Relevant bibliographies by topics / Okazaki fragments

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Academic literature on the topic 'Okazaki fragments'

Author: Grafiati
Published: 4 June 2021
Last updated: 26 April 2022

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Journal articles on the topic "Okazaki fragments"

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Padel, Ruth. "The Okazaki Fragments." Poem 1, no. 1 (January 2013): 114–23. http://dx.doi.org/10.1080/20519842.2013.11415334.

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Kumamoto, Soichiro, Atsuya Nishiyama, Yoshie Chiba, Ryota Miyashita, Chieko Konishi, Yoshiaki Azuma, and Makoto Nakanishi. "HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation." Nucleic Acids Research 49, no. 9 (April 19, 2021): 5003–16. http://dx.doi.org/10.1093/nar/gkab269.

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Abstract DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.
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Spiering, Michelle M., Philip Hanoian, Swathi Gannavaram, and Stephen J. Benkovic. "RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication." Proceedings of the National Academy of Sciences 114, no. 22 (May 15, 2017): 5635–40. http://dx.doi.org/10.1073/pnas.1620459114.

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The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior—that is, the signaling mechanism—have not been definitively identified. We examined the role of RNA primer–primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer–primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer–primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer–primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes.
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Chen, Danqi, Hongjun Yue, Michelle M. Spiering, and Stephen J. Benkovic. "Insights into Okazaki Fragment Synthesis by the T4 Replisome." Journal of Biological Chemistry 288, no. 29 (May 31, 2013): 20807–16. http://dx.doi.org/10.1074/jbc.m113.485961.

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In this study, we employed a circular replication substrate with a low priming site frequency (1 site/1.1 kb) to quantitatively examine the size distribution and formation pattern of Okazaki fragments. Replication reactions by the T4 replisome on this substrate yielded a patterned series of Okazaki fragments whose size distribution shifted through collision and signaling mechanisms as the gp44/62 clamp loader levels changed but was insensitive to changes in the gp43 polymerase concentration, as expected for a processive, recycled lagging-strand polymerase. In addition, we showed that only one gp45 clamp is continuously associated with the replisome and that no additional clamps accumulate on the DNA, providing further evidence that the clamp departs, whereas the polymerase is recycled upon completion of an Okazaki fragment synthesis cycle. We found no support for the participation of a third polymerase in Okazaki fragment synthesis.
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Cronan, Glen E., Elena A. Kouzminova, and Andrei Kuzminov. "Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision." Proceedings of the National Academy of Sciences 116, no. 4 (January 7, 2019): 1251–60. http://dx.doi.org/10.1073/pnas.1814512116.

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In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.
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Hernandez, Alfredo J., Seung-Joo Lee, and Charles C. Richardson. "Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome." Proceedings of the National Academy of Sciences 113, no. 21 (May 9, 2016): 5916–21. http://dx.doi.org/10.1073/pnas.1604894113.

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DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.
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Bartoszek, Krzysztof, and Wojciech Bartoszek. "On the time behaviour of Okazaki fragments." Journal of Applied Probability 43, no. 02 (June 2006): 500–509. http://dx.doi.org/10.1017/s0021900200001789.

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We find explicit analytical formulae for the time dependence of the probability of the number of Okazaki fragments produced during the process of DNA replication. This extends a result of Cowan on the asymptotic probability distribution of these fragments.
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Bartoszek, Krzysztof, and Wojciech Bartoszek. "On the time behaviour of Okazaki fragments." Journal of Applied Probability 43, no. 2 (June 2006): 500–509. http://dx.doi.org/10.1239/jap/1152413737.

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We find explicit analytical formulae for the time dependence of the probability of the number of Okazaki fragments produced during the process of DNA replication. This extends a result of Cowan on the asymptotic probability distribution of these fragments.
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Henneke, Ghislaine. "In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways." Biochemical Journal 447, no. 2 (September 26, 2012): 271–80. http://dx.doi.org/10.1042/bj20120959.

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Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi, I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5′-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5′-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by polymerase B and flap cleavage by Fen1 provided a nicked substrate for ligation by Lig1. The unique specificities of Okazaki fragment maturation enzymes and replicative DNA polymerases strongly support the existence of two pathways in the resolution of RNA fragments.
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Kang, Ho-Young, Eunjoo Choi, Sung-Ho Bae, Kyoung-Hwa Lee, Byung-Soo Gim, Hee-Dai Kim, Chankyu Park, Stuart A. MacNeill, and Yeon-Soo Seo. "Genetic Analyses of Schizosaccharomyces pombe dna2+ Reveal That Dna2 Plays an Essential Role in Okazaki Fragment Metabolism." Genetics 155, no. 3 (July 1, 2000): 1055–67. http://dx.doi.org/10.1093/genetics/155.3.1055.

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Abstract In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2+ of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2+ was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2+ led to chromosome fragmentation strikingly similar to that seen when cdc17+, the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase δ (cdc1+ and cdc27+), DNA ligase I (cdc17+), and Fen-1 (rad2+). Each of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2+ plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2+ acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.
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Dissertations / Theses on the topic "Okazaki fragments"

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Wu, Xia. "Determination of DNA replication program changes between cancer and normal cells by sequencing of Okazaki fragments." Thesis, Paris Sciences et Lettres (ComUE), 2016. http://www.theses.fr/2016PSLEE032.

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Jusqu'à présent, les modifications de la réplication de l'ADN entre cellules normales et cancéreuses ont été peu étudiées. Dans ce travail, nous avons utilisé le séquençage des fragments d'Okazaki, une technique récemment développée au laboratoire, pour déterminer la directionalité des fourches de réplication dans plusieurs lymphomes de Burkitt (LB), qui surexpriment l'oncoprotéine Myc à la suite de translocations chromosomiques spécifiques, ainsi que dans des lignées lymphoblastoides contrôles (LLC) et dans des léiomyosarcomes (LMS). Les profils de directionalité des fourches de réplication permettent de déduire la localisation et l'efficacité des sites d'initiation et de terminaison de la réplication le long du génome. Nous avons observé de nombreuses (~2000) différences de zones d'initiation entre les lignées Raji (LB) et GM06990 (LLC) ainsi qu'entre les lignées BL 79 et IARC385, une paire LB/LLC provenant d'un même patient. Nous avons détecté un nombre comparable de différences en comparant deux à deux les lignées étudiées. Cependant, les profils de BL79 et de Raji (deux LB) sont un peu plus proches l'un de l'autre que de la lignée contrôle GM06990. Ceci suggère l'existence de changements de la réplication récurrents dans les lignées LB. L'importance des différences observées entre les lignées IARC385 et GM06990 indique de façon surprenante une grande variabilité entre les LLC normales, provenant de différents individus. De façon intéressante, de nombreuses différences observées entre les lignées LB et LLC sont associées à des changements de l'expression des gènes ou de la liaison de l'oncoprotéine Myc. La comparaison des profils des deux LMS avec tous les profils disponibles au laboratoire montre que c'est à celui de fibroblastes normaux (IMR90) qu'ils ressemblent le plus. Ceci suggère que les cellules de tumeurs musculaires lisses auraient subi une transformation fibroblastique au cours de la tumorigénèse. Des données récentes suggèrent que les champs magnétiques peuvent perturber certains processus cellulaires comme l'assemblage du cytosquelette. Nous avons utilisé le séquençage de fragment d'Okazaki pour rechercher d'éventuels effets d'un champ magnétique sur la réplication de l'ADN chez la levure. Aucun effet du champ magnétique sur la directionalité des fourches de réplication n'a été détecté
Changes in DNA replication profiles between cancer and normal cells have been poorly explored. In this work, sequencing of Okazaki fragments, a novel methodology developed in the laboratory, was used to determine replication fork directionality (RFD) in several Burkitt's lymphomas (BL), which overexpress the Myc oncoprotein due to specific chromosomal translocations, and control normal lymphoblastoid cell lines (LCL), and in leiomyosarcomas (LMC). RFD profiles allow to infer the location and efficiency of replication initiation and termination sites genome-wide. A larger number (~2000) of differences in replication initiation zones were observed genome-wide between Raji (BL) and GM06990 (LCL), and between BL79 and IAR385, a BL / LCL pair of cell lines established from a single patient. Comparably large numbers of changes were slightly more similar to each other than to GM06990. This suggests the occurrence of some recurrent replication changes in BL cell lines. The large number of changes observed between IARC385 and GM06990 also indicates an unexpectedly large variation between normal LCLs of different individuals. Interestingly, many changes in RFD profiles between BLs and and LCLs are associated with cell-type specific gene expression and differential binding of the Myc oncoprotein. Comparison of the two LMS profiles with all RFD profiles available in the laboratory reveals that they most resemble normal fibroblasts (IMR90). This suggests that the smooth muscle cancer cells might have undergone a fibroblastic transformation during tumorigenesis. Magnetic fields have been reported to perturb cellular processes such as cytoskeleton assembly. Sequencing of Okazaki fragments was used in a preliminary investigation of the possible effects of magnetic fields on DNA replication in yeast cells. No effect of magnetic fields on replication fork directionality were observed
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Kemp, Harriet. "Lagging strand replication creates evolutionary hotspots throughout the genome." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17896.

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The rate of DNA mutation is known to fluctuate across the genome but the patterns of mutation rate variation and molecular causes are poorly defined. It is important to understand these patterns of mutation as they influence where deleterious mutations are likely to arise and how rapidly sequences are likely to accumulate change between species, a measure often used as a proxy for functional constraint. In this work I investigate the relationship between DNA replication and apparent mutation hotspots adjacent to transcription factor binding sites. In eukaryotes both DNA strands are replicated simultaneously, the leading strand as a continuous stretch and the lagging strand as a series of discrete Okazaki fragments that are subsequently ligated together. Some transcription factors are able to bind the DNA lagging strand during replication and act as a partial barrier to DNA polymerase, resulting in the accumulation of Okazaki fragment junctions adjacent to these sites. I find that mutation rate is correlated genome wide with Okazaki junction frequency, suggesting that Okazaki junction processing may be error-prone. We present a mechanistic hypothesis to explain this locally elevated mutation rate and propose a role for lagging strand replication and its error-prone Pol α tract retention in the formation of these hotspots. I test this hypothesis using Okazaki fragment sequencing data from the yeast Saccharomyces cerevisiae to identify peaks in Okazaki junctions. When these peaks are aligned and orientated, so that the direction of lagging strand replication is uniform, I find a peak in substitution rate immediately downstream of Okazaki junctions, precisely where Pol α tract retention is predicted to occur. Novel binding motifs are identified within the underlying DNA of these junctions that can be assigned to known strong and fast-binding transcription factors, previously implicated in the phasing of nucleosomes, such as Reb1. I show that mutation hotspots adjacent to transcription factor binding sites are a conserved feature of eukaryotic genomes. In the human genome I predict sites of preferential Pol α retention using DNase I hypersensitivity footprint data. We observe that those footprints predicted as germline-specific manifest an elevated mutation signature. I propose that the rapid binding of some transcription factors to DNA following replication is required for nucleosome positioning or other important functions, however this incurs a cost in terms of locally elevated mutation rate adjacent to and within the sequence specific binding site. As a consequence these binding sites are biologically important mutational hotspots whose functional significance has been systematically underestimated by standard measures of sequence constraint.
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Leriche, Mélissa. "Mise en évidence d’une interaction entre la protéine 53BP1 et les fragments d’Okazaki." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS065.

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Le maintien de l’intégrité du génome est un processus crucial à la vie cellulaire. Ce n’est que récemment que les protéines de liaison à l’ARN (« RNA-binding protein » ou RBP) ont été montré être impliquées dans ce processus. En présence d’ADN endommagé, les RBP régulent l’expression des gènes de réponse aux dommages de l’ADN et contrôlent le destin cellulaire. Elles jouent également un rôle plus direct dans la prévention et la réparation des dommages de l’ADN. De plus, des ARN sont présents aux sites de dommages de l’ADN et participent au maintien de l’intégrité du génome. Ainsi, le laboratoire recherche des acteurs protéiques capables de lier directement l’ARN au sein des protéines médiatrices de la réponse aux dommages de l’ADN. Un des candidats est la protéine 53BP1 (p53 binding protein 1) qui contient un domaine de liaison à l’ARN nommé domaine GAR (« Glycine-Arginine Rich »). 53BP1 est un acteur central de la signalisation des cassures double-brin de l’ADN et de la régulation de leur réparation par le processus de jonction d’extrémités non homologues pendant la phase G1 du cycle cellulaire. Le recrutement de 53BP1 aux sites de dommages de l'ADN dépend à la fois d'interactions directes entre 53BP1 et des marques d’histones, mais aussi d’une composante ARN.L’objectif était d’étudier l’interaction entre 53BP1 et l’ARN.Grâce aux méthodes de CLIP (« CrossLinking and ImmunoPrecipitation ») et de 2C (« Complex Capture »), nous avons montré que 53BP1 possède une activité de liaison directe à l'ARN, via son domaine GAR. Nous avons identifié l’acide nucléique interagissant avec 53BP1 comme étant une chimère ARN-ADN constituée d’une partie d’environ 10 ribonucléotides, suivie d’environ 100 désoxyribonucléotides. Ce type de molécule est très similaire à celle des fragments d’Okazaki qui sont impliqués dans l’initiation de la synthèse du brin retardé de la fourche de réplication. Par la méthode de SIRF (« In Situ Protein Interaction with Nascent DNA Replication Forks »), nous avons montré que 53BP1 est présent au niveau de l’ADN naissant, dans un contexte de réplication normal. De plus, la déplétion de la sous-unité catalytique de la primase (PRIM1) qui synthétise l’amorce ARN des fragments d’Okazaki, conduit à la diminution de la présence de 53BP1 à proximité d’ADN naissant. La déplétion de PRIM1 affecte également l’interaction de 53BP1 avec la chimère ARN-ADN in vivo. Ces résultats indiquent que 53BP1 est présent à la fourche de réplication via une interaction directe avec les fragments d’Okazaki. Enfin, sous stress réplicatif induit par l’hydroxyurée, la présence de 53BP1 au niveau de l’ADN naissant est fortement augmentée, indiquant que 53BP1 s’accumule aux fourches de réplication bloquées. L'ensemble de ces résultats montre que 53BP1 est une protéine de liaison aux ARN qui interagit directement avec les fragments d’Okazaki
Maintenance of genome integrity is essential for cell survival. It is only recently that RNA-binding proteins (RBPs) have been shown as fundamental actors in this process. In the presence of DNA damage, RBPs regulate the expression of DNA damage response (DDR) related genes and control cell fate. RBPs also have a more direct role in preventing and repairing DNA damage. Moreover, some RNAs are present at sites of DNA damage and, thus, participate in the maintenance of genome integrity. The laboratory is interested in proteins that are both able to directly bind RNA and involved in DDR. One candidate is the 53BP1 protein (p53 binding protein 1) that contains an RNA-binding domain called GAR domain (Glycin-Arginin Rich). 53BP1 is a key protein mediating the signalling of DNA double-strand breaks and channels DNA repair to the non-homologous end-joining pathway during the G1 phase of the cell cycle. The recruitment of 53BP1 to sites of DNA damage depends on both histones marks and an RNA component.The objective was to study the interaction between 53BP1 and RNA.By using CLIP (CrossLinking and Immunoprecipitation) and 2C (Complex Capture) technologies, we showed that 53BP1 presents a direct RNA-binding activity within its GAR domain. We identified the nucleic acid interacting with 53BP1 as being an RNA-DNA chimera composed of about 10 ribonucleotides, followed by about 100 dexoribonucleotides. This type of entity is highly similar to that of Okazaki fragments, that are involved in the initiation of lagging strand synthesis at replication forks. By using the SIRF method (In Situ Protein Interaction with Nascent DNA Replication Forks), we showed that 53BP1 is localized at sites of newly synthetized DNA, under normal conditions of replication. Furthermore, depletion of the catalytic sub-unit of the primase (PRIM1), that catalyzes the synthesis of the RNA primer of Okazaki fragments, results in a decrease in 53BP1 at sites of newly synthetized DNA. PRIM1 depletion also decreases the interaction between 53BP1 and RNA-DNA chimera in vivo. These results indicate that 53BP1 is localized at the replication fork through a direct interaction with Okazaki fragments. Likewise, under replicative stress induced by hydroxyurea, the presence of 53BP1 at the newly synthetized DNA is increased, indicating that 53BP1 accumulates at stalled replication forks. Altogether, these results show that 53BP1 is an RNA-binding protein that directly interacts with Okazaki fragments
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Kabalane, Hadi. "Caractérisation pangénomique et analyse comparative du programme de réplication de l'ADN dans 12 lignées cellulaires humaines." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSEN063.

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Le programme spatiotemporel de réplication de l'ADN est régulé au cours du développement et altéré durant la progression cancéreuse. Nous proposons une caractérisation originale de la plasticité du programme de réplication de l’ADN se basant sur le profilage de 12 lignées cellulaires humaines normales ou cancéreuses par la méthode Ok-seq de purification et séquençage des fragments d'Okazaki qui permet de déterminer l'orientation de la progression des fourches de réplication (OFR) à haute résolution (10 kilo bases). L'analyse comparative des profils OFR montre que les changements réplicatifs permettent la classification des lignées cellulaires en fonction de leur tissu d'origine, la nature cancéreuse ou non de la lignée n'intervenant qu'en second ordre. Il n’apparait pas de point chaud pour l’accumulation des changements réplicatifs, ceux-ci étant largement dispersés sur tout le génome. Néanmoins, les régions riches en G+C et en gènes actifs, répliquées précocement au cours de la phase S, ont le programme de réplication le plus stable, elles présentent une forte densité de zones d'initiation de la réplication (ZI) efficaces et conservées entre lignées cellulaires. En contraste, les dernières régions répliquées, à faible densité de gènes et pauvres en G+C, présentent peu de ZI efficaces, souvent spécifiques d'un tissu ou d'une lignée. Ceci nous conduit à quantifier le degré de dissociation entre ZI et activation de la transcription. Ce travail propose un panorama original des modifications du programme de réplication au cours de la différentiation normale ou pathologique, dont un contrôle lignée cellulaire spécifique des ZI dans les déserts de gènes à réplication tardive
The spatiotemporal program of DNA replication is regulated during development and altered during cancer progression. We propose an original characterization of the plasticity of the DNA replication program based on the profiling of 12 normal or cancerous human cell lines by the Ok-seq method of purification and sequencing of Okazaki fragments which allows to determine the orientation of the progression of replication forks (RFD) at high resolution (10 kilo bases). Comparative analysis of the RFD profiles shows that the replicative changes allow the classification of the cell lines according to their tissue of origin, the cancerous or non-cancerous nature of the cell line type intervening only in second order. There is no hotspot for the accumulation of replicative changes, they are widely dispersed throughout the genome. Nevertheless, the G+C rich and active gene regions, replicated early in the S phase, have the most stable replication program, they present a high density of efficient replication initiation zones (IZ) conserved between cell lines. In contrast, the late replicated, low gene density and low G+C content regions have few efficient IZs, often specific to a tissue or lineage. This leads us to quantify the degree of dissociation between IZ and activation of transcription. This work provides an original overview of replication program changes during normal or pathological differentiation, including a cell line specific control of IZ in late-replication gene deserts
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Beattie, Thomas R. "The molecular biology of DNA replication in the archaeon Sulfolobus solfataricus." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:99d668a5-2d7a-4c7f-a1f8-b514e699347e.

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DNA replication is essential for the propagation of all living organisms. The ability of a cell to accurately duplicate its entire genome is dependent upon the activity of numerous proteins. Identifying the molecular mechanisms by which these proteins act, and determining how they are physically and functionally coordinated at sites of active DNA replication, is central to understanding this essential cellular process. Archaea possess a DNA replication machinery which is ancestral to the one present in eukaryotes, and thus these organisms serve as simplified model systems for understanding the complexities of eukaryotic DNA replication. This thesis investigates the molecular mechanisms underlying Okazaki fragment maturation in the crenarchaeon Sulfolobus solfataricus, which is essential to the completion of lagging strand DNA replication. Reconstitution of Okazaki fragment maturation in vitro demonstrated that the activities of three enzymes – PolB1, Fen1, and Lig1 – are required for this process in S. solfataricus. Furthermore, it was shown that optimum coordination of their three distinct activities is dependent on the ability of PolB1, Fen1 and Lig1 to simultaneously interact with a single PCNA ring, providing evidence for a mechanism of multi-enzyme coordination which may be universally employed by DNA sliding clamp proteins. The importance of protein flexibility in the accommodation of multiple proteins around a single PCNA was also investigated. Finally, the physical coordination of one of these key maturation enzymes – PolB1 – with other replisome proteins was examined. It was demonstrated that PolB1 exists in a trimeric complex in vivo with two previously unidentified factors, raising the possibility of uncharacterised activities and interactions for this crucial enzyme. Taken together, these data provide new insights into functionally important protein-protein interactions within the archaeal replisome, and facilitate a greater understanding of the DNA replication machinery in both archaea and eukaryotes.
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Cannone, Giuseppe. "Structural investigation of the archaeal replicative machinery by electron microscopy and digital image processing." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17070.

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Previous studies suggest a degree of homology between eukaryotic replication, transcription and translation proteins and archaeal ones. Hence, Archaea are considered a simplified model for understanding the complex molecular machinery involved in eukaryotic DNA metabolism. DNA replication in eukaryotic cells is widely studied. In recent years, DNA replication studies expanded on the archaeal DNA replication machinery. P. abyssi was the first archaeon whose genome was fully sequenced. Genome sequencing and comparative genomics have highlighted an MCM-like protein in P. abyssi. In this study, I report the biochemical and structural characterisation of PabMCM. PabMCM is explored as model for understanding more complex eukaryotic MCM proteins and unravelling the biochemical mechanism by which MCM proteins release their helicase activity. The crenarchaeon Sulfolobus solfataricus possesses a simplified toolset for DNA replication compared to Eukaryotes. In particular, S. solfataricus has a subset of the eukaryotic Okazaki fragment maturation factors, among which there are a heterotrimeric DNA sliding clamp, (the proliferating cell nuclear antigen, PCNA), the DNA polymerase B1 (PolB1), the flap endonuclease (Fen1) and the ATP-dependent DNA ligase I (LigI). PCNA functions as a scaffold with each subunit having a specific binding affinity for each of the factors involved in Okazaki fragment maturation. Here, the 3D reconstruction of PCNA in complex with the Okazaki fragment maturation proteins PolB1, LigI and Fen1 is reported.
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Johnson, Vinu. "Structural and Biophysical Studies of Single-Stranded DNA Binding Proteins and dnaB Helicases, Proteins Involved in DNA Replication and Repair." University of Toledo / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1198939056.

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Sabouri, Nasim. "Structure of eukaryotic DNA polymerase epsilon and lesion bypass capability." Doctoral thesis, Umeå : Univ, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1477.

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Joudeh, Luay. "Toward The Reconstitution of the Maturation of Okazaki Fragments Multiprotein Complex in Human At The Single Molecule Level." Diss., 2017. http://hdl.handle.net/10754/623664.

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The maturation of Okazaki fragments on the lagging strand in eukaryotes is mediated by a highly coordinated multistep process involving several proteins that ensure the accurate and efficient replication of genomic DNA. Human proliferating cell nuclear antigen (PCNA) that slides on double-stranded DNA is the key player that coordinates the access of various proteins to the different intermediary steps in this process. In this study, I am focusing on characterizing how PCNA recruits and stimulates the structure specific flap endonuclease 1 (FEN1) to process the aberrant double flap (DF) structures that are produced during maturation of Okazaki fragments. FEN1 distorts the DF structures into a bent conformer to place the scissile phosphate into the active site for cleavage. The product is a nick substrate that can be sealed by DNA ligase I whose recruitment is also mediated by its interaction with PCNA. Using single-molecule Förster resonance energy transfer (smFRET) measurements that simultaneously monitored bending and cleavage of various DF substrates by FEN1 alone or in the presence of PCNA, we found that FEN1 and PCNA bends cognate and non-cognate substrates but display remarkable selectivity to stabilize the bent conformer in cognate substrate while promoting the dissociation of non-cognate substrates. This mechanism provides efficiency and accuracy for FEN1 and PCNA to cleave the correct substrate while avoiding the deleterious cleavage of incorrect substrates. This work provides a true molecular level understanding of the key step during the maturation of Okazaki fragment and contributes towards the reconstitution of its entire activity at the single molecule level.
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DeNapoli, Leyna. "DNA Replication of the Male X Chromosome Is Influenced by the Dosage Compensation Complex in Drosophila melanogaster." Diss., 2013. http://hdl.handle.net/10161/7238.

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Abstract

DNA replication is an integral part of the cell cycle. Every time a cell divides, the entire genome has to be copied once and only once in a timely manner. In order to accomplish this, DNA replication begins at many points throughout the genome. These start sites are called origins of replication, and they are initiated in a temporal manner throughout S phase. How these origins are selected and regulated is poorly understood. Saccharomyces cerevisiae and Schizosaccharomyces pombe have autonomously replicating sequences (ARS) that can replicate plasmids extrachromosomally and function as origins in the genome. Metazoans, however, have shown no evidence of ARS activity.

DNA replication is a multistep process with several opportunities for regulation. Potential origins are marked with the origin recognition complex (ORC), a six subunit complex. In S. cerevisiae, ORC binds to the ARS consensus sequence (ACS), but no sequence specificity is seen in S. pombe or in metazoans. Therefore, factors other than sequence play a role in origin selection.

In G1, the pre-replicative (pre-RC) complex assembles at potential origins. This involves the recruitment of Cdc6 and Cdt1 to ORC, which then recruits MCM2-7 to the origin. In S phase, a subset of these pre-RC marked origins are initiated for replication. These origins are not fired simultaneously; instead, origins are fired in a temporal manner, with some firing early, some firing late, and some not firing at all.

The temporal firing of origins leads to wide regions of the genome being copied at different times during S phase. , which makes up the replication timing profile of the genome. These regions are not random, and several correlations between replication timing and both transcriptional activity and chromosomal landscape. Regions of the genome with high transcriptional activity tend to replicate earlier in S phase, and it is well know that the gene rich euchromatin replicates earlier than the gene poor heterochromatin. Additionally, areas of the genome with activating chromatin marks also replicate earlier than regions with repressive marks. Though many correlations have been observed, no single mark or transcriptional player has been shown to directly influence replication timing.

We mapped the replication timing profiles of three cell lines derived from Drosophila melanogaster by pulsing cells with the nucleotide analog bromodeoxyuridine (BrdU), enriching for actively replicating DNA labeled with BrdU, sequencing with high throughput sequencing and mapping the sequences back to the genome. We found that the X chromosome of the male cell lines replicated earlier than the X chromosome in the female cell line or the autosomes. We were then able to compare the replication timing profiles to data sets for chromatin marks acquired through the modENCODE (model organism Encyclopedia Of DNA Elements). We found that the early replicating regions of the male X chromosomes correlates with acetylation of lysine 16 on histone 4 (H4K16).

Hyperacetylation of H4K16 on the X chromosome in males is a consequence of dosage compensation in D. melanogaster. Like many organisms, D. melanogaster females have two X chromosomes while males have one. To compensate for this difference, males upregulate the genes on the X chromosome two-fold. This upregulation is regulated by the dosage compensation complex (DCC), which is restricted to the X chromosome. This complex includes a histone acetyl transferase, MOF, which acetylates H4K16. This hyperacetylation allows for increased transcription of the X chromosome.

We hypothesized that the activities of the DCC and the hyperacetylation of H4K16 also influences DNA replication timing. To test this, I knocked down components of the DCC (MSL2 and MOF) using RNAi. Cells were arrested in early S phase with hydroxyurea, released, and pulsed with the nucleotide analog EdU. The cells were arrested in metaphase and labeled for H4K16 acetylation and EdU. We found that male cells were preferentially labeled with EdU on the X chromosome, which corresponded with H4k16 acetylation. When the DCC was knocked down, H4K16 acetylation was lost along with preferential EdU labeling on the X chromosome. These results suggest that the DCC and H4K16 acetylation are necessary for early replication of the X chromosome. Additionally, early origin mapping of different cell lines showed that while ORC density does not differ between male and female cell lines, early origin usage is increased on the X chromosome of males, suggesting that this phenomenon is regulated at the level of activation, not pre-RC formation. Other experiments in female cell lines have been unclear about whether the DCC and subsequent H4K16Ac is sufficient for early X replication. However, these results are exciting because this is, to our knowledge, the first mark that has been found to directly influence replication timing.

In addition to these timing studies, I attempted to design a new way to map origins. A consequence of unidirectional replication with bidirectional replication fork movement is Okazaki fragments. These are short nascent strands on the lagging strand of replicating DNA. Because these fragments are small, we can isolate them by size and map them back to the genome. Okazaki density could tell us about origin usage and any directional preferences of origins. The process proved to be tedious, and although they mapped back with a higher density around ORC binding sites than randomly sheared DNA, little information about origin usage was garnered from the data. Additionally, the process proved difficult to repeat.

In these studies, we examined the replication timing program in D. melanogaster. We found that the male X chromosome replicates earlier in S phase, and this early replication is regulated by the DCC. However, it is unclear if the change in chromatin landscape directly influences replication or if the replication program is responding to other dosage compensation cues on the X chromosome. Regardless, we have found one the first conditions in which a mark directly influences the DNA replication timing program. 


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Books on the topic "Okazaki fragments"

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Rosyjskie ścieżki Klio: Wybór szkiców i esejów historycznych oraz fragmentów "Dziennika" Autora : wydany z okazji Jego 75. rocznicy urodzin. Pułtusk: Akademia Humanistyczna im. Aleksandra Gieysztora, 2007.

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Book chapters on the topic "Okazaki fragments"

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Gooch, Jan W. "Okazaki Fragments." In Encyclopedic Dictionary of Polymers, 912. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4419-6247-8_14380.

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Smith, Duncan J., Tejas Yadav, and Iestyn Whitehouse. "Detection and Sequencing of Okazaki Fragments in S. cerevisiae." In Methods in Molecular Biology, 141–53. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2596-4_10.

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Gooch, Jan W. "Okazaki Fragment." In Encyclopedic Dictionary of Polymers, 911. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4419-6247-8_14379.

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McHenry, Charles S. "Cycling of the Lagging Strand Replicase During Okazaki Fragment Synthesis." In Molecular Life Sciences, 146–53. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4614-1531-2_132.

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McHenry, Charles. "Cycling of the Lagging Strand Replicase During Okazaki Fragment Synthesis." In Molecular Life Sciences, 1–9. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4614-6436-5_132-1.

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"Okazaki Fragments." In Encyclopedia of Genetics, Genomics, Proteomics and Informatics, 1389–90. Dordrecht: Springer Netherlands, 2008. http://dx.doi.org/10.1007/978-1-4020-6754-9_11795.

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"Replication of DNA: formation of Okazaki fragments." In Biochemical Basis of Medicine, 596. Elsevier, 1985. http://dx.doi.org/10.1016/b978-0-7236-0722-9.50075-4.

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Reha-Krantz, L. J. "Okazaki Fragment." In Brenner's Encyclopedia of Genetics, 158–60. Elsevier, 2013. http://dx.doi.org/10.1016/b978-0-12-374984-0.01087-1.

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"Okazaki Fragment." In Encyclopedia of Genetics, 1367. Elsevier, 2001. http://dx.doi.org/10.1006/rwgn.2001.1945.

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Lucchesi, John C. "Chromatin replication." In Epigenetics, Nuclear Organization & Gene Function, 165–72. Oxford University Press, 2019. http://dx.doi.org/10.1093/oso/9780198831204.003.0014.

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During chromosome replication, each strand of the DNA serves as a template for the synthesis of a new strand (semiconservative replication). Replication originates with the binding of pre-replication complexes (MCM2–7) at multiple sites, termed origins of replication (ORIs). One strand of the parent DNA molecule (the leading strand) is replicated continuously in the 5´ to 3´ direction; the other, or lagging strand, is replicated one short segment at a time. These segments are referred to as Okazaki fragments, and their generation requires the synthesis of short complementary RNA primers. For replication to proceed, DNA must be unwound and freed from nucleosomes. After replication, the nucleosomal structure is re-established using a mixture of old and newly synthesized histones. DNA replication can encounter problems such as nucleotide depletion, DNA damage or topologically unfavorable structures that generate replication stress and replication fork stalling. The DNA replication process itself can also be subjected to errors. Cells have evolved a battery of mechanisms designed to bypass or repair damaged DNA strands.
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