Dissertations / Theses on the topic 'DNA Gyrase DNA Gyrase DNA Replication DNA'

Argonautes (AGOs) are present in all domains of life. Like their eukaryotic counterparts, archaeal and eubacterial AGOs adopt a similar global architecture and bind small nucleic acids. In many eukaryotes, AGOs, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. We find that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15–18 nt DNA guides derived from the chromosomal region where replication terminates, and TtAgo complexed to short DNA guides enhances target finding and prefers to bind targets with full complementarity. Additionally, TtAgo associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of both gyrase and TtAgo activity slows growth and produces long, segmented filaments in which the individual bacteria are linked by DNA. Furthermore, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. Finally, at physiologic temperature in vitro, we find TtAgo possesses highest affinity for fully complementary targets. We propose that terminus-derived guides binding in such a fashion localize TtAgo, and that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.

DNA gyrase, a type II topoisomerase, catalyses the introduction of negative supercoils into closed-circular DNA, using the energy from ATP hydrolysis. The reaction mechanism involves the breakage of one DNA double strand (the DNA gate) and the passing of another DNA strand (the passage helix) through that break and finally the re-sealing of the DNA gate. The strand-passage reaction was studied by the use of novel DNA substrates and by site-directed mutagenesis of one of the gyrase proteins. The DNA substrates were used to attempt to define the DNA segments used by the enzyme as the DNA gate and passage helix in a catenation reaction. This was achieved by using oligonucleotides to form partial duplex regions in single-stranded DNA. A high-affinity gyrase cleavage site from the plasmid pBR322 was cloned into M13mpl8 and generated both the single and double-stranded circular forms of the molecule (MAT1). It was shown that gyrase could form a specific DNA gate in a short duplex region in single-stranded MAT1 when quinolone drugs were present. This DNA gate was much smaller than that normally utilised by the enzyme. The catenation and decatenation reactions were examined in detail with normal duplex substrates; reactions using a non-hydrolysable ATP analogue gave different results to those previously reported for the eukaryotic homologue of gyrase, indicating a possible mechanistic difference between the enzymes. Conditions under which the partial duplex substrates would be catenated were not found. Site-directed mutagenesis was used to alter arginine residues thought to interact with the passage helix during the reaction cycle. Assays of the mutant protein revealed that supercoiling activity was markedly reduced, but that partial activities of gyrase, such as the ATPase and DNA cleavage reactions, were close to wild-type levels.

DNA gyrase is a molecular machine comprising a series of protein gates. The opening and closing of these gates enables the passage of one segment of double-stranded DNA (the T segment) through a transient break in another (the G segment). We have blocked the passage of DNA through each of three dimer interfaces within gyrase and investigated the effects on gyrase mechanism. This has been achieved by cross-linking novel cysteine residues on either side of the dimer interface, or trapping the dimer interface in a closed conformation using a non-hydrolysable ATP analogue. Cross-linking a pair of novel cysteine residues on either side of the bottom dimer interface of DNA gyrase blocks catalytic supercoiling. Limited strand passage is allowed, but T-segment release is prevented. In contrast, ATP-independent relaxation of negatively supercoiled DNA is completely abolished, suggesting that T-segment entry via the bottom gate is blocked. These findings support a two-gate model for supercoiling in by DNA gyrase and suggest that relaxation by gyrase is the reversal of supercoiling. Cross-linking a truncated version of gyrase, (A642B2) that lacks the DNA wrapping domains, does not block ATP-dependent relaxation. This indicates that passage of DNA through the bottom dimer interface is not essential for this reaction. Using a similar approach, we have locked the DNA gate of gyrase using cysteine cross-linking. We show that this locked-gate mutant can bind quinolone drugs and perform DNA cleavage. However, locking the DNA gate prevents strand passage and the ability of DNA to stimulate ATP hydrolysis.

Purification of the DNA gyrase B protein consistently led to two contaminating bands of 47kDa and 43kDa molecular masses. These were found to be the C- and N-terminal fragments of GyrB. The specific supercoiling activity of GyrB was found to be consistently lower than the specific supercoiling activity of GyrA. This was due to about 90% of GyrB being in an uncoupled form. The uncoupled GyrB was found to have a relatively high ATPase activity, therefore an in-depth kinetic study on DNA gyrase was not possible. Kinetic studies were carried out on the 43kDa protein, which is a cloned N-terminal fragment of GyrB. The 43kDa protein was found to hydrolyse ATP at a relatively low rate, with 10 muM 43kDa having and apparent kcat of 0.01 s-1, and 20 muM a kcat of 0.02 s-1. A greater than first order dependence of rate upon 43kDa concentration was observed in the concentration range of 2-40 muM. Hyperbolic type kinetics were observed at constant 43kDa concentration (5, 10, 20 and 40 ?M) for the rate with respect to ATP concentration. A model which was found to be consistent with molecular weight studies and the kinetic data has been proposed. The 43kDa monomer can bind ATP but is not competent to hydrolyse ATP. Hydrolysis can only occur in the context of a 43kDa2ATP2 dimer, which leads to the collapse of the dimer into monomers and release of products. The rate limiting step at the protein concentration range used is the dimerisation step. Novobiocin and coumermycin inhibit the ATPase reaction, with novobiocin binding at a stoichiometry of 1 novobiocin molecule to 1 43kDa monomer and coumermycin binding with a stoichiometry of 1 molecule to two molecules of 43kDa protein. The inhibition by coumarin drugs appears non-competitive. ADPNP binding to the 43kDa protein was found to be slow, with a second order rate constant of 0.86 M-1s-1 to 9.9 M-1s-1. ADPNP seems to bind with stoichiometries varying from 2 per 43kDa dimer to 1 per 43kDa dimer. ATP and ADP inhibit the amount of ADPNP bound, with ATP having no effect on the rate of ADPNP binding and ADP decreasing the rate of ADPNP binding. Novobiocin and coumermycin inhibit ADPNP binding to the 43kDa protein. ADPNP dissociates from the 43kDa protein at a very slow rate, with a half life of about 8 days. ATP and ADP have little effect on this rate. However high concentrations of novobiocin (10-100 mM) dramatically increase the rate of ADPNP dissociation from the 43kDa protein, indicating different coumarin and nucleotide binding sites.

A study has been conducted aimed at the generation and characterisation of mutations in the Escherichia coli gyrA gene, resistant to the quinolone group of antibacterial agents. Preliminary studies on quinolone-resistant mutants of strains that over-express the DNA gyrA gene, revealed the over-production of a 60 KDa protein which was partially purified. This 60 KDa protein was found to be similar, but not identical to the E. coli heat shock protein GroEL. The gyrA gene has been recloned in the 8.0 kb plasmid pPH3, which contains the gene under the stringent control of the hybrid tac promoter. The E. coli strain JMtacA containing pPH3 exhibits no expression of the gyrA gene in the absence of the inducer (IPTG), but over-produces the protein at greater than 20% of the total soluble cell protein after induction. The optimisation and purification of the GyrA subunit from JMtacA is also described. A fragment was subjected to site-directed mutagenesis which contained the TCG codon for serine-83, which was mutated to alanine (GCG). The mutant showed a 15x increase in MIC50 compared to wild-type. The mutated GyrA subunit was over-produced, complexed with wild-type GyrB subunit and used in various assays for reactions performed by DNA gyrase. The ID50 was determined for supercoiling, decatenation, relaxation of negatively supercoiled DNA, and cleavage of supercoiled DNA. The cleavage reaction mediated by Ca++ was also investigated. The technique of gap-misrepair mutagenesis, geared to the generation of single, random point mutations on a plasmid was also used on the plasmid pPH3, to generate a quinolone-resistant mutant of the gyrA gene. The mutant isolated (GMIOO) was also over-produced and compared to wild-type in various assays. The mutation was determined by DNA sequencing to be glutamine-106 to arginine.

Methoden zur direkten Detektion oder Anreicherung von doppelsträngiger DNA (dsDNA) bieten ein hohes Potential zum Einsatz in der molekularen Diagnostik. Bereits etablierte Methoden für die Nukleinsäure - Detektion (NAD) basieren in der Regel auf der Hybridisierung des komplementären Stranges gefolgt von der optischen Detektion oder enzymatischer Amplifikation. DNA - bindende oder organisierende Proteine (z.B. endogene Transkriptionsfaktoren) bieten im Kontrast zu den Hybridisierungsreaktionen eine überaus interessante Alternative um dsDNA direkt und zugleich spezifisch zu detektieren oder diese aus einem komplexen Gemisch heraus anzureichern. Im Rahmen der Entwicklung von neuartigen NAD - Assays zur direkten Detektion oder Anreicherung von Nukleinsäuren wurden vier DNA - bindende Proteine kloniert und in HEK293 und E. coli exprimiert. Der Cys2His2 - Zinkfinger (ZFD) vom humanen Transkriptionsfaktor Sp1 wurde mit MBP und 9×Lys - MBP fusioniert. Das MBP - Derivat 9×Lys - MBP ist eine erweiterte Variante mit neun aufeinanderfolgenden Lysinen im N - terminalen Bereich, welche eine regioselektive Immobilisierung ermöglichen soll. Der humane Sp3 - ZFD wurde mit EGFP fusioniert. Die Mitglieder der Sp - Familie binden spezifisch die Konsensussequenz 5’ - GGG GCG GGG - 3’ (GC - Box). Zusätzlich wurde die C - terminale DNA - bindende Domäne der E. coli DNA - Gyrase Untereinheit A (gyrA - CTD) ebenfalls mit MBP fusioniert. Die Domäne bindet spezifisch repetitive extragene Palindrome (REP), welche bislang nur auf bakteriellen Chromosomen vorkommen. Sämtliche MBP - Fusionsproteine liegen nach der Expression löslich vor und konnten über eine native Strategie aufgereinigt werden. Transiente Transfektionsexperimente in HEK293 zeigten einen destabilisierenden Effekt der Sp3 - ZFD und eine massive einhergehende Degradierung des EGFP - Fusionsproteins nach 120 h. Die Analyse der mRNA - Integrität nach Transfektion des Expressionsplasmids, sowie zellbiologische und proteinbiochemische Untersuchungen mit Durchflusszytometrie bzw. Western Blots deuten auf eine posttranslationale Modulierung von EGFP - Sp3 hin. Um die Hypothese der proteasomalen Degradierung von EGFP - Sp3 zu belegen, wurden transfizierte HEK293 mit dem reversiblen Proteasominhibitor MG132 behandelt. In Gegenwart von 1 µM MG132 konnte das zytosolische Fusionsprotein stabilisiert werden. Die hier präsentierten Daten offenbaren die humane Sp3 - ZFD als ein neues Substrat für das 26S - Proteasom. Lediglich die SUMOylierung von Wildtyp - Sp3 im Bereich der inhibitorischen Domäne (ID) ist bislang beschrieben worden. Die Funktionalität, Affinität und kinetische Parameter der mit MBP fusionierten Sp1 - ZFD und gyrA - CTD wurden anhand von Oberflächenplasmonresonanz (BIAcore) bzw. EMSAs analysiert. Sämtliche gewonnenen MBP - Fusionsproteine sind funktionell und interagieren mit dsDNA. Fusionsproteine mit Sp1 - Domäne zeigten in EMSAs ebenso eine Bindung an unspezifische dsDNA. In sensitiveren BIAcore - Assays mit immobilisierter dsDNA wurden (um den Faktor 2) geringere Assoziations (ka) - und Dissoziationsraten (kd) von MBP - Sp1 ermittelt, wenn bestimmte Basen innerhalb der GC - Box ausgetauscht wurden. Die Affinität (Kd) von MBP - Sp1 mit 4×10 - 9 M zur GC - Box und deren Derivate ist vergleichbar mit der Kd von nativem Sp1. Die EMSA - Experimente für MBP - gyrA zeigen eine deutliche Präferenz zum spezifischen dsDNA - Oligo in Gegenwart von humaner gDNA, eine interessante Eigenschaft die durchaus zur Anwendung in einem Assay zur Anreicherung von bakterieller DNA dienen kann. Nach der vorausgehenden Charakterisierung der MBP - Fusionsproteine wurden diese auf verschiedenen gängigen festen und semifesten Substraten über physische Adsorption, kovalent oder Affinität immobilisiert um das Konzept der direkten Detektion von dsDNA mit funktionellen Proteinen als neuartige Komponente in NAD - Assays umzusetzen. Lediglich MBP - Sp1 zeigte auf Glas und Polystyren - Mikrotiterplatten nach kovalenter oder adsorptiver Immobilisierung eine ausgeprägte Funktionalität hinsichtlich der Bindung von dsDNA. Die Immobilisierung von 9×Lys - MBP - Sp1 über identische Strategien führten zum massiven Verlust der ZFD - Funktion. Aus dieser Datenlage heraus wurde erfolgreich ein simples Lumineszenz - basiertes Mikrotiterplatten - Assay mit MBP - Sp1 entwickelt um PCR - Amplikons direkt aus einer analytischen PCR auf gDNA von S. aureus, welche die GC - Box beinhalten, nachzuweisen. Das spezifische Amplikon konnte mittels des simplen Assays in Gegenwart von 100fachem Überschuss an humaner gDNA nachgewiesen werden. Mit einem höheren Anteil an humaner gDNA wurde die PCR massiv inhibiert, ein negativer Effekt der bislang im Bereich der diagnostischen NAD - Assays nicht optimal adressiert wurde. Die magnetische Separation von bakterieller und humaner gDNA wurde dazu mit MBP - gyrA umgesetzt. Zunächst erfolgte die regioselektive Immobilisierung von MBP - gyrA auf Protein A - funktionalisierte magnetische Nanopartikel mittels MBP - Antikörper, wodurch die Funktionalität hinsichtlich der Bindung von dsDNA gewährleistet werden konnte. Dieses System eignet sich insbesondere für die Separation von bakterieller DNA (E. coli oder S. aureus) aus einem komplexen Gemisch mit bis zu 100fachem Überschuss an humaner gDNA. Die Kombination von MBP - gyrA - basierter magnetischer Separation mit NAD - Assays könnte deren Sensitivität signifikant erhöhen. Durch simple Verfahrensweise bietet das System einen wesentlichen Beitrag zur Verringerung des zeitlichen Aufwands für die Generierung therapierelevanter Resultate
Methods for direct detection or enrichment of double - stranded DNA (dsDNA) possess tremendous potential for use in molecular diagnostics. Already established methods for nucleic acid detection (NAD) are generally based on the hybridization of two complementary strands followed by optical detection or enzymatic amplification. In contrast, DNA - binding or organizing proteins (e.g. endogenous transcriptions factors) are able to read the sequence information directly from dsDNA without prior denaturation of the double strand and subsequent hybridization. In order to develop novel NAD assays or assays for sample preparation, four artificial DNA - binding proteins were cloned, expressed and purified in HEK293 cells or E. coli. The Cys2His2 zinc finger domains (ZFD) from human Sp1 were fused to maltose binding protein (MBP) and its derivate 9×Lys - MBP, an extended variant with nine successive lysine residues in the N - terminal region of the protein to facilitate site - directed immobilization. The human Sp3 - ZFD was fused to green fluorescent protein (EGFP). The family of Sp - transcription factors was known to bind specifically the consensus sequence 5\' - GGG GCG GGG - 3 \'(GC - box). Moreover, the C - terminal DNA - binding domain of E. coli DNA Gyrase subunit A (gyrA - CTD) was fused to MBP. The CTD binds specifically repetitive extragenic palindromes (REP), which were only found on prokaryotic chromosomes. All MBP fusion proteins were soluble after expression and could be purified to homogeneity. Surprisingly, transient transfection experiments in HEK293 revealed a destabilizing effect of the Sp3 - ZFD accompanied by massive degradation of the EGFP fusion protein after 120 h post transfection. Analysis of mRNA integrity in combination with western blots indicates a posttranslational modulation of EGFP - Sp3. To confirm the hypothesis of proteasomal degradation of EGFP - Sp3, transfected cells were treated with the reversible proteasome inhibitor MG132. In the presence of 1µM MG132 the fusion protein could be stabilized. Taken together, the data presented here identified the human Sp3 - CTD as a new substrate for the 26S proteasome. Only SUMOylation of wild type human Sp3 within the inhibitory domain (ID) has been described so far. Initial EMSA experiments showed that purified MBP - ZFD fusion proteins were functional in terms of interacting with dsDNA containing the specific sequence motiv. However, all proteins bound to unspecific dsDNA as well. Therefore MBP - Sp1 was subjected to BIAcore analysis to determine the rate constants for association ka, dissociation kd and the dissociation constant Kd of the GC - Box - Protein complex as well as mutants of the GC - Box. The determined Kd (4 × 10 - 9 M) for MBP - Sp1 associated with GC - box or its derivatives were found to be comparable with the Kd of native Sp1, however the rate constants were reduced 2 fold in presence of the modified GC - boxes. EMSA experiments with MBP - gyrA revealed functionality and a clear preference for specific dsDNA in the presence of unspecific human genomic DNA (gDNA). After preliminary functional characterization, MBP fusion proteins were immobilized by physical adsorption, covalent or by affinity on various solid substrates or nanoscaled magnetic beads to implement the concept of direct detection of dsDNA or specific enrichment of bacterial DNA, respectively. MBP - Sp1 remains functional after adsorptive or covalent immobilization on different chemical modified glas surfaces. 9×Lys - MBP - Sp1 shows significantly reduced functionality after immobilization on the same glas substrates by similar strategies. Moreover, a simple NAD - assay with adsorptive immobilized MBP - Sp1 on polystyrene in microtiter format was established for direct detection of GC - boxes within PCR - products from S. aureus gDNA. By using the assay, specific PCR - products could be detected in presence up to 100 - fold excess of human gDNA in relation to 10 ng bacterial DNA. Separation of bacterial DNA from human DNA from clinical samples may have an important impact on downstream applications, involving NAD assays. To address this often underestimated technical problem, a new functional protein MBP - gyrA was introduced to overcome some limitations of already established methods. MBP - gyrA was site - directed coupled on nanoscaled magnetic beads by affinity. This system enabled the fast and specific separation of gDNA of E. coli or S.aureus from a huge background of human gDNA. The combination of MBP - gyrA - based magnetic separation with NAD assays could significantly increase the sensitivity and shorten the time for initiation of effective treatment

Increases in Staphylococcus aureus resistance against existing treatment options and the shortage of new antibiotics signals an urgent need for new treatments for the ongoing battle against the development of antibiotic resistance. DNA gyrase is an essential bacterial type II DNA topoisomerase that manipulates DNA topology by performing transient double-strand breaks and DNA strand passage. As gyrase is vital for bacterial survival, it is an effective antibacterial target. By understanding the mechanistic differences between S. aureus gyrase and the better studied Escherichia coli counterpart, we aim to better utilise S. aureus gyrase as an antibacterial target and improve the design of future antibacterial drug. This study has investigated features unique to S. aureus DNA gyrase: the potassium glutamate (KGlu) salt-dependent and salt-specific supercoiling. This KGlu dependency in S. aureus, but not E. coli gyrase, was partially attributed to the differences in the Cterminal domain of the gyrase A-subunit (GyrA). The discovery of the two novel monovalent alkali metal cation (M+) binding sites located in N-terminal domain of GyrB by protein crystallography has suggested a novel role of these M+ ions in the supercoiling functions of DNA gyrase, providing theoretical links to the unique KGlu dependency in S. aureus gyrase and the dependency of monovalent ions in E. coli and B.subtilis gyrase. Diospyrin, a phyto-naphthoquinone, was found to be active against S. aureus in vivo. It inhibits S. aureus gyrase and topo IV in vitro, with gyrase as the preferred target. Further studies suggested the binding site of Diospyrin to be located in the N-terminal domain of GyrB. Diospyrin also partially inhibits the ATPase activity of GyrB in an allosteric manner. Diospyrin is hypothesized to bind to a novel binding site between the ATPase domain and the transducer domain. Diospyrin also inhibits both the relaxation and the DNA cleavage ability of gyrase, suggesting it inhibits gyrase with a novel mechanism.

DNA, due to its double-helical structure, is subject to changes in topology due to the nature of transcription and replication. To overcome this, cells have processes and enzymes that ameliorate these changes. One such group of enzymes are the DNA topoisomerases, which are responsible for the maintenance of DNA topology. Despite this important role, these enzymes participate in illegitimate recombination (IR), which is genetic recombination between regions of DNA that share little or no homology. This can result in chromosomal rearrangements and is often a consequence of DNA-damaging agents. A consequence of topoisomerase-induced IR is thought to be therapy-related acute myeloid leukaemia (tAML). Analogously, there is evidence that exposure to sublethal concentrations of ciprofloxacin, a topoisomerase inhibitor, can cause resistance to non-quinolone antibiotics. This may work by a similar mechanism as that proposed for t-AML. This project centres around the examination of DNA gyrase-mediated IR focussing on the proposed subunit-exchange model. Using Blue-Native PAGE, I set up an assay to examine subunit exchange in topoisomerases. I have also characterised previously identified gyrase hyper-recombination mutations, known to increase the frequency of IR. Furthermore, I have investigated quinolone-induced antibiotic resistance and what the mechanism is. Here, I show that DNA gyrase can undergo subunit exchange, and that this seems to occur within higherorder oligomers of the enzyme, which have not been investigated before. Biochemical characterisation of the hyper-recombination mutations shows that they impair DNA gyrase activity which, in vivo, may have downstream consequences that may lead to IR. Using an in vivo assay where E. coli is treated with subinhibitory levels of quinolones, I have seen resistance to other non-quinolone antibiotics. This is not seen when other antibiotics, including other topoisomerase inhibitors, are tested. Whole genome sequencing has revealed point mutations that explain the resistances seen, however other larger chromosomal modifications have been observed as well.

Quinolone drags are a clinically significant family of antibacterial compounds that are known to affect the activity of DNA gyrase, an essential bacterial enzyme involved in controlling the topological state of DNA, Gyrase holoenzyme, a complex of two A and two B subunits, can introduce negative supercoils into DNA using energy derived from the hydrolysis of ATP. Although addition of quinolones rapidly inhibits the supercoiling activity of gyrase, it was found that quinolone-dependent DNA cleavage was a slow process, leading to the suggestion that there may be two levels of interaction between quinolones and the gyrase-DNA complex. Rapid gel-filtration experiments have shown that stable quinolone binding requires the presence of both gyrase and DNA; no significant binding was found to either gyrase or DNA alone. Enzyme containing gyrase A protein with the mutation Ser83 to Trp (which is known to confer quinolone resistance) showed greatly reduced drug binding. It is concluded that efficiency of binding is primarily determined by the gyrase A subunits. Investigation of transcription by T7 and Escherichia coli RNA polymerases has found that quinolone-mediated stabilisation of a gyrase-DNA complex prevents passage of polymerase along the template. Inhibition of transcription required the presence of gyrase and quinolone together; RNA polymerase was unaffected by either quinolone or gyrase alone, implying that polymerase can normally pass or displace gyrase. In the presence of ciprofloxacin, gyrase was found to shield a region of about 26 bp of DNA from transcription by T7 RNA polymerase, with especially strong protection of a 20 bp core. Preliminary experiments performed using an in vitro DNA replication system suggest that DNA polymerases may be similarly interrupted by a gyrase-quinolone-DNA complex.

DNA gyrase is the enzyme from bacteria which is unique among type II topoisomerases in its ability to introduce negative supercoils into DNA. The enzyme acts as an A2B2 tetramer of molecular weight 374 kDa. The supercoiling reaction of gyrase involves wrapping of DNA around the enzyme and the coupling of ATP binding and hydrolysis to the passage of a DNA segment through a transient double strand break stabilised by gyrase, although its details are undefined. This reaction of gyrase is inhibited by two classes of anti-bacterial compounds, the quinolones and the coumarins. Hydroxyl radical footprinting was used to probe the complex between gyrase and a 198 bp DNA fragment containing the preferred gyrase cleavage site from plasmid pBR322. Gyrase protects 128 bp from the hydroxyl radical with the central 13 bp (adjacent to the gyrase cleavage site) being most strongly protected. Flanking the central region are arms showing periodic protection from the reagent suggesting a helical repeat of 10.6 bp, consistent with the DNA being wrapped upon the enzyme surface. The presence of 5'-adenylyl,Y-imidodiphosphate (ADPNP) or a quinolone drag causes alteration of the protection pattern consistent with a conformational change in the complex involving one arm of the wrapped DNA. This is thought to represent an intermediate in the supercoiling cycle of gyrase. Protein-DNA crosslinking using the photoactivatable thymine analogue 4-thiothymidine was used in an attempt to identify regions of gyrase involved in DNA binding. Complexes containing gyrase and 4-thiothymidine-substituted DNA were irradiated with long-wave UV light to activate the photoreactive reagent. However, no protein-DNA crosslinks were detected. An attempt to radiolabel DNA-binding lysine residues of gyrase was also unsuccessful due to the dissociation of the gyrase-DNA complex upon modification of its lysines. Irradiation of gyrase DNA complexes with short-wave laser-UV light results in the formation of covalent protein-DNA complexes at an efficiency of ~1%. Primer extension analysis was used to tentatively assign the crosslinks to two positions along the 147 bp DNA fragment used. The 43 kDa N-terminal domain of the gyrase B protein is responsible for ATP hydrolysis and also interacts with the coumarin class of gyrase inhibitors. To gain insight into the nature of the ATP-induced conformational change in the gyrase A2B2 tetramer, the effect of the non-hydrolysable analogue ADPNP on the conformation and oligomeric state of the 43 kDa domain was examined. Protein crosslinking studies suggest that the protein exists as a monomer but dimerises in the presence of ADPNP. Limited trypsin proteolysis in the presence of ADPNP results in the protection of a 33 kDa N-terminal fragment of the protein (residues 2-307), consistent with an altered conformation of the 43 kDa domain in the presence of the nucleotide. The effects of the coumarin drugs novobiocin and coumermycin A1 on the 43 kDa domain were also investigated. Novobiocin does not cause oligomerisation of the 43 kDa protein but coumermycin A1 induces dimerisation when present at molar concentrations approaching half that of the 43 kDa protein. Limited trypsin digestion in the presence of either drug results in the protection of a 16 kDa proteolytic fragment from digestion (residues 111-247). Moreover, the products of trypsin digestion in the presence of novobiocin can stably bind novobiocin when denatured and renatured. The 16 kDa fragment was cloned and overexpressed as a direct gene product but was found to be incapable of stable novobiocin binding.

DNA gyrase is an essential bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. This study concentrates on the interaction of the enzyme with ATP and on the way the free energy of hydrolysis of the nucleotide is coupled to the supercoiling reaction. Positional isotope exchange experiments, using ATP labelled with 18O in the ?-?-bridge position, have shown that no detectable scrambling of the label occurs during the gyrase ATPase. This is interpreted in terms of a slow ATP off-rate in gyrase. Gyrase-catalysed turnover of ATP?S was found to be 300 to 1000-fold slower than ATP from supercoiling and hydrolysis measurements. This precluded the use of isotopically chiral ATP?S to determine the stereochemistry of the gyrase ATPase, although the availability of more protein in the future may permit such a study. Various aspects of the interaction of gyrase with diastereoisomers of ATP?S and ATP?S have been explored which has yielded information on the type of MgATP complex handled by the enzyme and on the relationship between hydrolysis of the ATP analogues and their ability to support DNA supercoiling. Gyrase showed a strong preference for the Rp epimers of ATP?S and ATP?S in the presence of Mg2+ which is consistent with the Mg2+ ion being coordinated to the pro-S oxygens of the a and ?-phosphates in ATP bound to the enzyme; an ?,?,?-tridentate Mg-ATP complex with A-exo geometry is proposed. ATP?S(Rp) hydrolysis appears to be well coupled to DNA supercoiling whereas ATP?S(Rp) hydrolysis is only poorly coupled to the supercoiling reaction. Two hypotheses are proposed to account for this phenomenon. The divalent metal ion specificities of the DNA supercoiling, relaxation, and cleavage reactions of gyrase have been characterised. It was found that the cleavage reaction exhibited a greater tolerance towards different metal ions than the relaxation and supercoiling reactions; the mechanistic implications of this observation are discussed. An N-terminal fragment of the gyrase B protein was used as a simple model for gyrase-ATP interactions. This truncated protein showed the same stereospecificity towards the diastereoisomers of ATP?S and ATP?S as intact gyrase but the kinetics of hydrolysis of the two phosphorothioate ATP analogues showed significant differences from those with gyrase, which may be due to differences in the mechanisms of ATP hydrolysis for the two enzymes. Attempts to demonstrate reversal of stereospecifity on switching from a hard metal ion such as Mg2+ to a soft metal centre such as Cd2+ were thwarted by the inhibitory effects of Cd2+. The extent of supercoiling of pBR322 was investigated with ATP, ATP?S(Rp), and ATP?S(Rp). With ATP?S(Rp) the rate of DNA supercoiling was very slow (since its hydrolysis is inefficiently coupled to supercoiling; see above) and the reaction did not reach a limit even after long time-courses. With ATP and ATP?S(Rp), the supercoiling reaction was faster and did reach a limit: ATP gave a ?Lk of -46 and ATP?S(Rp) gave a ?Lk of -47. Measurements of the displacement of the equilibrium of the reaction catalysed by arginine kinase showed that ATP?S(RP) has a greater free energy of hydrolysis than ATP. This energy difference corresponds closely to the extra free energy required for the additional supercoiling seen with ATP?S(Rp). It is proposed that the extent of supercoiling in gyrase is limited by the free energy available from nucleotide hydrolysis.

DNA gyrase is an essential bacterial topoisomerase with a central role in many cellular processes. Its lack of homology to eukaryotic topoisomerases has been the basis of its role as an important drug target. The quinolone antibacterials have previously been shown to "poison" DNA gyrase of Escherichia coli, resulting in rapid bacterial cell death by a pathway which has been a subject of controversy. Following on from the finding that the complex of quinolone and gyrase on DNA results in truncated mRNAs in vitro, the results presented in this thesis show that in vivo, the addition of quinolone drugs results in the production of truncated mRNAs which are presumably translated into truncated proteins. This is expected to cause total deregulation of cellular processes and result in cell death. It has also been shown that the newer more potent quinolone drugs such as ciprofloxacin and ofloxacin have an additional effect on the bacterial cells in that the chromosome is broken down into fragments of DNA, some of which are estimated to be as small as 4kb in length. This process appears similar to the degradation of chromosomal DNA that occurs during apoptosis of some eukaryotic cells. The acquisition of quinolone-resistance in E. coli and Salmonella has also been investigated. Firstly, part of gyrA of S. typghimurium NCTC5710 was sequenced and found to be 94% homologous at the nucleotide level to the corresponding sequence of E.coli. Following on from this, clinical isolates were screened by DNA sequencing and amino acid mutations in the "quinolone resistance determining region" (QRDR) of gyrA identified. Mutations in gyrA such as serine-83 to leucine, serine-83 to phenylalanine, aspartate-87 to tyrosine, aspartate-87 to asparagine and aspartate-87 to glycine were found to have arisen in the QRDR of gyrA in common with other quinolone resistance mutations previously found. Cloning of gyrA into a vector in which the copy number could be altered confirmed that high level expression of gyrA is detrimental to the cell.

DNA gyrase is the bacterial type II topoisom erase w hich couples the free energy o f ATP hydrolysis to the introduction of negative supercoils into DNA. The active enzym is composed of two GyrA and two GyrB subunits forming an A2B2 complex. The specific supercoiling activity of GyrB was found to be consistently lower than the specific supercoiling activity of GyrA and this is believed to be due to mis-folding of the subunit. Expression as a thioredoxin-fusion protein did not improve the specific supercoiling activity of GyrB. The C-terminal 47 kDa domain of GyrB (GyrB47) was over-expressed as a soluble protein when fused to thioredoxin. This domain interacts with GyrA and DNA. In complex with GyrA, GyrB47 supports quinolone- and Ca2+-induced DNA cleavage and has ATP-independent relaxation activity which is comparable with that of full-length GyrB. GyrB47 also supports low level ATP-independent decatenation. Protein cross linking was used to investigate nucleotide-, DNA- and drug-induced conformational changes during the reaction cycle of the enzyme. Upon addition of the non-hydrolysable ATP analogue, ADPNP, there was an increase in crosslinking between the GyrB subunits. In the presence of DNA, crosslinks between the GyrA subunits were identified. Using limited proteolysis and immunodetection, these crosslinks were shown to be between the N-terminal 64 kDa domains of GyrA. Examination of the X-ray crystal structure of the 43 kDa domain of GyrB (GyrB43) shows that the side chains of Gln335 and Lys337 interact with the gamma-phosphate of the ATP. Both these residues are highly conserved among type II topoisomerases. The proposed role of these residues is in nucleotide binding, transition-state stabilisation and triggering conformational changes following ATP hydrolysis. Site-directed mutagenesis was used to convert Gln335 to Asn and Ala and Lys337 to Gln and Ala. No clear role for Gln335 has been established in nucleotide binding, ATP hydrolysis or triggering conformational changes. However mutations at Lys337 lead to a modest decrease in nucleotide binding and a large reduction in ATP hydrolysis (~103-fold decrease in k cat). Therefore Lys337 is a critical residue for ATP turnover and the results are consistent with its involvement in transition-state stabilisation.

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.

L’ADN gyrase est une enzyme vitale pour la bactérie grâce à sa capacité de manipuler les molécules d’ADN dans la cellule vivante. Cette capacité fait de l’ADN gyrase une cible idéale pour des composés anti-infectieux. Dans ce travail, l’ADN gyrase a été étudié par des méthodes de modélisatoin moléculaire. Une approche de conception de ligands basée sur la structure a été entreprise sur le sous-domaine N-terminal de 24 kDa de l’ADN gyrase B (domaine GHKL). La flexibilité de deux boucles du site actif du domaine GHKL a été étudiée par des simulations de dynamiques moléculaires en présence de différents ligands. Dans une dernière partie, une analyse des modes normaux du dimère du domaine N-terminal de 43 kDa a été entreprise
DNA gyrase is a vital bacterial enzyme necessary for the handling of the large DNA molecules in the living cell. Therefore DNA gyrase is an ideal target enzyme for anti-infectious compounds. In this work DNA gyrase has been studied by molecular modelling methods. A computational structure-based ligand design approach has been carried out on the N-terminal 24 kDa subdomain of DNA gyrase B (GHKL domain). To further examine the flexibility of two active site loops, molecular dynamics simulations have been carried out on the GHKL domain in different ligand binding conditions. In a final part, normal mode analysis has been carried out on the dimer of the 43 kDa domain of DNA gyrase B

Quinolones are a clinically-useful class of antibacterial agents known to target DNA gyrase, a bacterial type II topoisomerase. Gyrase is unique among topoisomerases in its ability to introduce negative supercoils into DNA using the energy derived from ATP hydrolysis. The active enzyme is composed of two GyrA and two GyrB subunits, forming an A2B2 tetramer of molecular weight 374 kDa. The mechanism of supercoiling by gyrase involves the ATP-driven passage of one segment of DNA through a gyrase-stabilised double-stranded break in another. Tyrosine 122 of E. coli GyrA becomes covalently attached to DNA when gyrase breaks the phosphodiester bonds of DNA during supercoiling. When this residue is mutated to serine or phenylalanine, gyrase can no longer cleave or supercoil DNA, but can bind DNA normally. Rapid-gel filtration experiments have shown that quinolones can still bind to proteins bearing these mutations, suggesting that DNA cleavage by gyrase is not required for quinolone binding. Transcription by T7 and E. coli RNA polymerases is blocked by the presence of a gyrase-quinolone-DNA complex. Mapping of the transcription termination sites in the presence of gyrase and quinolones shows that blocking occurs about 10 to 20 base-pairs upstream of the gyrase cleavage site. Blocking of transcription by T7 RNA polymerase by a gyrase-quinolone complex on DNA does not occur when the active-site tyrosine of gyrase is mutated to serine, which indicates that the polymerase blocking requires DNA cleavage. Analysis of transcription in the absence of drug suggest that RNA polymerase does not displace gyrase from the template. DNA gyrase is also the target of the CcdB protein which is encoded by the F plasmid. When its action is not prevented by CcdA protein, CcdB is a potent cytotoxin. Using in vitro transcription by T7 RNA polymerase, it has been shown that CcdB complexed with gyrase can block transcription in a similar manner to the gyrase-quinolone complex. Furthermore, in the presence of CcdA, CcdB can no longer induce gyrase to block transcription.

The type II topoisomerase subfamily of enzymes has been clinically targeted by the widely used, broad-spectrum quinolone class of antibacterials. Due to emerging drug-resistant strains of bacteria, the quinolones’ effectiveness is threatened. The natural product simocyclinone D8 (SD8) has shown the ability to inhibit the type II topoisomerase, DNA gyrase, even when mutated to be resistant to the quinolones. In order to determine the pharmacophore required for SD8 binding to DNA gyrase, 16 compounds were synthesized. These compounds were then tested by surface plasmon resonance for their ability to inhibit the DNA – DNA gyrase binding interaction. It was found that three compounds were able to inhibit the DNA – DNA gyrase binding interaction, while another showed partial inhibition of the interaction. From this data, a minimum pharmacophore was able to be determined. The pharmacophore required a coumarin scaffold bonded to a carboxylic acid group through an approximately 15 Å hydrocarbon linker. Functional supercoiling assays determined that while the compounds were able to bind the enzyme, the binding did not inhibit DNA gyrase’s ability to supercoil DNA.

Cyclothialidine, a natural product isolated from Streptomyces .filipinensis NR0484, has been proven to be a potent and selective inhibitor of the bacterial enzyme DNA gyrase. Gyrase inhibition results in cell death, the enzyme being the target of several currently used antibiotics. Cyclothialidine showed poor activity against whole bacterial cells, highlighting scope for improvement regarding cell membrane pemeability in order for the full potential of this new class of antibiotics to be realised, Structurally, cyclothialidine contains a 12-membered lactone ring which is partly integrated into a pentapeptide chain, with a substituted aromatic moiety bordering the lactone, Retrosynthetically it can be traced back to cis-3-hydroxyproline, 3,5-dihydroxy-2,6-dimethylbenzoic acid and four commercially available amino acids; two serine, one cysteine and one alanine. In this work, a model of cyclothialidine was synthesised in order to establish the methodology for more complex compounds. Analogues with hydroxy, dihydroxy and dihydroxymethyl substituted aromatic moieties were then prepared to ensure successful protection methods could be performed and the pharmacophore synthesised. The key aromatic moiety, 2,6-dimethyl-3,5-dihydroxybenzoic acid was produced via two successive Mannich reaction/reduction steps. Acid protection using 4-nitrobenzyl bromide and TBDMS hydroxyl protection followed by bromination of one methyl afforded the desired intermediate. Reaction with a serine/cysteine dipeptide, followed by deprotection and cyclisation under Mitsunobu conditions lead to the 12-membered lactone. An amine substituted aromatic analogue and also replacement of the cysteine sulphur by oxygen were attempted but without success. In an effort to improve cell permeability, a conjugate was synthesised between the pharmacophore and a cholesterol moiety. It was hoped the steroid fragment would serve to increase potency by escorting the molecule through the lipid environment of the cell membrane. The pharmacophore and conjugate were tested against a variety of bacterial strains but the conjugate failed to improve activity.

Thesis (Honors paper)--Florida State University, 2008.
Advisor: David L. Balkwill, Ph.D, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Includes bibliographical references.

Since cyclothialidine was discovered as the most active DNA gyrase inhibitor in 1994, enormous efforts have been devoted to make it into a commercial medicine by a number of pharmaceutical companies and research groups worldwide. However, no serious breakthrough has been made up to now. An essential problem involved with cyclothialidine is that though it demonstrated the potent inhibition of DNA gyrase, it showed little activity against bacteria. This probably is attributable to its inability to penetrate bacterial cell walls and membranes. We applied the TSAR programme to generate a QSAR equation to the gram-negative organisms. In that equation, LogP is profoundly indicated as the key factor influencing the cyclothialidine activity against bacteria. However, the synthesized new analogues have failed to prove that. In the structure based drug design stage, we designed a group of open chain cyclothialidine derivatives by applying the SPROUT programme and completed the syntheses. Improved activity is found in a few analogues and a 3D pharmacophore of the DNA gyrase B is proposed to lead to synthesis of the new derivatives for development of potent antibiotics.

Treatment of Escherichia coli DNA gyrase A protein with trypsin generates two large, stable fragments of molecular masses 64 kDa and 33 kDa which are derived respectively from the N-and C-terminus of GyrA. The trypsin-cleaved A protein (A'), can support DNA supercoiling, relaxation and other reactions of gyrase. The isolated 64 kDa fragment will also catalyse DNA supercoiling but the 33 kDa fragment shows no enzymic activity. An amber mutation, introduced into gyrA near the point which corresponds to the tryptic cleavage site, yields GyrA(1-573) which shares the same properties as the 64 kDa tryptic fragment. Using genetic engineering, large numbers of 3'-gyrA deletion mutants have been produced; those encoding a protein smaller than 58 kDa (GyrA(1-523)) did not obviously overproduce truncated GyrA. GyrA(1-523) shows similar enzymic properties to GyrA(1-573) but cannot perform DNA supercoiling. Deletion of fifty C-terminal residues from GyrA(1-573) has the effect of disrupting part of the protein essential for supercoiling. I propose that the N-terminal 64 kDa represents the DNA breakage/reunion domain of the A protein, while the 33 kDa fragment contributes to gyrase-DNA complex stability. Certain N-terminal deletion mutants of the GyrA protein were also constructed. Removal of the N-terminal 6 amino acids had no effect on the properties compared to GyrA. Removal of the N-terminal 69 amino acids yields a protein with no supercoiling or cleavage ability. The start of the N-terminal breakage-reunion domain is probably located within this 63 amino acid region. The domains of GyrA were investigated by microcalorimetry. GyrA yields two unfolding transitions. GyrA(1-573) and GyrA(1-523) both yield a single unfolding transition corresponding to one of the GyrA transitions. Therefore GyrA contains two structural domains that can be assigned to the functional moieties described above. GyrA(1-573) has been crystallized, and four crystal forms identified. A diffraction pattern to 7 A has been obtained.

The purpose of this study was (1) to determine whether a correlation exists among the protein profiles, extracted from cell membranes of mutants belonging to five pigment cluster groups, (2) to locate the protein moiety and cartenoprotein complex in the membranes of wild type and colorless mutant (designated W-19) of C. poinsettae and to show whether there are any structural differences between cell membranes of the wild type and a colorless mutant, (3) to determine the effect of six antibiotics on cartenoid gene expression.

The spread of antibiotic resistance is a great threat to medicine. We are in desperate need of new antibiotics to replace those for which resistance is widespread. The bacterial type two topoisomerases, DNA gyrase and topoisomerase IV, are well validated antimicrobial targets. The fluoroquinolone antibiotics target the DNA binding site of these enzymes, but no currently available antibiotics target the ATP binding site. It is hoped that simultaneously targeting the ATP binding sites of these two bacterial enzymes will slow the emergence of resistance. This thesis describes the investigation of a series of pyridine 3-carboxamide DNA gyrase inhibitors, extending knowledge of the structure activity relationships of these compounds and making assessment of their inhibition of the topoisomerase IV enzyme in addition to their inhibition of DNA gyrase. Many of the inhibitors proved to be highly potent inhibitors of both DNA gyrase and topoisomerase IV with the most potent compound having IC50s of 24 nM and 87 nM against the DNA gyrase and topoisomerase IV enzymes respectively. These compounds also showed promising antibacterial activity with MICs as low as 1 μg/ml against S. aureus. Additionally, in vitro ADME profiling of key compounds from this series was carried out. A subunit of the DNA gyrase protein was produced for use in crystallisation studies. Cocrystallisation of the protein with four inhibitor molecules was carried out and four crystal structures were solved revealing the binding poses of the pyridine 3-carboxamide compounds bound in the ATP site of GyrB. Efforts were also made to identify a new series of DNA gyrase inhibitors. De novo design and virtual high throughput screening were utilised and a number of compounds were purchased and assessed for their ability to inhibit DNA gyrase. Two of the compounds showed activity in the enzyme assay. A scaffold hopping approach was also used to rationally design new series of potential inhibitors. Three compound series were designed and synthesis was carried out towards them. A series of quinazolinones proved to be the most synthetically tractable and a small library was produced and assessed for their biological activity. Several of the compounds were active against the DNA gyrase enzyme with IC50s as low as 1.1 μM.

Compositae plants of the genus Erigeron have ca. 390 species, widely distributed in most temperate regions around the world. Erigeron anl7UUS (L.) Pers. (fleabane) is one of the most valuable plants in this genus, used in Chinese foll< medicine to treat indigestion, malaria, enteritis, hepatitis and hematuria increasingly since the 1970s. However, it is not an indigenous species in China and has not been officially recorded in the Chinese Pharmacopeia. Very little research has been published on its biological activity and no activity against MRSA has been reported . In this research, whole plant material was collected in Shanghai, China and its chemical composition , antibacterial activity, DNA gyrase inhibitory activity and mutagenicity assessment were evaluated on isolated compounds and extracts.

The inevitability of bacterial drug resistance to all marketed antibiotic drug classes warrants continual research into the development of novel chemotype antibacterial agents. Drug resistant `superbugs' such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE) account for >70% of US hospital bound bacteremias. Such infections are associated with vastly increased rates of morbidity and mortality resulting in a heavy economic burden on health care authorities. The bacterial topoisomerase enzymes DNA gyrase and topoisomerase IV are highly conserved amongst almost all bacterial species on account of their unique and essential function in the conservation of chromosomal integrity during DNA replication and transcription. Furthermore, structural similarity between gyrase and topo IV, and distinction from human homologues, makes the selective dual-inhibition of these enzymes a realistic goal. In this thesis, three distinct approaches were utilized in the discovery of novel inhibitors of gyrase (GyrB) and topoisomerase IV (ParE) acting at the ATP binding domains of these enzymes. In the initial strategy, structural variants based on known inhibitors were modelled within the GyrB ATPase site in a bid to displace a highly conserved water molecule at the ligand-enzyme interface. Synthesis and biological screening of these variants proved that such changes were to the detriment of activity, although the work did yield a novel, convenient preparation of Nsubstituted thieno[2,3-d]pyrimidinones. In an alternative strategy, the de novo molecular design software SPROUT was used in concert with crystallographic data on the GyrB ATP binding site. Putative inhibitors were thus generated 'from scratch', the most attractive of which were further modelled using docking software (AUTODOCK), synthesised and screened for enzyme inhibitory and antimicrobial activity. A number of early series were derived which demonstrated modest (GyrB IC50 <100 μM) enzyme activity. The most promising of these, a series of pyridine-3-carboxamides, was optimized to offer potent (GyrB IC50 <100 nM) enzyme inhibitory activity, dual target specific antibacterial activity (MICs <0.5 μg / mL) and a low potential for resistance development. Finally, using the GLIDE docking software, virtual compound libraries were screened in silico against the ATP binding domain of GyrB. Though largely unsuccessful, the process did identify a moderate inhibitor (GyrB IC50 118 μM, MIC 32 μg / mL), in a cost and time effective manner relative to high-throughput screening.

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'ADN gyrase est l'unique topoisomérase de type II présente chez M. Tuberculosis. De ce fait elle constitue l'unique cible des fluoroquinolones dans le traitement de la tuberculose. Il s'agit d'un hétérotétramère d'environ 350 kDa assemblées à partir de deux sous unités (A et B). Chaque sous-unité est constituée de deux domaines structuraux, un domaine de coupure-ligation de l'ADN (DCL) et un domaine CTD, pour la sous-unité A, un domaine ATPase et un domaine TOPRIM pour la sous-unité B. Les fluoroquinolones sont des antibiotiques clefs pour le traitement des tuberculoses à bacilles multirésistant, c'est-à-dire résistant au moins à l'isoniazide et à la rifampicine. Des souches ultrarésistantes (XDR), résistantes au fluoroquinolones et aux aminosides, apparaissent dans le monde, y compris en France où elles représentent 10 % des tuberculoses multiresistantes. Ces souches XDR inquiètent la communauté internationale car elles sont difficiles à traiter et sont à l'origine de 65 à 100 % de décès. A l'heure actuelle plusieurs programmes scientifiques à l'échelle mondiale sont en cours pour tenter d'optimiser l'efficacité des fluoroquinolones dans le traitement de la tuberculose, en particulier dans le but de raccourcir la durée du traitement. Le laboratoire y contribue directement en orientant une partie de ses recherches à la compréhension des mécanismes de résistance aux quinolones chez M. Tuberculosis. Un second axe concerne un aspect plus fondamental et vise à une meilleure compréhension à l'échelle atomique du mode de fonctionnement des topoisomérases de type II en général, mais aussi des spécificités de l'ADN gyrase de M tuberculosis, liées notamment à sa caractéristique de topoisomérase de type II unique dans cet organisme. Dans ce contexte, le projet de thèse à consister à étudier d'un point de vue structural par cristallographie des rayons X et fonctionnel, le domaine CTD de l'ADN gyrase de M. Tuberculosis. Nous avons résolu la structure cristallographique du domaine CTD à une résolution de 1. 4 À. L'analyse de la séquence du domaine CTD a permis de révéler une caractéristique inédite : la présence d'un second motif GyrA-box. Ce second motif GyrA-box est situé d'un point de vue structural au niveau de la pale 5 du repliement de type ,l3-pinwheel. Les études fonctionnelles menées en collaboration avec l'équipe d'Alexandra Aubry (Pitié-Salpêtrière) ont permis de montrer que ce second motif GyrA-box joue un rôle essentiel dans l'activité de décaténation de l'ADN gyrase de M tuberculosis
Mycobacterium tuberculosis possesses only one type IIA DNA topoisomerase, DNA gyrase, in contrast to most other bacteria which also possess DNA topoisomerase IV. It has been shown previously that die unique M tuberculosis type IIA DNA topoisomerase is functionally a hybrid enzyme, exhibiting the classical activity of DNA gyrase for supercoiling, but enhanced relaxation, cleavage and decatenation activities close to those of topoisomerase IV. Functional differences between the two type IIA DNA topoisomerases are thought to be specified by a C-terminal DNA binding domain (CTD) which controls DNA recognition. To explore the molecular mechanism responsible for the hybrid functions of the M. Tuberculosis DNA gyrase, we conducted a series of sequence analyses and structural and biochemical experiments with the isolated GyrA CTD and the holoenzyme. While the CTD displayed a global structure similar to that of bona fide GyrA and ParC paralogs, it harbors a second key motif similar in all respects to that of the GyrA-box. Biochemical assays showed that the canonical GyrA-box is responsible for DNA supercoiling and also relaxation, whereas the second GyrA-box-like motif (GyrA-box-1) is responsible for the enhanced decatenation activity of M. Tuberculosis DNA gyrase. Our results suggest that the mechanistic originality of M tuberculosis DNA gyrase depends largely on the particular DNA path around the CTD which is allowed for by the presence of eyrA-box-1. They reveal how the M tuberculosis DNA gyrase operates in detail, and provide also, through phylogenetic exploration of the entire Corynebacterineae suborder, new and broader insight into the functional stuctural and functional studies of the mycobacterium tuberculosis DNA gyrase C-terminal domain

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O sistema ParE-ParD é um sistema Toxina-Antitoxina (TA) do tipo II (composto por duas proteínas) encontrado no plasmídeo RK2 de uma gama de bactérias. A antitoxina ParD (9kDa) é capaz de neutralizar a citotoxicidade da toxina ParE, pela formação de um complexo estável, e também é eficaz na auto-repressão do operon parDE. A toxina (12kDa) apresenta atividade citotóxica no processo de replicação do DNA por interferir diretamente na ação da DNA girase. Estudos prévios sugeriram que a região C-terminal da antitoxina é responsável pelo processo de interação com ParE. Embora esta toxina possa ser encontrada em um grande número de microrganismos, ainda apresenta mecanismos de citotoxicidade e funções celulares a serem elucidadas. Neste contexto, este trabalho teve como objetivo a tentativa de expressão das duas proteínas ParE e ParD, bem como o design e a síntese de peptídeos análogos da antitoxina, para a realização de estudos de interação molecular, a fim de encontrar uma estrutura mínima de ParD capaz de inativar a função toxica de ParE. Com base nas informações estruturais, obtidas por modelagem e dinâmica molecular, quatro sequências peptídicas análogas de ParD foram projetadas e sintetizadas pela metodologia da fase sólida. As sequências foram analisadas e purificadas por cromatografia líquida de alta eficiência e caracterizadas por espectrometria de massas. Os estudos de interação foram realizados através de ensaios de cromatografia de afinidade e supressão de fluorescência. A fluorescência intrínseca de ParEAC2 foi suprimida pelos análogos de ParD (ParDTB1, ParDTB3, ParDTB5 e ParDTB6), evidenciando a formação de complexos estáveis entre as espécies, resultados confirmados pelos ensaios de cromatografia de afinidade. Resultados semelhantes foram obtidos empregando a proteína ParD obtida por expressão heteróloga. Com base nos resultados obtidos, foi possível concluir que o análogo ParDTB1 representa uma estrutura peptídica mínima com potencial para neutralizar o efeito da toxina ParE.
The ParE-ParD system is a toxin-antitoxin (TA) type II module (composed of two proteins) of the plasmid RK2 of a range of bacteria. The ParD antitoxin (9 kDa) is able to neutralize the cytotoxicity of the ParE toxin by forming a stable complex and is effective in the auto repression of the parDE operon. The toxin (12 kDa) exhibits cytotoxic activity by blocking DNA replication, acting directly in the DNA gyrase action. Previous studies have been suggest that the C-terminal region of the antitoxin is responsible for the interaction process with ParE. Although this toxin can be find in a large number of microorganisms, still have cytotoxicity mechanisms and cellular functions to be elucidate. In this context, this work aimed at the expression of ParE and ParD proteins, as well as the design and synthesis of antitoxin analog peptides, to perform molecular interaction studies in order to find a minimum ParD structure able to inactivate the toxic function of ParE. Based on the structural information obtained by modeling and molecular dynamics, four analogous peptide sequences of ParD were designed and synthesized by the solid phase methodology. The peptide sequences were analyzed and purified by high performance liquid chromatography and characterized by mass spectrometry. Interaction studies were performed by affinity chromatography and fluorescence suppression assays. The intrinsic fluorescence of ParEAC2 was suppressed by ParD analogs (ParDTB1, ParDTB3, ParDTB5 and ParDTB6) addition, evidencing the formation of stable complexes between the species, results confirmed by the affinity chromatography assays. Similar results were obtained using ParD protein obtained by heterologous expression. Based on the results obtained, it was possible to conclude that the ParDTB1 analog represents a minimal peptide structure with potential to neutralize the effect of the ParE toxin.

Background: Sexually transmitted diseases (STD's) have a major impact on sexual and reproductive health worldwide. Each year, the World Health Organization (WHO) estimates 448 million new cases of curable STD's are diagnosed. The emergence of drug resistance in STD related microorganisms and potential side effects demand the discovery of newer drugs. The exploration of newer anti-microbial substances from natural sources may serve as promising alternatives. In this study, twelve medicinal plant species used traditionally in the treatment of STD's are investigated in this regard. Methods: Ethanol plant extracts and three flavonoids were evaluated for their antimicrobial properties against one fungi and three bacteria, through the micro-dilution assay. To determine the anti-inflammatory activities of the extracts and compounds, the inhibitory effect was measured on the pro-inflammatory enzyme lipoxygenase, 15-LOX. Extracts were further evaluated for their inhibitory effect on the supercoiling activity of bacterial DNA gyrase by using the DNA gyrase kit. The extracts and compounds were lastly investigated for their anti-HIV activities against recombinant HIV-1 enzyme using non-radioactive HIV-RT colorimetric assay. Results: Acacia karroo and Rhoicissus tridentata extracts showed good antimicrobial activity with MIC values ranging between 0.4 and 3.1 mg/ml. Extracts of Jasminum fluminense, Solanum tomentosum and flavonoid 2 and 3 had good anti-inflammatory activity with IC50 less than the positive control quercetin (IC50 = 48.86 ug/ml). Extracts of Diospyros mespiliformis, Peltophorum africanum, Rhoicissus tridentata and flavonoids 1 and 2 showed the best inhibitory activity against the bacterial DNA gyrase. A. karroo and flavonoid 3 exhibited moderate HIV RT inhibition activity of 66.8 and 63.7 % respectively. R. tridentata and Terminalia sericea had the best RT inhibition activity (75.7 and 100 %) compared to the positive control doxorubicin (96.5%) at 100 ug/ml concentration. Conclusion: The observed activities may lead to new multi-target drugs against sexually transmitted diseases.
Dissertation (MSc)--University of Pretoria, 2017.
Plant Science
MSc
Unrestricted

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

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

Deux cent mille cas de lèpre surviennent chaque année dans le monde, surtout dans les pays en voie de développement. Le laboratoire d’accueil ayant une double activité de surveillance de la lèpre et de recherche centrée sur l’étude des mécanismes de résistance aux antituberculeux, les objectifs du travail s’inscrivent dans des enjeux médicaux et fondamentaux. Un 1er travail de génotypage de M. leprae nous a permis de montrer le caractère acquis dans des territoires hors France métropolitaine des cas de lèpre diagnostiqués sur le territoire national. L’équipement enzymatique de M. leprae est remarquable, par exemple, il n’existe qu’une seule topoisomérase de type II, l’ADN gyrase. Cette enzyme est la cible unique des fluoroquinolones (FQ) chez M. leprae. Cette bactérie n’étant pas cultivable in vitro, mettre au point des outils moléculaires fiables de dépistage de la résistance aux antibiotiques est essentiel. Concernant les FQ, nous avons dans un 1er temps, réalisé une revue systématique des mutations publiées dans gyrA/gyrB et dans un 2ème temps étudié l’impact dans la résistance de 5 mutations inconnues grâce à un test biochimique (mesure de l’inhibition par les FQ du surenroulement de l’ADN par l’ADN gyrase). Un dernier travail avait pour objectif de caractériser l’ADN gyrase sauvage de M. leprae. Si nous avons réussi à mettre au point un protocole de purification de l’enzyme optimisé, nous n’avons pas pu caractériser son fonctionnement mais proposons des perspectives d’étude de cette enzyme
Two hundred thousand cases of leprosy occur each year worldwide, especially in developing countries. Our laboratory having a double activity, leprosy surveillance and research focused on studying resistance mechanisms to antituberculosis drugs, the objectives of the work have medical and fundamental issues. A first genotyping study of M. leprae allowed us to show that all the leprosy cases diagnosed on the national territory are acquired outside metropolitan France. M. leprae enzymatic equipment is remarkable, for example, it encodes only a single type II topoisomerase, DNA gyrase. The only known FQ resistance mechanism in M. leprae is occurrence of mutations in DNA gyrase genes, the sole target of FQ in this species. As M. leprae is not cultivable in vitro, no antibiogram can be performed. It is thus essential to evaluate precisely the impact on resistance of all the DNA gyrase gene (gyrA / gyrB) mutations that have been observed in clinical strains. A systematic review of all the mutations published was performed. We investigated the impact on FQ resistance of 5 new mutants through a DNA gyrase biochemical assay (DNA supercoiling inhibition by FQ). A final work aimed to characterize the wild-type M. leprae DNA gyrase. We developed an optimized enzyme purification protocol. We did not manage to characterize how the enzyme functions, however we offer several study perspectives to achieve this goal

L’instabilité génétique de la bactérie streptomyces ambofaciens atcc23877 se manifeste par l'apparition de mutants dépigmentés à une fréquence voisine de 0,5%. Certains mutants sont pléiotropes. La majorité des mutants dépigmentés présentent des délétions et des amplifications de grandes séquences d’ADN. Deux mutants présentant une instabilité génétique augmentée ont été isoles. Les rayonnements ultraviolets, la mitomycine c et l'acide nitreux augmentent la fréquence des mutants dépigmentés. De plus, cinq antibiotiques inhibant l'ADN gyrase, la topoisomérase ii bactérienne, induisent de nombreux secteurs mutants (phénotype patchwork). La fréquence des mutants dépigmentés induits peut atteindre des valeurs proches de 100%. Ces mutants présentent les mêmes caractéristiques que les spontanés. Par contre, la rifampicine, inhibant la transcription, et la streptomycine, agissant au niveau de la traduction, n'ont aucun effet sur le phénomène. Des mutants résistants à la novobiocine ou à l'acide oxolinique ont été isolés. Chez ceux-ci, ces antibiotiques n'induisent plus l'instabilité génétique. D’autre part, les gènes codant les deux sous-unités de la gyrase ont été clonés, partiellement séquences et localises proches du gène DNAA et de l'origine de réplication chromosomique ORIC. L’intervention dans le phénomène de fonctions inductibles de type SOS ou de cassures double-brins est discutée. De plus, un nouveau mécanisme de plasticité génomique impliquant l’ADN gyrase est provoqué

Clostridium difficile est un bacille à Gram positif anaérobie strict, sporulé et producteur des toxines A et B. Il représente la principale étiologie des diarrhées nosocomiales de l'adulte. Le taux de résistance acquise aux quinolones en France était de 7 % entre 1991 et 1997. Cette résistance était toujours associée à la présence d'une mutation dans les gènes de l'ADN gyrase. C. Difficile ne possède qu'une seule cible des quinolones, l'ADN gyrase, et est dénué d'ADN topoisomérase IV. Nous avons cloné une protéine d'efflux appelée CdeA appartenant au cluster 3 de la famille MATE. Elle est capable de conférer une résistance aux fluoroquinolones lorsqu'elle est surexprimée chez E. Coli. L'exposition au bromure d'éthidium augmente la transcription du gène cdeA. Nous avons réussi à introduire par conjugaison un nouveau plasmide réplicatif exprimant un ARN antisens chez C. Difficile. Cependant, le rôle de CdeA dans la résistance aux fluoroquinolones et aux toxiques n'a pu être démontré
C. Difficile represents the main cause of nosocomial diarrhea in adults. The prevalence of acquired resistance to quinolones in France was 7% and identical for the years 1991 and 1997. All isolates with decreased susceptibility carried a mutation in the DNA gyrase gyrA or gyrB genes. We found that C. Difficile lacks the genes coding for DNA topoisomerase IV. CdeA is the first multidrug efflux transporter identified in C. Difficile. CdeA belongs to the cluster 3 of the MATE family. It was responsible for quinolone resistance in E. Coli when overexpressed. The presence of subinhibitory concentration of ethidium bromide significantly increased the transcription of cdeA. We have successfully introduced by conjugation between E. Coli and C. Difficile a new replicative vector which was used to introduce antisens RNA for cdeA. However, there was no significant difference in the susceptibility to quinolones or toxics between the recombinant and the parental strain