer de répondre aux questions fondamentales mécanistiques encore non élucidées à propos des ADN topoisomérases de type II et à la découverte de nouveaux inhibiteurs « anti-­‐Topo » face à l’émergence de populations bactériennes résistantes aux traitements
Type II DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. Most TopoIIA are able to perform ATP-­‐dependent DNA relaxation or decatenation but the bacterial DNA gyraseis the sole type II DNA topoisomerase able to introduce negative supercoils. Several biochemical and structural studies haverevealed a highly sophisticated supercoiling catalytic mechanism but despite a wealth of information, the full architectureof Topo2A and the structural basis for DNA supercoiling remain elusive. Due to their physiological roles, topoisomerasesare also important targets for antibiotics targeting the bacterial enzyme but also anti-­‐cancer molecules inhibiting the humanprotein. This presented work has combinedboth structural and functional approach to answer the fundamental mechanisticquestions still unveiled and to discover new inhibitors against the emergence of resistant bacterial population

L’entérobactérie Dickeya dadantii est responsable de la maladie de la pourriture molle sur de nombreux hôtes végétaux. Ce symptôme est essentiellement dû à la production d’un arsenal d’enzymes qui dégradent la pectine, ciment des parois des cellules végétales. Parmi ces enzymes, les pectate lyases (Pels) ont un rôle majeur dans le pouvoir pathogène en raison de leur capacité àreproduire, sous forme purifiée, le symptôme de la pourriture molle. La synthèse des Pels est soumise à un contrôle très fin qui fait intervenir différents régulateurs agissant de manière intégrée via un réseau de régulation. De nombreuses conditions environnementales modulent la synthèse des Pels via l’action de ces régulateurs. La température est un facteur qui agit sur leur synthèse et pour lequel les mécanismes moléculaires restaient non élucidés. Lors de cette étude, nous avons montré que le régulateur PecT, un répresseur du réseau de régulation, intervient dans la thermorégulation de la synthèse des Pels. PecT s’est avéré être également impliqué dans la thermorégulation de deux autres fonctions de virulence : la mobilité et la synthèse desexopolysaccharides de surface. La quantification des transcrits des gènes de ces 3 fonctions de virulence a permis de montrer que l’action de PecT dans ce contrôle a lieu au niveau transcriptionnel. Le mécanisme moléculaire de la thermorégulation exercée par PecT a été étudié plus en détail sur les gènes pel. Des résultats obtenus in vivo ont montré que la fixation de PecT sur les régionsrégulatrices des gènes pel est plus efficace quand la température augmente. La croissance de D. dadantii à hautes températures induit un relâchement de l’ADN. De manière remarquable, un relâchement artificiel de l’ADN par un traitement inhibant la gyrase entraine une augmentation de la fixation de PecT sur les gènes pel même pour des cellules cultivées à basses températures. De plus, la délétion de PecT se traduit par une augmentation de la capacité de D. dadantii à induire la pourriture molle à hautes températures. Ainsi la topologie de l’ADN et PecT agissent de manière concertée pour moduler la synthèse des Pels en fonction de la température.L’ensemble de ces données apporte une preuve supplémentaire de l’importance de la dynamique structurale de la chromatine dans l’ajustement de la physiologie bactérienne en réponse aux variations des conditions environnementales
Bacteria are colonizers of various environments and host organisms, and they are often subjected to drastic temperature variations. Dickeya dadantii is a Gram-negative pathogen infecting a wide range of plant species. Soft rot, the visible symptom, is mainly due to the production of pectate lyases (Pels) that can destroy the plant cell walls. Production of Pels is controlled by a complex regulation system and responds tovarious stimuli, such as presence of pectin, plant extracts, growth phase, temperature or iron concentration. Although many studies have been carried out, the mechanisms of control of Pels production by temperature have not yet been elucidated. In bacteria, thermoregulation acting at the level of transcription initiation occurs usually both via transcription factors and DNA topology. We show that PecT, a previously identified repressor, is involved in the thermoregulation of the pel gene expression. Using in vivo Chromatin ImmunoPrecipitation (ChIP) coupled to quantitative RT-PCR(qRT-PCR), we reveal that PecT binding to the pel gene promoters is modulated by temperature. By manipulating the DNA topology in vivo, we further show that DNA supercoiling state is involved in the thermoregulation of pel gene expression by PecT. In addition, we show that the development of the pathogenicity of the pecT mutant according to changes in temperature is different from that of the parental strain. This report presents a new example of how plant pathogenic bacteria use transcription factor and DNA topology to adjust synthesis of virulence factors in response to temperature variation

The traditional boundary between hard sciences (physics and mathematics) and soft sciences (chemistry and biology) is progressively fading away as the complexity inherent in the biological world is understood and mapped out thanks to a joint attack on two fronts. On the one side more quantitative experiments allow to investigate the details of the atomic structures of biological molecules and to measure with greater precision the laws of interaction among dierent molecules; on the other side, the massive introduction of information technology in the management and catalogation of the multitude of molecular components found inside a cell is allowing to gain deep insights in the complex dynamic equilibrium that regulates the network of interactions amog different molecules. The work described in this thesis concerns the first side of the battlefield: the development of new techniques to allow quantitative measurements of biologically relevant quantities. The work consisted in the design, construction and validation of three different experiments dealing with proteins and DNA mechanics. Key components of the cellular microcosmos, DNA and proteins are large macromolecules that constantly interact and accomplish most of the tasks needed by the cell to survive. The first part of the thesis summarises the known properties of these molecules and introduces the motivations driving the designed experiments. Proteins catalyse chemical reactions in the cell and their threedimensional conguration gives each of them its specic function. The connections between structural and chemical properties of a protein are a subject largely unexplored.The second part of the thesis describes an experiment based on single molecule fluorescence microscopy designed to explore the dynamics of fluctuations of catalytic activity of a single enzyme. The experiments described in this part have not yet given the hoped results. However part of the preliminary considerations done when building these setup were used to write the article F. Mosconi et al. "Some nonlinear challenges in biology", Nonlinearity 21 (2008) T131-T147. DNA stores the genetic information needed by the cell to accomplish its tasks, and such information must be physically stored, read, written and restored in different times during the cell cycle. The importance of a proper knowledge of its mechanical properties is fundamental if its interaction with proteins is to be understood. The third part of this thesis describes two different experiments based on the magnetic tweezers micro-manipulation technique, that attempt to measure some not yet entirely characterised mechanical properties of DNA. The two experiments presented in this part gave interesting results. A new determination of the biologically relevant parameter C, the twist modulus of DNA was obtained developing a novel type of analysis of data collected using the standard magnetic tweezers apparatus. Also, a new type of "soft" magnetic tweezers that allows the simultaneous application of an external force and an external torque has been developed and validated to measure the torque response of a DNA molecule. The results described in this part of the thesis are summarised in two papers that are ready to be submitted.

Les ADN topoisomerérasessont des enzymes présentent chez tous les organismes vivant et elles résolvent les problèmes topologique de l’ADN grâce à des réactions de clivage et de religation. Ces enzymes ubiquitaires jouent un rôle essentiel dans de nombreux processus du métabolisme de l’ADN tel que la réplication et la reparation de l’ADN, la transcription, la recombinaison, la séparation des chromatides et la stabilité des génomes. Les topoisomérases sont classées en famille de type I et de type II selon leur structure et leur méchanisme. Parmi les topoisomérases de type I, une sous-famille a une structure en forme de cadenas et une reaction fondamentale de passage de brin qui supprime les surenroulement négatif un par un. Elles sont divisées en trois groupes : les Topo I, les TopoIII et les reverse gyrase (RG). Les TopoI sont très efficace pour relaché les ADN sous-enroulés alors que les Topo III sont plus apte à traiter les problèmes de caténation de l’ADN. La reverse gyrase est une enzyme chimérique composé d’une hélicase de type RecQ et d’une Top IA. L’interaction helicase–topoisomérase confère à la reverse gyrase une nouvelle fonctionnalité lui permettant d’augmenter l’enchevêtrement des deux brins d’ADN en s’opposant à la torsion. Nous avons effectué des expériences en molécule unique pour disséquer le mécanisme de RG2 (TopR2) de Sulfolobus solfataricus dont l’activité est strictement dépendante de la présence d’ATP. Nous avons observé que la fixation initiale de TopR2 génère une bulle d’ADN de 20 paires de base et que la fixation de l’ATP referme l’ADN sur 10 paires de base. L’hydrolyse d’ATP entraine ensuite le passage de brin d’ADN et la reformation de la bulle initiale d’ADN de 20 paires de base aboutissant à une augmentation de l’enchevêtrement des brins d’ADN de un. Nous avons également décrit une caractéristique unique de TopA de S. solfataricus, une Topo III hyperthermophile. Cette enzyme peut séparer efficacement des caténanes fermés covalement et cette activité tire parti de la présence de régions d’ADN simple brin qui est favorisée haute température et qui peut être également stabilisée par des protéines liant l’ADN simple brin
DNA topoisomerases are present among all the living organisms and they resolve DNA topological problems through strand cleavage and re-ligation reactions. These ubiquitous enzymes play crucial roles in plenty of processes of DNA metabolism such as DNA replication and repair, transcription, recombination and chromatid segregation. Topoisomerases are categorized into type I and type II families concerning their differences in protein structure and mechanism. Among type I topoisomerases, Top IA sub-family enzymes share a padlock shape and a strand-passage core reaction which removes DNA negative supercoils one by one. They are subdivided into three groups: Topo I, Topo III and reverse gyrase (RG). Topo I is efficient at relaxing underwound DNA while Topo III ismore adept at dealing with DNA catenation issues. RG is a chimera composed of a RecQ-like helicase and a Top IA. The helicase-topoisomerase interplay provides new functionality to RG which allows it to increase DNA linking number against torque. We applied single-molecule experimentation to dissect the mechanism of a Sulfolobus solfataricus RG (TopR2) which catalysis is strictly dependent on the presence of ATP. We observed that the initial binding of TopR2 on DNA generates a 20 base pair DNA bubble and ATP binding rewinds about 10 base pair within the bubble. The following ATP hydrolysis leads to DNA strand-passage and reforming of the initial 20 base pair DNA bubble, resulting in one unit increase of DNA linking number. We also describe the unique functionality of S. solfataricus TopA, a hyperthermophilic Topo III, to efficiently unlink covalently closed DNA catenanes. This activity is found benefited from the single-strand DNA region generated with high thermal energy and also promoted by single-strand DNA binding proteins

Les bactéries pathogènes coordonnent de manière très stricte l’expression de leurs facteurs de virulence en fonction de leur état métabolique, des conditions externes et de l’état de l’hôte. La topologie du chromosome bactérien est également modulée par les conditions environnementales et l’état métabolique des cellules. Le surenroulement de l’ADN, est considéré désormais comme un élément clé de la régulation de l’expression génique. L’objectif de cette thèse est d’identifier les acteurs essentiels de la réponse aux conditions rencontrées durant l’infection chez la bactérie phytopathogène Dickeya dadantii, responsable de la pourriture molle d’une large gamme d’hôtes. Les symptômes de pourriture molle sont principalement associés à la synthèse d'enzymes extracellulaires, en particulier les pectinases qui vont dégrader la paroi des cellules végétales. Cependant une colonisation efficace de la plante requiert de nombreux facteurs additionnels. L’analyse du transcriptome de D. dadantii dans 32 conditions de culture proches de celles rencontrées lors du cycle infectieux a révélé qu’en moyenne 63% des gènes sont exprimés dans chacune des conditions testées alors que 82% des gènes sont exprimés dans au moins une des conditions analysées. Deux facteurs modifient profondément l’expression génique: la phase de croissance et la nature des stress appliqués. L’analyse des gènes différentiellement exprimés a permis d’établir des signatures transcriptionnelles et fonctionnelles spécifiques de chaque stress et d’identifier de nouveaux régulateurs potentiellement impliqués dans la survie aux stress. L’adaptation au stress acide étant peu connue chez les pathogènes de plante, la régulation de quelques gènes spécifiquement induits en stress acide a été approfondie. Ces études ont révélé que le régulateur OmpR est un élèment clé de la réponse au stress acide chez Dickeya. Afin d’établir un lien entre réponse aux stress et impact de la topologie de l’ADN et des NAPs, les profils transcriptionnels obtenus lors des stress ont été comparés à ceux obtenus dans des conditions de relaxation artificielle de l’ADN et chez des mutants fis ou hns. La distribution chromosomique des GDE a révélé des profils cohérents de gènes activés ou réprimés lors des variations de conditions environnementales quelque soit le milieu de culture utilisé et l’existence de patchs d’expression qui illustre l’organisation en domaines du chromosome bactérien.L’expression des gènes au sein des domaines est dépendante de leur propriété thermodynamique, de leur préférence vis à vis du surenroulement, et de leur réponse aux NAPs. Ainsi, Dickeya tire partie des variations topologiques de l’ADN au cours de l’infection pour coordonner son programme de virulence. Ces résultats illustrent la complexité des processus utilisés par D. dadantii pour s’adapter aux conditions de l’infection et coloniser ses hôtes
Pathogenic bacteria strictly coordinate the expression of their virulence factors according to their metabolic states, external conditions and the host environment. The topology of the bacterial chromosome is also modulated by environmental conditions and the metabolic state of the cells. The DNA supercoiling is now considered as a key factor in the regulation of gene expression. The objective of this thesis is to provide a comprehensive picture of the Dickeya infection process by integrated analyses of gene expression patterns obtained under various stress conditions encountered by this pathogen in the course of infection. Dickeya dadantii is a plant pathogenic bacterium that causes soft-rot disease in a wide range of plant species. Soft rot symptoms are mainly associated with the synthesis of extracellular enzymes, particularly pectinases which degrade the plant cell wall. However, an effective colonization of the plant requires a number of additional factors. The transcriptome analysis of D. dadantii, grown in a suite of thirty-two different growth conditions similar to those conditions encountered during the infection cycle revealed that an average of 63% of genes was expressed in each individual condition, while 82% of genes are expressed in at least one of the analyzed conditions. Two factors profoundly alter gene expression: the growth phase and the nature of applied stress. Analysis of differentially expressed genes in this work has established specific transcriptional and functional signatures of each stress and proposed new regulators potentially involved in survival under stress conditions. In this way, we obtained the apparent « temporal map » of the bacterial responses to sequential stress conditions encountered during the infection. The chromosomal distribution of DEG revealed coherent patches of genes activated or repressed during changes in environmental conditions and highlighted a rational organization of the DEG in the chromosomal space. Gene expression within the chromosomal domains is dependent on primary sequence organisation, DNA thermodynamic stability, supercoil dynamics, and binding effects of two abundant nucleoid associated proteins, FIS and H-NS. Therefore, Dickeya takes advantage of DNA topological variations during the infection to coordinate its virulence program. These results illustrate the complexity of mechanisms used by D. dadantii to adapt to stress conditions and colonize its hosts

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

Chez les bactéries, le chromosome est situé dans le cytoplasme, sous une forme très compacte. Cette compaction résulte notamment du surenroulement de l'ADN (SC), c'est à dire de la déformation torsionnelle de la double-hélice de l’ADN. Selon les conditions environnementales rencontrées par les bactéries, le niveau de SC peut varier. Ce niveau est principalement contrôlé par la gyrase, qui augmente le niveau de SC, et la topoisomérase I, qui relâche l'ADN. La régulation du niveau de SC est très importante car le SC est un régulateur de l'expression des gènes. L'objectif de ma thèse est de caractériser la régulation globale de la transcription bactérienne par le SC. Nous avons obtenu le premier transcriptome d'une bactérie à Gram négatif à une inhibition de sa topoisomérase I avec un antibiotique. Nous avons mis une nouvelle fois en évidence une régulation globale et complexe de la transcription par le SC et nous avons découvert que la réponse des gènes à une variation de SC dépend du niveau d'expression et du contexte génomique des gènes, de la direction de la variation de SC et du contexte physiologique de la bactérie. J’ai ensuite développé un package Python facilitant les analyses statistiques reproductibles de données expérimentales (ChIP-Seq, RNA-Seq…) et d'annotations dans le cadre de l'étude de l'expression d'un génome bactérien selon ses propriétés spatiales. Avec ce package j’ai pu exploiter des données publiées sur la fixation de la topoisomérase I et de la gyrase le long du chromosome. En analysant ces données qui donnent un aperçu du niveau de SC local, j'ai pu, d’une part, étudier l'effet de la transcription sur le niveau de SC à une échelle de 10 à 20 kb autour des unités de transcription et, d’autre part, observer une organisation méconnue du chromosome en domaines de 100 kb environ
The bacterial chromosome is highly compacted in the cytoplasm. This compaction is largely the result of DNA supercoiling (SC), which involves the torsional deformation of the DNA double helix and is dependent on the environmental conditions encountered by bacteria. SC levels are primarily controlled by gyrase, which increases SC, and topoisomerase I, which relaxes the DNA. The regulation of SC levels is crucial because SC serves as a regulator of gene expression. The objective of my thesis is to characterize the global regulation of bacterial transcription by SC. We obtained the first transcriptome of a Gram-negative bacterium under inhibition of its topoisomerase I using an antibiotic. We confirmed a global and complex regulation of transcription by SC and highlighted that the response of genes to SC variation depends on the gene's expression level, genomic context, the direction of SC change, and the physiological context of the bacterium. I also developed a Python package to facilitate reproducible statistical analyses of experimental data (ChIP-Seq, RNA-Seq, etc.) and annotations for studying the expression of a bacterial genome based on its spatial properties. With this package, I was able to analyze publicly available ChIP-Seq data of Escherichia coli topoisomerase I and gyrase along the chromosome. By examining this data, which provides insight into local SC levels, I was able to investigate, on one hand, the effect of transcription on SC levels within a 10 kb region around transcription units and, on the other hand, observe a previously unknown organization of the chromosome into approximately 100 kb domains

碩士
國立交通大學
生物科技研究所
84
Escherichia coli exhibits diverse respiratory abilities. It synthesizes atleast two distinctive cytochrome oxidase (cytochrome o oxidase and cytochrome d oxidase) during aerobic growth and can produce an additional terminal oxidoreductase, nitrate reductase(narGHIJ), DMSO/TMAO reductase( dmsABC), and fumarate reductase(frdABCD), for anaerobic respiration with the alternative electron acceptors. Although the anaerobic genes are activated by the Fnr, they still have two to five folds induction during anaerobic growth in fnr mutants. Hence Fnr might not be the sole requirement for the anaerobic induction of anaerobic genes. A topological state of the bacterial chromosome is important for transcription, replication and recombination. To determine how the anaerobic genes are regulated in response to a variety of DNA supercoiling, including inhibitors of gyrase and topoisomerase mutants, we examined their expression by using lacZ repoter fusions in wild-type and fnr mutant strains. The effect of DNA supercoling on the expression of anaerobic respiratory genes was more conspicuous when cells were growth under aerobic condition and fnr mutants. These results infer that Fnr may be a factor for the balance of DNA superhelicity. When the DNA supercoling was relaxed, expression of frdA-lacZ and dmsA-lacZ fusion were activated under anaerobic growth, but narG-lacZ expression was repressed. In contrast, the expression of frdA-lacZ and dmsA-lacZ fusion were repressed and narG-lacZ expression increased when DNA supercoiling became more negative. These findings suggested that in addition to Fnr, DNA︿ upercoiling was another factor for the regulation of anaerobic respiratory gene expression.

碩士
國立交通大學
生物科技研究所
86
LacZ operon is regulated by DNA supercoiling which expression increases on the negative supercoiling template. In previously study, we found that lacZ expression was growth rate-dependent in Escherichia coli . The intracellular ATP/ATP ratio has been proved directly related to DNA supercoiling because gyrase activity is ATP dependent. Therefore, the growth rate control of lacZ expression might be dependent on the intracellular ATP/ADP ratio and DNA superhelicity changes. In this study, we determined the pla We also examined the total cAMP concentration during exponential growth of Escherichia coli on different carbon medium and at different growth rate in chemostat cultures. The cAMP concentration decreased with increasing cell growth rate. Further introduction of cya mutant into lacZ fusion strain, we found that growth rate-dependent regulation of lacZ expression pattern was changed. Therefore, we proposed that growth rate control of lacZ expression is cAMP dependent .

碩士
國立交通大學
生物科技研究所
84
We determined the plasmid pBR322 superhelicity at different cell growth rate in Escherichia coli and found that DNA supercoiling was growth rate depedent. The DNA supercoiling of plasmid pBR322 was more negitive when the cell growth rate increassed. Since the expression of lac promoter increased with increasing superhelical density and gyrA promoter was a reciprocalresponse to changes in superhelical density; therefore, we also examined theexpression of lac and gyrA promoters during expontial growth of Escherichia coli on different medium and at different growth rate in chemostat cultures.The expression of lac promoter decreased monotonically with increasing cell growth rate, gyrA promoter expression showed a reciprocal response. When theDNA supercoiling was perturbed by topA10 mutant or gyrA inhibitor novobiocin,the effect of cell growth rate on lacZ operon expression pattern was changed.In the topA10 mutant, the lacZ operon expression did not regulate by cell growth rate when the specific growth rate was lower than 0.72 1/hr. If mediumcontained 25 ug/ml novobiocin and the specific growth rate is higher than 0.61/hr, the lac promoter expression became growth rate independent. Thus, changesin cell growth rate affected the DNA supercoiling of Escherichia coli as well as the growth rate depent genes, lacZ and gyrA, expression.

碩士
國立交通大學
生物科技研究所
85
Fumarase , one of the tricarboxylic acid cycle enzymes, catalyzes the interconversion of fumarate and L-malate. Three biochemically distinct fumarase have been reported in E.coli. While the fumA and fumB genes encode heat-labile , iron- containing fumarases , the fumC gene product is a heat-stable fumarase which does not reguire iron for its activity. Although three fumarase genes are regulated by Fnr and ArcA proteins , there still has some induction or repression during anaerobic cell growth in fnr and ArcA proteins , there still has some induction or repression during anaerobic cell growth in fnr and arcA double mutant. Hence Fnr and ArcA might not be the only reguirement for the anaerobic regulators of fumarase genes. A topological state of the bacterial chromosome is important for transcription , replication and recombination. To determine how the fumA , fumB and fumC genes are regulated in response to a variety of DNA supercoiling , inhibitors of gyrase and topoisomerase mutation were induced into cells and examined. The results indicated that the effect of supercoiling on expression of three fumarase genes were more conspicious when cells were grown under aerobic conditions. The existance of Fnr and ArcA would affect the effect of varied DNA supercoiling structures on fumA and fumB genes expression , while topA10 mutant caused an increasement of fumC-lacZ expression. Therefore , the change of DNA supercoiling structure will affect the expression of three fumarase genes , and this control is part of mediated by Fnr and ArcA regulators.

碩士
國立交通大學
生物科技研究所
87
Escherichia coli exhibits diverse respiratory abilities. It synthesizes at least two distinctive cytochrome oxidases during aerobic growth and additional three terminal oxidoreductases for anaerobic respiration. Although two broadly acting regulators, Fnr and ArcA, independent control of oxygen-regulated genes have been identified, in fnr and arcA mutants they still show the difference during anaerobiosis. A topological state of the bacterial chromosome is important for transcription and replication. DNA superhelicity becomes more negative under anaerobic condition in E. coli. To determine how the respiratory genes are regulated in response to a variety of DNA supercoiling during aerobiosis the DNA gyrase inhibitors, salt and topoisomerase I mutants were examined on the lacZ reporter fusions in the wild-type, fnr and arcA mutants. When DNA supercoiling was relaxed, the expression of cyo-lacZ, cyd-lacZ, narG-lacZ, dmsA-lacZ, and frdA-lacZ decreased under aerobic and anaerobic growth. In contrast, the expression of five respiratory genes were activated when negative DNA supercoiling was introduced into the strains. These findings suggestted that in addition to Fnr and ArcA, DNA supercoiling was another factor for regulation of respiratory genes expression. Fumarase is one of the TCA cycle enzyme. fumA and fumC genes are induced under aerobic growth condition and are repressed by Fnr and ArcA. In this study, we demonstrated that the change of DNA supercoiling structure would activate their expression. Because fumA and fumC genes was controlled by growth rate, therefore, the growth rate might affect DNA supercoiling and result in changing fumA and fumC genes expression.

Aufgrund einer komplexen Wechselwirkung mit Proteinen ist das Genom in einer Zelle ständig mechanischer Spannung und Torsion ausgesetzt. Daher ist es wichtig die Mechanik und die Dynamik von verdrillter DNA unter Spannung zu verstehen. Diese Situation wurde experimentell mittels einer sog. magnetischen Pinzette nachgestellt, indem sowohl Kraft als auch Drehmoment auf ein einzelnes DNA Molekül ausgeübt und gleichzeitig die mechanische Antwort des Polymers aufgezeichnet wurde. Als erstes Beispiel wurde der Übergang von linearer zu sog. plectonemischer DNA untersucht, d.h. die Absorption eines Teils der induzierten Verdrillung in einer superhelikalen Struktur. Eine abrupte Längenänderung am Anfang dieses Übergangs wurde bereits im Vorfeld publiziert. In der vorliegenden Arbeit wird gezeigt, dass diese abrupte DNA Verkürzung insbesondere von der Länge der DNA und der Ionenkonzentration der Lösung abhängt. Dieses Verhalten kann mittels eines Modells verstanden werden, in dem die Energie pro Verwringung der ersten Schlinge innerhalb der Superhelix größer ist als die aller nachfolgenden. Des Weiteren wurden DNA-DNA Wechselwirkungen in der Umgebung monovalenter Ionen durch die Analyse des Superspiralisierungsverhaltens einzelner DNA Moleküle bei konstanter Kraft charakterisiert. Solche Wechselwirkungen sind für die Kompaktierung des Genoms und die Regulation der Transkription wichtig. Oft wird DNA als gleichmäßig geladener Zylinder modelliert und ihre elektrostatischen Wechselwirkungen im Rahmen der Poisson-Boltzmann-Gleichung mit einem Ladungsanpassungsfaktor berechnet. Trotz erheblicher Anstrengung ist eine präzise Bestimmung dieses Parameters bisher nicht gelungen. Ein theoretisches Modell dieses Prozesses zeigte nun eine erstaunlich kleine effektive DNA Ladung von ~40% der nominalen Ladungsdichte. Abgesehen von Gleichgewichtsprozessen wurde auch die Dynamik eines Faltungsvorgangs von DNA untersucht. Spontane Branch Migration einer homologen Holliday-Struktur wurde genutzt, um die intramolekulare Reibung der DNA zu erforschen. Mittels einer magnetischen Pinzette wurde eine torsionslimitierte Holliday-Struktur gestreckt während die Längenfluktuationen der Zweige mit schneller Videomikroskopie bei ~3 kHz aufgezeichnet wurden. Einzelne diffusive Schritte der Basenpaare sollten auf einer sub-Millisekunden Zeitskala auftreten und viel kleiner als die Gesamtfluktuationen der DNA sein. Eine Analyse der spektralen Leistungsdichte der Längenfluktuationen ermöglicht eine eindeutige Beschreibung der Dynamik der Branch Migration. Die Holliday-Struktur wurde außerdem als nanomechanischer Linearversteller eingesetzt, um einen einzelnen fluoreszierenden Quantenpunkt durch ein exponentiell abfallendes evaneszentes Feld zu bewegen. Durch die Aufzeichnung der Emission des Quantenpunkts sowohl in dem evaneszenten Feld als auch unter gleichmäßiger Beleuchtung kann die Intensitätsverteilung des Anregungsfelds ohne weitere Dekonvolution bestimmt werden. Diese neue Technik ist von besonderem wissenschaftlichen Interesse, weil die Beschreibung dreidimensionaler inhomogener Beleuchtungsfelder eine große Herausforderung in der modernen Mikroskopie darstellt. Die Ergebnisse dieser Arbeit werden dem besseren Verständnis einer Vielzahl biologischer Prozesse, die in Verbindung mit DNA Superspiralisierung stehen, dienen und weitere technische Anwendungen des DNA-basierten Linearverstellers hervorbringen.
The genome inside the cell is continuously subjected to tension and torsion primarily due to a complex interplay with a large variety of proteins. To gain insight into these processes it is crucial to understand the mechanics and dynamics of twisted DNA under tension. Here, this situation is mimicked experimentally by applying force and torque to a single DNA molecule with so called magnetic tweezers and measuring its mechanical response. As a first example a transition from a linear to a plectonemic DNA configuration is studied, i.e. the absorption of part of the applied twist in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. Here, it is found that this abrupt DNA shortening strongly depends on the length of the DNA molecule and the ionic strength of the solution. This behavior can be well understood in the framework of a model in which the energy per writhe for the initial plectonemic loop is larger than for subsequent turns of the superhelix. Furthermore DNA-DNA interactions in the presence of monovalent ions were comprehensively characterized by analyzing the supercoiling behavior of single DNA molecules held under constant tension. These interactions are important for genome compaction and transcription regulation. So far DNA is often modeled as a homogeneously charged cylinder and its electrostatic interactions are calculated within the framework of the Poisson-Boltzmann equation including a charge adaptation factor. Despite considerable efforts, until now a rigorous quantitative assessment of this parameter has been lacking. A theoretical model of this process revealed a surprisingly small effective DNA charge of ~40% of the nominal charge density. Besides describing equilibrium processes, also the dynamics during refolding of nucleic acids is investigated. Spontaneous branch migration of a homologous Holliday junction serves as an ideal system where the friction within the biomolecule can be studied. This is realized by stretching a torsionally constrained Holliday junction using magnetic tweezers and recording the length fluctuations of the arms with high-speed videomicroscopy at ~3 kHz. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Apart from studying intramolecular friction, the four-arm DNA junction was also used as a nanomechanical translation stage to move a single fluorescent quantum dot through an exponentially decaying evanescent field. Recording the emission of the quantum dot within the evanescent field as well as under homogeneous illumination allows to directly obtain the intensity distribution of the excitation field without additional deconvolution. This new technique is of particular scientific interest because the characterization of three-dimensional inhomogeneous illumination fields is a challenge in modern microscopy. The results presented in this work will help to better understand a large variety of biological processes related to DNA supercoiling and inspire further technical applications of the nanomechanical DNA gear.

碩士
國立交通大學
生物科技研究所
89
Bacterial can generate energy by using different carbon sources. The energy state of Escherichia coli varied with nutrient and growth conditions. In this study we used continuous culture to study how the different cell growth rates and different carbon sources affect the energy state of E. coli. The result showed that the ATP/ADP ration and energy charge were all raised with cell growth rate when glucose, acetate, succinate, glycerol were used as carbon sources. In the study of relationship between ATP/ADP ratio and DNA supercoiling, had observed that ATP/ADP ratio raise with increasing cell growth rate and more negative DNA supercoiling. In this study, we also used the continuous culture and control the cell at constant growth rate (k = 1.2), to determine the effect of carbon source concentration on the ATP/ADP ratio and the DNA supercoiling. The result suggested that DNA supercoiling is dependent on ATP/ADP ratio but not on cell growth rate. It has been reported that the TCA cycle enzymes, such as succinate dehydrogenase, malat dehydrogenase, fumarate dehydrogenase, expression decreased with increasing cell growth rate. Since the NADH+H+ is the major product of TCA cycle which will into the respiratory chain to generate ATP. In this study, we observed that the ATP concentration increased with increasing cell growth rate. Also, we measured expression of the two respiratory genes (cyoABCDE, cydAB), and we found these two genes expression decreased with increasing cell growth rate. This results suggested that at the high growth rate, the energy may be generate from substrate level phosphorylation. In addition, cyo and cyd genes showed the phenomenon of catabolite repression, and the phenomenon was disappeared in the cya mutant strain (the strain can not generate cAMP). Therefore, it suggested that cAMP is an important factor related to cyo and cyd gene expression at different growth rate.

博士
國立交通大學
生物科技研究所
92
Bacteria can generate energy by using different carbon sources. The energy state of Escherichia coli varies with nutrient and growth rate conditions as well as regulated gene expressions. In contrast to ribosomal RNA gene expression, we use lac operon as the model to demonstrate gene expression in E. coli, and the lac operon is down regulated by cell growth rate. We found growth rate regulation of lac promoter was independent of both carbon substrate used and its location on the chromosome in continuous culture. In cAMP mutant, but not ppGpp mutant, expression of plac-lacZ was both lower and growth rate independent. Thus, ours results indicate that cAMP mediates the growth rate-dependent regulation of lac operon expression in E. coli. In anaerobiosis, we used fumarate reductase gene (frdABCD) as the model system. Fumarate reductase carries out the reduction of fumarate to succinate and is repressed in the presence of oxygen or nitrate. Expression of frdABCD operon in Escherichia coli is regulated by oxygen and cell growth rate. Early study has shown that FNR is an anaerobic activator of frdABCD operon expression. Although FNR is responsible for the anaerobic activation of frdABCD operon expression, a three to four-fold aerobic/anaerobic control of frdA-lacZ expression has still been seen in the fnr deletion strain suggesting that the cells have an alternative means to regulate the frdABCD operon in response to anaerobiosis. Besides, expression of the frdABCD operon has been found to be growth rate-dependent in aerobic continuous culture but the regulation involved is still unclear. Since intracellular cAMP also plays an important role on gene expression under anaerobic conditions and its concentration decreased at high cell growth rates in glucose-limited chemostats. In addition, the structure of CRP shows significant homology to FNR. So cAMP-CRP may be another factor regulating the expression of frdABCD operon during anaerobic growth and at different cell growth rates. To determine whether cAMP is involved in anaerobiosis and growth rate-dependent regulation of frdABCD operon, we examined the role of cya and crp gene products on frdA-lacZ fusion expression. In batch culture, frdA-lacZ expression was repressed by the presence of glucose and decreased in cya mutant under anaerobic conditions whereas it was slightly (< 20%) regulated by the crp gene product. In continuous culture, we further demonstrate that cAMP in addition to FNR was needed to activate frdA-lacZ expression in response to anaerobiosis. Gel retardation assay directly shows that cAMP-CRP complex could bind to the FNR binding-site of frdABCD promoter and that this binding is cAMP dependent. Thus, our studies have revealed that cAMP is involved in glucose, oxygen, and growth rate-dependent regulation of frdABCD operon expression in E. coli. DNA supercoiling, which is highly related to the ATP/ADP ratio, is another global regulator of gene expression. The enzymes that have the potential to control the level of DNA supercoiling are topoisomerase I (topA) and DNA gyrase (gyrA and gyrB). Our previous studies demonstrated that different growth rate conditions will change DNA supercoiling, and that cellular energy plays a role in its control. The ATP/ADP ratio increased with cell growth rate, and simultaneously the DNA supercoiling became more negative at a high growth rate. Although the growth rate regulation of the ATP/ADP ratio and DNA supercoiling was carbon source utilization independent, the DNA supercoiling was dependent on the ATP/ADP ratio but not on cell growth rate. In addition, cAMP-CRP complex regulated the expression of gyrA gene in E. coli. To deetrmine the effect of cAMP and cell growth rate on DNA spercoiling, we examined DNA supercoiling in cya mtant. Interestingly, in this study we found that the ATP/ADP ratio and DNA supercoiling were decontrolled by cell growth rate in cya deletion strain. This indicates that the thermodynamic ATP/ADP ratio regulation of DNA supercoiling was dependent on cAMP in E. coli. On transcription level, the cAMP could effect the expression of gyrA, gyrB and topA genes, which regulated DNA supercoiling. For cultures grown in glucose medium under aerobic conditions at a low growth rate (k = 0.24/h), expression of topA gene decreased by 40% in cya mutant compared to wild-type strain. These results show that DNA supercoiling was more negative in cya mutant than in wild-type strain at a low cell growth rate. In addition, DNA supercoiling was not changed in cya mutant relative to wild-type strain at a high growth rate (k=0.96), and this may result from the increased expression of gyrA, gyrB, and topA genes in cya mutant.

In the current dissertation, efforts have been made to probe the in vivo role of DNA gyrase to determine its importance in the growth, physiology and gene expression in mycobacteria. In this dissertation, the role of DNA gyrase in gene expression were explored. The thesis is divided into four chapters. In Chapter I, a general introduction to DNA topology, genome supercoiling, DNA topoisomerases, their mechanism of action Synopsis xvi and regulation is provided. It covers a description of the central player- DNA gyrase followed by its functions in vitro and various factors involved in the regulation of supercoiling. Further, the regulation of topoisomerase activity and the role in gene regulation has been described. In Chapter 2, the studies are aimed at generation and characterization of conditional gyrase mutant in M. smegmatis. Depletion of gyrase level beyond the 50% threshold drastically impaired cell growth and viability indicating the minimal gyrase level is required for cell sustenance. Various pleiotropic effects, altered colony morphology, elongated cells and diffused nucleoid were observed in the gyrase-depleted cells. The perturbation in the gyrase level resulted in a reduction of FtsZ levels in elongated cells suggesting the link between gyrase and cell division. Further, transcript analysis indicated gyrase as a global regulator modulating the expression of the genes involved in encoding topology modulators, transcription and core DNA transaction processes. Altered transcription pattern was a result of impaired occupancy of RNAP at the promoters and coding sequences. The gyrase knockdown strain acquired increased sensitivity to drugs used in TB treatment demonstrating its utility to screen new anti-tubercular drugs. The study thus establishes the essentiality of gyrase for mycobacterial growth, physiology and illustrates the consequences of perturbing intracellular gyrase levels on gene expression in mycobacteria. In Chapter 3, studies have been carried out to validate the in vivo functionality of the MtGyrBA fusion protein. Since gyrB and gyrA of M. tuberculosis are separated by a 34 bp stretch and transcription is controlled by the presence of 3 promoters, efforts have been directed towards constructing the fusion gyrase by linking the gyrB and gyrA genes together with a 6 bp linker sequence. The gyrBA fusion has been expressed in a mycobacterial vector under an inducible promoter. MtGyrBA rescued the growth defect showed by the M. smegmatis gyrase-depleted cells and partially complemented the E. coli ts mutants. The utility of the complemented strain has been tested to screen for drugs that target the M. tuberculosis gyrase in the background of fast growing M. smegmatis. Chapter 4 of the thesis focuses on the transcriptomic landscape of M. tuberculosis gene expression using novobiocin as an agent to bring about the relaxation of the genome at 6 hr and restoration of supercoiling due to relaxation-stimulated transcription (RST) at 24 hr of treatment. Treatment with the inhibitor changed the expression of a large number of genes. 53% of the genome exhibited relaxation-dependent transcription while 41% showed supercoiling-sensitive transcription. Genes with altered expression are distributed as large clusters across the chromosome with a distinct pattern observed during chromosome relaxation. The presence supercoil-sensitive genes interspersed between the clusters were also detected. The downregulated genes had higher AT percentage both upstream and downstream of transcription start site. Three major groups of supercoiling-sensitive genes have been identified throughout the genome: up-regulated (U), down-regulated (D) and less-responsive (LR). Thus, organization of supercoil-sensitive genes in the M. tuberculosis genome reveals DNA supercoiling as a major controller of gene expression in pathogenic mycobacteria. In conclusion, the results presented in this dissertation indicate a close connection between transcription regulation and topology by DNA gyrase mediated supercoiling

碩士
國立交通大學
生物科技系所
92
To understand the expression of ATP generation and cell division genes in different carbon sources, we individually used acetate, glucose, glycerol or succinate as a sole carbon for energy source. The results of this study showed that the expression of ATP generating genes in metabolic pathway varied with carbon sources and ATP concentration increased with cell growth rate. Comparision with the wild-type strain and relA spoT double mutant, the growth rate and ATP yields were changed, but ATP/ADP ratio remained at the same level. DNA supercoiling was dependent on ATP/ADP ratio. Whereas ppGpp did not change DNA supercoiling. Under various growth conditions, fast-growing E. coli cells were larger than slowly growing ones. Starvation of the cells resulted in filamentous morphology. In addition, the results clearly showed that relA spoT double mutant had more filamentous than wild-type cells regardless acetate or glucose as carbon substrates. It was also notable that the filamentous features provided the phenotypic clues for ppGpp function. However, the morphology raised the possibility of indirect, rather than direct, effects of ppGpp. It indicated that a link between the levels of ppGpp and cell division, which ppGpp could act as a positive regulator of the expression of ftsZ gene. Deficiency of ppGpp (relA spoT double mutant) drastically reduced the expression of minC and minD. These results also suggested that ppGpp was important factor involved in the regulation of cell cycle of E. coli under starvation condition.

Le surenroulement de l’ADN est important pour tous les processus cellulaires qui requièrent la séparation des brins de l’ADN. Il est régulé par l’activité enzymatique des topoisomérases. La gyrase (gyrA et gyrB) utilise l’ATP pour introduire des supertours négatifs dans l’ADN, alors que la topoisomérase I (topA) et la topoisomérase IV (parC et parE) les éliminent. Les cellules déficientes pour la topoisomérase I sont viables si elles ont des mutations compensatoires dans un des gènes codant pour une sous-unité de la gyrase. Ces mutations réduisent le niveau de surenroulement négatif du chromosome et permettent la croissance bactérienne. Une de ces mutations engendre la production d'une gyrase thermosensible. L’activité de surenroulement de la gyrase en absence de la topoisomérase I cause l’accumulation d’ADN hyper-surenroulé négativement à cause de la formation de R-loops. La surproduction de la RNase HI (rnhA), une enzyme qui dégrade l’ARN des R-loops, permet de prévenir l’accumulation d’un excès de surenroulement négatif. En absence de RNase HI, des R-loops sont aussi formés et peuvent être utilisés pour déclencher la réplication de l’ADN indépendamment du système normal oriC/DnaA, un phénomène connu sous le nom de « constitutive stable DNA replication » (cSDR). Pour mieux comprendre le lien entre la formation de R-loops et l’excès de surenroulement négatif, nous avons construit un mutant conditionnel topA rnhA gyrB(Ts) avec l’expression inductible de la RNase HI à partir d’un plasmide. Nous avons trouvé que l’ADN des cellules de ce mutant était excessivement relâché au lieu d'être hypersurenroulé négativement en conditions de pénurie de RNase HI. La relaxation de l’ADN a été montrée comme étant indépendante de l'activité de la topoisomérase IV. Les cellules du triple mutant topA rnhA gyrB(Ts) forment de très longs filaments remplis d’ADN, montrant ainsi un défaut de ségrégation des chromosomes. La surproduction de la topoisomérase III (topB), une enzyme qui peut effectuer la décaténation de l’ADN, a corrigé les problèmes de ségrégation sans toutefois restaurer le niveau de surenroulement de l’ADN. Nous avons constaté que des extraits protéiques du mutant topA rnhA gyrB(Ts) pouvaient inhiber l’activité de surenroulement négatif de la gyrase dans des extraits d’une souche sauvage, suggérant ainsi que la pénurie de RNase HI avait déclenché une réponse cellulaire d’inhibition de cette activité de la gyrase. De plus, des expériences in vivo et in vitro ont montré qu’en absence de RNase HI, l’activité ATP-dépendante de surenroulement négatif de la gyrase était inhibée, alors que l’activité ATP-indépendante de cette enzyme demeurait intacte. Des suppresseurs extragéniques du défaut de croissance du triple mutant topA rnhA gyrB(Ts) qui corrigent également les problèmes de surenroulement et de ségrégation des chromosomes ont pour la plupart été cartographiés dans des gènes impliqués dans la réplication de l’ADN, le métabolisme des R-loops, ou la formation de fimbriae. La deuxième partie de ce projet avait pour but de comprendre les rôles des topoisomérases de type IA (topoisomérase I et topoisomérase III) dans la ségrégation et la stabilité du génome de Escherichia coli. Pour étudier ces rôles, nous avons utilisé des approches de génétique combinées avec la cytométrie en flux, l’analyse de type Western blot et la microscopie. Nous avons constaté que le phénotype Par- et les défauts de ségrégation des chromosomes d’un mutant gyrB(Ts) avaient été corrigés en inactivant topA, mais uniquement en présence du gène topB. En outre, nous avons démontré que la surproduction de la topoisomérase III pouvait corriger le phénotype Par- du mutant gyrB(Ts) sans toutefois corriger les défauts de croissance de ce dernier. La surproduction de topoisomérase IV, enzyme responsable de la décaténation des chromosomes chez E. coli, ne pouvait pas remplacer la topoisomérase III. Nos résultats suggèrent que les topoisomérases de type IA jouent un rôle important dans la ségrégation des chromosomes lorsque la gyrase est inefficace. Pour étudier le rôle des topoisomérases de type IA dans la stabilité du génome, la troisième partie du projet, nous avons utilisé des approches génétiques combinées avec des tests de « spot » et la microscopie. Nous avons constaté que les cellules déficientes en topoisomérase I avaient des défauts de ségrégation de chromosomes et de croissance liés à un excès de surenroulement négatif, et que ces défauts pouvaient être corrigés en inactivant recQ, recA ou par la surproduction de la topoisomérase III. Le suppresseur extragénique oriC15::aph isolé dans la première partie du projet pouvait également corriger ces problèmes. Les cellules déficientes en topoisomérases de type IA formaient des très longs filaments remplis d’ADN d’apparence diffuse et réparti inégalement dans la cellule. Ces phénotypes pouvaient être partiellement corrigés par la surproduction de la RNase HI ou en inactivant recA, ou encore par des suppresseurs isolés dans la première partie du projet et impliques dans le cSDR (dnaT18::aph et rne59::aph). Donc, dans E. coli, les topoisomérases de type IA jouent un rôle dans la stabilité du génome en inhibant la réplication inappropriée à partir de oriC et de R-loops, et en empêchant les défauts de ségrégation liés à la recombinaison RecA-dépendante, par leur action avec RecQ. Les travaux rapportés ici révèlent que la réplication inappropriée et dérégulée est une source majeure de l’instabilité génomique. Empêcher la réplication inappropriée permet la ségrégation des chromosomes et le maintien d’un génome stable. La RNase HI et les topoisomérases de type IA jouent un rôle majeur dans la prévention de la réplication inappropriée. La RNase HI réalise cette tâche en modulant l’activité de surenroulement ATP-dependante de la gyrase, et en empêchant la réplication à partir des R-loops. Les topoisomérases de type IA assurent le maintien de la stabilité du génome en empêchant la réplication inappropriée à partir de oriC et des R-loops et en agissant avec RecQ pour résoudre des intermédiaires de recombinaison RecA-dépendants afin de permettre la ségrégation des chromosomes.
DNA supercoiling is important for all cellular processes that require strand separation and is regulated by the opposing enzymatic effects of DNA topoisomerases. Gyrase uses ATP to introduce negative supercoils while topoisomerase I (topA) and topoisomerase IV relax negative supercoils. Cells lacking topoisomerase I are only viable if they have compensatory mutations in gyrase genes that reduce the negative supercoiling level of the chromosome to allow bacterial growth. One such mutation leads to the production of a thermosensitive gyrase (gyrB(Ts)). Gyrase driven supercoiling during transcription in the absence of topoisomerase I causes the accumulation of hypernegatively supercoiled plasmid DNAs due to the formation of R-loops. Overproducing RNase HI (rnhA), an enzyme that degrades the RNA moiety of R-loops, prevents the accumulation of hypernegative supercoils. In the absence of RNase HI alone, R-loops are equally formed and can be used to prime DNA replication independently of oriC/DnaA, a phenomenon known as constitutive stable DNA replication (cSDR). To better understand the link between R-loop formation and hypernegative supercoiling, we constructed a conditional topA rnhA gyrB(Ts) mutant with RNase HI being conditionally expressed from a plasmid borne gene. We found that the DNA of topA rnhA gyrB(Ts) cells was extensively relaxed instead of being hypernegatively supercoiled following the depletion of RNase HI. Relaxation was found to be unrelated to the activity of topoisomerase IV. Cells of topA rnhA gyrB(Ts) formed long filaments full of DNA, consistent with segregation defect. Overproducing topoisomerase III (topB), an enzyme that can perform decatenation, corrected the segregation problems without restoring supercoiling. We found that extracts of topA rnhA gyrB(Ts) cells inhibited gyrase supercoiling activity of wild type cells extracts in vitro, suggesting that the depletion of RNase HI triggered a cell response that inhibited the supercoiling activity of gyrase. Gyrase supercoiling assays in vivo as well as in crude cell extracts revealed that the ATP dependent supercoiling reaction of gyrase was inhibited while the ATP independent relaxation reaction was unaffected. Genetic suppressors of a triple topA rnhA gyrB(Ts) strain that restored supercoiling and corrected the chromosome segregation defects mostly mapped to genes that affected DNA replication, R-loop metabolism and fimbriae formation. The second part of this project aimed at understanding the roles of type IA DNA topoisomerases (topoisomerase I and topoisomerase III) in chromosome segregation and genome maintenance in E. coli. To investigate the role of type IA DNA topoisomerases in chromosome segregation we employed genetic approaches combined with flow cytometry, Western blot analysis and microscopy (for the examination of cell morphology). We found that the Par- phenotypes (formation of large unsegregated nucleoid in midcell) and chromosome segregation defects of a gyrB(Ts) mutant at the nonpermissive temperature were corrected by deleting topA only in the presence of topB. Moreover, overproducing topoisomerase III was shown to correct the Par- phenotype without correcting the growth defect, but overproducing topoisomerase IV, the major cellular decatenase, failed to correct the defects. Our results suggest that type IA topoisomerases play a role in chromosome segregation when gyrase is inefficient. To investigate the role of type IA DNA topoisomerases in genome maintenance, in the third part of the project, we employed genetic approaches combined with suppressor screens, spot assays and microscopy. We found that cells lacking topoisomerase I suffered from supercoiling-dependent growth defects and chromosome segregation defects that could be corrected by deleting recQ, recA or overproducing topoisomerase III and by an oriC15::aph suppressor mutation isolated in the first part of the project. Cells lacking both type 1A topoisomerases formed very long filaments packed with diffuse and unsegregated DNA. Such phenotypes could be partially corrected by overproducing RNase HI or deleting recA, or by suppressor mutations isolated in the first part of the project, that affected cSDR (dnaT18::aph and rne59::aph). Thus, in E. coli, type IA DNA topoisomerases play a role in genome maintenance by inhibiting inappropriate replication from oriC and R-loops and by preventing RecA-dependent chromosome segregation defect through their action with RecQ. The work reported here reveals that inappropriate and unregulated replication is a major source of genome instability. Preventing such replication will ensures proper chromosome segregation leading to a stable genome. RNase HI and type IA DNA topoisomerases play a leading role in preventing unregulated replication. RNase HI achieves this role by modulating ATP dependent gyrase activity and by preventing replication from R-loops (cSDR). Type IA DNA topoisomerases ensure the maintenance of a stable genome by preventing inappropriate replication from oriC and R-loops and by acting with RecQ to prevent RecA dependent-chromosome segregation defects.

Genus Mycobacterium comprises a large number of species including many pathogens such as Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one is the causative agent of the fatal disease tuberculosis. The unique features of the deadly organism viz, slow growing, tough cell wall, latency and resistance to various drugs demand a systematic understanding of many essential molecular processes including transcription. Studies have been undertaken to understand several aspects of transcription in mycobacteria which revealed its machinery to be conserved with other eubacteria? However, many facets of transcription in mycobacteria and regulation are different. The transcription regulators and them regulation are the basic counter stones which govern gene expression. The present study is aimed to understand better the mechanistic regulation of transcription of important housekeeping functions, DNA gyrase and also to obtain further insights into the role of transcription elongation factor Gre. Chapter 1 of the thesis provides a general introduction of the bacterial transcription machinery, associated transcription regulators and their regulation. It covers the description of the central player- the RNA polymerase (RNAP) followed by each step of the transcription initiation, elongation and various factors involved in their regulation. Finally, an overview of the emerging information on several aspects of mycobacterial transcription is discussed emphasizing on RNAP, promoter architecture, and its regulation. In Chapter 2, the studies are directed to understand the mechanism for topology-dependent regulation of Mtb Gyrase. The gyrase is encoded by two genes gyrB and gyrA which form a bicistronicity operon in Mtb and harbor multiple promoters. The principal promoter PgyrB1 drives the transcription of the dicistron and the weaker divergent promoter PgyrR is engaged in transcription in the opposite direction. The divergent and overlapping PgyrR show decrease in activity when the PgyrB1 was induced upon relaxation of the genome by a phenomenon termed relaxation stimulated transcription (RST). PgyrR plays a role in the fine tuning of gyr gene expression by reiterative transcription (RT), a regulatory mechanism hitherto not described in Mtb. In vitro transcription assays show that RT at PgyrR is dependent on the negatively supercoiled status of the DNA. The principal promoter PgyrB1 is also regulated by DNA topology but does not exhibit RT. It is elucidated that the RNAP binding is efficient at PgyrB1 when the DNA is relaxed whereas binding to PgyrR is preferred when DNA is supercoiled. Thus, a collaboration between RST and RT govern the regulation of gyr operon; the differential topology sensitivity of the overlapping promoters determines and dictate the efficiency of transcription initiation at gyr promoters. In addition, this study suggests a new mechanism of RST distinct from the one observed for other bacteria, such as E. coli or M. smegmatis. Chapter 3 describes studies that have been carried out to delineate the mechanism underlying the differential function of transcription regulator MtbGreA and its homolog Rv3788 (MtbGfh1). MtbGreA binds to RNAP and induces the intrinsic transcript cleavage activity of RNAP thereby allowing RNAP to resume transcription from paused and arrested sites. In spite of having Gre like domains, MtbGfh1 does not stimulate RNA cleavage. Instead, it inhibits transcription by binding to RNAP. Homology modeling and docking data suggest that Gre and MtbGfh1 bind to RNAP in a different orientation. MtbGreA coordinate with the Mg2+ present in the catalytic center of the RNAP while MtbGfh1 was observed to be facing away from Mg2+ Swapping of a stretch of residues from the N-terminus of MtbGreA into MtbGfh1 acquire GreA like transcript cleavage stimulatory activity and enhance promoter clearance for MtbGfh1. Bioinformatics analysis and biochemical assays demonstrate the significance of a stretch of residues in the N-terminus of MtbGreA and MtbGfh1 for their functions. Also, the orientation of the MtbGreA and MtbGfh1 while binding to RNAP is a crucial determinant in governing their respective function. Being the general inhibitor of transcription, overexpression of MtbGfh1 led to the appearance of tiny colonies and slow growth of cells suggesting its regulatory role to maintain the physiology of Mtb. In Chapter 4, the influence of perturbation of GreA level on Mtb growth and physiology has been studied. Mtb contains a single Gre protein (Rv1080c), unlike many other bacteria where both GreA and GreB are present. Further, the GC-rich genome of Mtb may pose an additional challenge to the transcribing RNAP. Hence the role of GreA could be essential to maintain high fidelity of transcription and RNAP distribution in Mtb genome. To validate this, the conditional knockdown strain of MtbGreA was generated. GreA depleted strain exhibited slow growth and caused phenotypical changes in Mtb cells. Moreover, the occupancy of RNAP on the promoter and gene body of candidate gene tested was found to be disrupted upon MtbGreA depletion, suggesting the regulatory role of GreA in modulating Mtb physiology.

Des variations importantes du surenroulement de l’ADN peuvent être générées durant la phase d’élongation de la transcription selon le modèle du « twin supercoiled domain ». Selon ce modèle, le déplacement du complexe de transcription génère du surenroulement positif à l’avant, et du surenroulement négatif à l’arrière de l’ARN polymérase. Le rôle essentiel de la topoisomérase I chez Escherichia coli est de prévenir l’accumulation de ce surenroulement négatif générée durant la transcription. En absence de topoisomérase I, l’accumulation de ce surenroulement négatif favorise la formation de R-loops qui ont pour conséquence d’inhiber la croissance bactérienne. Les R-loops sont des hybrides ARN-ADN qui se forment entre l’ARN nouvellement synthétisé et le simple brin d’ADN complémentaire. Dans les cellules déficientes en topoisomérase I, des mutations compensatoires s’accumulent dans les gènes qui codent pour la gyrase, réduisant le niveau de surenroulement négatif du chromosome et favorisant la croissance. Une des ces mutations est une gyrase thermosensible qui s’exprime à 37 °C. La RNase HI, une enzyme qui dégrade la partie ARN d’un R-loop, peut aussi restaurer la croissance en absence de topoisomérase I lorsqu’elle est produite en très grande quantité par rapport à sa concentration physiologique. En présence de topoisomérase I, des R-loops peuvent aussi se former lorsque la RNase HI est inactive. Dans ces souches mutantes, les R-loops induisent la réponse SOS et la réplication constitutive de l’ADN (cSDR). Dans notre étude, nous montrons comment les R-loops formés en absence de topoisomérase I ou RNase HI peuvent affecter négativement la croissance des cellules. Lorsque la topoisomérase I est inactivée, l’accumulation d’hypersurenroulement négatif conduit à la formation de nombreux R-loops, ce qui déclenche la dégradation de l’ARN synthétisé. Issus de la dégradation de l’ARNm de pleine longueur, des ARNm incomplets et traductibles s’accumulent et causent l’inhibition de la synthèse protéique et de la croissance. Le processus par lequel l’ARN est dégradé n’est pas encore complètement élucidé, mais nos résultats soutiennent fortement que la RNase HI présente en concentration physiologique est responsable de ce phénotype. Chose importante, la RNase E qui est l’endoribonuclease majeure de la cellule n’est pas impliquée dans ce processus, et la dégradation de l’ARN survient avant son action. Nous montrons aussi qu’une corrélation parfaite existe entre la concentration de RNase HI, l’accumulation d’hypersurenroulement négatif et l’inhibition de la croissance bactérienne. Lorsque la RNase HI est en excès, l’accumulation de surenroulement négatif est inhibée et la croissance n’est pas affectée. L’inverse se produit Lorsque la RNase HI est en concentration physiologique. En limitant l’accumulation d’hypersurenroulement négatif, la surproduction de la RNase HI prévient alors la dégradation de l’ARN et permet la croissance. Quand la RNase HI est inactivée en présence de topoisomérase I, les R-loops réduisent le niveau d’expression de nombreux gènes, incluant des gènes de résistance aux stress comme rpoH et grpE. Cette inhibition de l’expression génique n’est pas accompagnée de la dégradation de l’ARN contrairement à ce qui se produit en absence de topoisomérase I. Dans le mutant déficient en RNase HI, la diminution de l’expression génique réduit la concentration cellulaire de différentes protéines, ce qui altère négativement le taux de croissance et affecte dramatiquement la survie des cellules exposées aux stress de hautes températures et oxydatifs. Une inactivation de RecA, le facteur essentiel qui déclenche la réponse SOS et le cSDR, ne restaure pas l’expression génique. Ceci démontre que la réponse SOS et le cSDR ne sont pas impliqués dans l’inhibition de l’expression génique en absence de RNase HI. La croissance bactérienne qui est inhibée en absence de topoisomérase I, reprend lorsque l’excès de surenroulement négatif est éliminé. En absence de RNase HI et de topoisomérase I, le surenroulement négatif est très relaxé. Il semble que la réponse cellulaire suite à la formation de R-loops, soit la relaxation du surenroulement négatif. Selon le même principe, des mutations compensatoires dans la gyrase apparaissent en absence de topoisomérase I et réduisent l’accumulation de surenroulement négatif. Ceci supporte fortement l’idée que le surenroulement négatif joue un rôle primordial dans la formation de R-loop. La régulation du surenroulement négatif de l’ADN est donc une tâche essentielle pour la cellule. Elle favorise notamment l’expression génique optimale durant la croissance et l’exposition aux stress, en limitant la formation de R-loops. La topoisomérase I et la RNase HI jouent un rôle important et complémentaire dans ce processus.
Important fluctuations of DNA supercoiling occur during transcription in the frame of the “twin supercoiled domain” model. In this model, transcription elongation generates negative and positive supercoiling respectively, upstream and downstream of the moving RNA polymerase. The major role of bacterial topoisomerase I is to prevent the accumulation of transcription-induced negative supercoiling. In its absence, the accumulation of negative supercoiling triggers R-loop formation which inhibits bacterial growth. R-loops are DNA/RNA hybrids formed during transcription when the nascent RNA hybridizes with the template strand thus, leaving the non-template strand single stranded. In cells lacking DNA topoisomerase I, a constant and selective pressure for the acquisition of compensatory mutations in gyrase genes reduces the negative supercoiling level of the chromosome and allows growth. One of these mutations is a thermosensitive gyrase expressed at 37 °C. The overexpression of RNase HI, an enzyme that degrades the RNA moiety of an R-loop, is also able to correct growth inhibition in absence of topoisomerase I. In the presence of topoisomerase I, R-loops can also form when RNase HI is lacking. In these mutants, R-loop formation induces SOS and constitutive stable DNA replication (cSDR). In our study, we show how R-loops formed in cells lacking topoisomerase I or RNase HI can affect bacterial growth. When topoisomerase I is inactivated, the accumulation of hypernegative supercoiling inhibits growth by causing extensive R-loop formation which, in turn, can lead to RNA degradation. As a result of RNA degradation, the accumulation of truncated and functional mRNA instead of full length ones, is responsible for protein synthesis inhibition that alters bacterial growth. The mechanism by which RNA is degraded is not completely clear but our results strongly suggest that RNase HI is involved in this process. More importantly, the major endoribonuclease, RNase E, is not involved in RNA degradation because RNA is degraded before its action. We show also that there is a perfect correlation between RNase HI concentration, the accumulation of hypernegative supercoiling and bacterial growth inhibition. When RNase HI is in excess, no accumulation of hypernegative supercoiling and growth inhibition are observed. The opposite is true when RNase HI is at its wild type level. By preventing the accumulation of hypernegative supercoiling, the overproduction of RNase HI inhibits extensive R-loop formation and RNA degradation, thus, allowing growth. In absence of RNase HI (rnhA) and in presence of topoisomerase I, R-loops are also responsible for an inhibition in gene expression, including stress genes such as rpoH and grpE. The inhibition of gene expression is not related to RNA degradation as seen in absence of topoisomerase I but it is rather related to a reduction in gene expression. In absence of RNase HI, the diminution of genes expression is responsible for a reduction in the cellular level of proteins, which negatively affects bacterial growth and bacterial survival to heat shock and oxydative stress. Additional mutations in RecA, the protein that activates SOS and cSDR after R-loop formation in rnhA, do not correct this phenotype in rnhA. Thus, SOS and cSDR are not directly involved in the inhibition of gene expression in the absence of RNase HI. In absence of topoisomerase I, growth inhibition resumes when hypernegative supercoiling is reduced. When compared to wild type strains, DNA is very relaxed in absence of RNase HI and topoisomerase I. It seems that R-loop formation induces the relaxation of negatively supercoiled DNA. All this strongly supports the idea that negative supercoiling plays an important role in R-loop formation. Finally, our work shows how essential negative supercoiling regulation is for cell physiology. By preventing R-loop formation, regulation of negative supercoiling allows optimal gene expression, which is crucial for cellular growth and for stress survival. Both topoisomerase I and RNase HI play an important and complementary role in this process.

The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication. The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication.

The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication. The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication.

Les topoisomérases I (topA) et III (topB) sont les deux topoisomérases (topos) de type IA d’Escherichia coli. La fonction principale de la topo I est la relaxation de l’excès de surenroulement négatif, tandis que peu d’information est disponible sur le rôle de la topo III. Les cellules pour lesquelles les deux topoisomérases de type IA sont manquantes souffrent d’une croissance difficile ainsi que de défauts de ségrégation sévères. Nous démontrons que ces problèmes sont majoritairement attribuables à des mutations dans la gyrase qui empêchent l’accumulation d’excès de surenroulement négatif chez les mutants sans topA. L’augmentation de l’activité de la gyrase réalisée par le remplacement de l’allèle gyrB(Ts) par le gène de type sauvage ou par l’exposition des souches gyrB(Ts) à une température permissive, permet la correction significative de la croissance et de la ségrégation des cellules topos de type IA. Nous démontrons également que les mutants topB sont hypersensibles à l’inhibition de la gyrase par la novobiocine. La réplication non-régulée en l’absence de topA et de rnhA (RNase HI) augmente la nécessité de l’activité de la topoisomérase III. De plus, en l’absence de topA et de rnhA, la surproduction de la topoisomérase III permet de réduire la dégradation importante d’ADN qui est observée en l’absence de recA (RecA). Nous proposons un rôle pour la topoisomérase III dans la ségrégation des chromosomes lorsque l’activité de la gyrase n’est pas optimale, par la réduction des collisions fourches de réplication s’observant particulièrement en l’absence de la topo I et de la RNase HI.
E. coli possesses two type IA topoisomerases (topos), namely topo I (topA) and topo III (topB). The major function of topo I is the relaxation of excess negative supercoiling. Much less is known about the function of topo III. Cells lacking both type IA topos suffer from severe chromosome segregation and growth defects. We show that these defects are mostly related to the presence of gyrase mutations that prevent excess negative supercoiling in topA null mutants. Indeed, increasing gyrase activity by spontaneous mutations, by substituting a gyrB(Ts) allele for a wild-type one or by exposing cells carrying the gyrB(Ts) allele to permissive temperatures, significantly corrected the growth and segregation defects of cells lacking type IA topo activity. We also found that topB mutants are hypersensitive to novobiocin due to gyrase inhibition. Our data also suggest that unregulated replication occurring in the absence of topA and rnhA (RNase HI) exacerbates the need for topo III activity. Moreover, when topA and rnhA were absent, we found that topo III overproduction reduced the extensive DNA degradation that took place in the absence of recA (RecA). All together, our results lead us to propose a role for topo III in chromosome segregation when gyrase activity is suboptimal, thus reducing replication forks collapse, especially when replication is unregulated due to the absence of topo I and RNase HI.

Les topoisomérases (topos) de type IA jouent un rôle primordial dans le maintien et l’organisation du génome. Cependant, les mécanismes par lesquels elles contrôlent cette stabilité génomique sont encore à approfondir. Chez E. coli, les deux principales topoisomérases de type IA sont la topo I (codée par le gène topA) et la topo III (codée par le gène topB). Il a déjà été montré que les cellules dépourvues des topos I et III formaient de très longs filaments dans lesquels les chromosomes ne sont pas bien séparés. Comme ces défauts de ségrégation des chromosomes sont corrigés par l’inactivation de la protéine RecA qui est responsable de la recombinaison homologue, il a été émis comme hypothèse que les topoisomérases de type IA avaient un rôle dans la résolution des intermédiaires de recombinaison afin de permettre la séparation des chromosomes. D’autre part, des études réalisées dans notre laboratoire démontrent que le rôle majeur de la topoisomérase I est d’empêcher la formation des R-loops durant la transcription, surtout au niveau des opérons rrn. Ces R-loops on été récemment identifiés comme des obstacles majeurs à l’avancement des fourches de réplication, ce qui peut provoquer une instabilité génomique. Nous avons des évidences génétiques montrant qu’il en serait de même chez nos mutants topA. Tout récemment, des études ont montré le rôle majeur de certaines hélicases dans le soutien aux fourches de réplication bloquées, mais aussi une aide afin de supprimer les R-loops. Chez E. coli, ces hélicases ont été identifiées et sont DinG, Rep et UvrD. Ces hélicases jouent un rôle dans la suppression de certains obstacles à la réplication. Le but de ce projet était de vérifier l’implication de ces hélicases chez le mutant topA en utilisant une approche génétique. Étonnamment, nos résultats montrent que la délétion de certains de ces gènes d’hélicases a pour effet de corriger plutôt que d’exacerber des phénotypes du mutants topA qui sont liés à la croissance et à la morphologie des nucléoides et des cellules. Ces résultats sont interprétés à la lumière de nouvelles fonctions attribuées aux topoisomérases de types IA dans la stabilité du génome.
Type 1A topoisomerases (topos) play a vital role in the maintenance and organization of the genome. However, the mechanisms by which they control genome stability still remain to be explored. In E. coli, the two type IA topoisomerases are topo I (encoded by topA) and topo III (encoded by topB). It has been shown that cells lacking topo I and III form very long filaments in which the chromosomes are not well separated. As the chromosome segregation defects are corrected by inactivation of the RecA protein, that is responsible for homologous recombination, it has been hypothesized that type IA topoisomerases have a role in the resolution of recombination intermediates to allow chromosome segregation. On the other hand, studies in our laboratory have shown that the major role of topoisomerase I is to prevent the formation of R-loops during transcription, especially at the rrn operons. These R-loops have been recently identified as major roadblocks to the progression of replication forks, which can cause genomic instability. We have genetic evidence suggesting similar effects may occur in our topA mutants. More recently, studies have shown the important role of certain helicases in eliminating roadblocks for replication forks that could sometimes be R-loops. In E. coli, these helicases have been identified and they are DinG, Rep and UvrD. The purpose of this project was to study the roles of these helicases in our topA mutant, using a genetic approach. Surprisingly, our results show that deletions of some of these genes have the effect of correcting rather than exacerbating topA mutant phenotypes that are related to the growth and cell and nucleoid morphology. These results are interpreted in the light of new functions assigned to the type IA topoisomerases in genome stability.