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Carrington, James T. "Post-replicative resolution of under-replication." Thesis, University of Dundee, 2017. https://discovery.dundee.ac.uk/en/studentTheses/f0a89d2a-6ee2-4175-ba65-d58aaaee4e24.
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The evolutionary pressure to prevent re-replication by inactivating licensing during S phase leaves higher-eukaryotes with large genomes, such as human cells, vulnerable to replication stresses. Origins licensed in G1 must be sufficient to complete replication as new origins cannot be licensed in response to irreversible replication fork stalling. Interdisciplinary approaches between cellular biology and biophysics predict that replication of the genome is routinely incomplete in G2, even in the absence of external stressors. The frequency of converging replication forks that never terminate due to irreversible stalling (double fork stall), which result in a segment of unreplicated DNA, was modelled using high quality origin-mapping data in HeLa and IMR-90 cells. From this, hypotheses were generated that related an increase in unreplicated segments of DNA to reduced functional origin number. Presented in this thesis is the confirmation of this relation by quantifying chromosome mis-segregation and DNA damage responses when origin number was reduced using RNAi against licensing factors. The number of ultrafine anaphase bridges and 53BP1 nuclear bodies are in remarkable concordance with the theoretical predictions for the number of double fork stalls, indicating that cells are able to tolerate under-replication through such post-replicative cellular responses. 53BP1 preferentially binds to chromatin associated with large replicons, and functions synergistically with dormant origins to protect the stability of the genome. Additional candidates, inspired by common fragile site research, have also been characterised as responders to spontaneous under-replication, and include FANCD2 and MiDAS, which function in early mitosis to facilitate completion of replication before cells enter anaphase. In conclusion, a series of mechanisms that sequentially function throughout the cell cycle protects the stability of the human genome against inevitable spontaneous under-replication brought about by its large size.
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Rytkönen, A. (Anna). "The role of human replicative DNA polymerases in DNA repair and replication." Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514281381.
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Abstract
The maintenance of integrity of the genome is essential for a cell. DNA repair and faithful DNA replication ensure the stability of the genome. DNA polymerases (pols) are the enzymes that synthesise DNA, a process important both in DNA replication and repair. In DNA replication DNA polymerases duplicate the genome during S phase prior to cell division. Pols α, δ, and ε are implicated in chromosomal DNA replication, but their exact function in replication is not yet completely clear. The mechanisms of different repair pathways and proteins involved are not yet completely characterised either. The deeper understanding of DNA repair and replication mechanisms is crucial for our understanding on the function of the cell.
The mechanism of repair of DNA double strand breaks (DSBs) by non-homologous end joining (NHEJ) was studied with an in vitro assay. DNA polymerase activity was found to be involved in NHEJ and important in stabilising DNA ends. Antibodies against pol α, but not pol β or ε, decreased NHEJ significantly, which indicates the involvement of pol α in NHEJ. In addition, the removal of proliferating cell nuclear antigen (PCNA) slightly decreased NHEJ activity.
The division of labour between pols α, δ, and ε during DNA replication was studied. Results from UV-crosslinking, chromatin association, replication in isolated nuclei, and immunoelectron microscopy (IEM) studies showed that there are temporal differences between the activities and localisations of the pols during S phase. Pol α was active throughout S phase, pol ε was more active at early S phase, whereas the activity of pol δ increased as S phase advanced. These results suggest that pols δ and ε function independently during DNA replication.
Pol ε could be crosslinked to nascent RNA, and this labelling was not linked to DNA replication, but rather to transcription. Immunoprecipitation studies indicated that pol ε, but not pols α and δ, associated with RNA polymerase II (RNA pol II). Only the hyperphosphorylated, transcriptionally active RNA pol II was found to associate with pol ε. A large proportion of pol ε and RNA pol II colocalised in cells as determined with immunoelectron microscopy. The interaction between pol ε and RNA pol II suggests that they are involved in a global regulation of transcription and DNA replication.
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Chen, Shuhua. "Multiple mechanisms regulate the human replication factors : replication protein A and DNA polymerase alpha-during DNA replication and DNA repair /." [S.l. : s.n.], 2003.
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Anderson, Mary E. Ph D. (Mary Elizabeth)Massachusetts Institute of Technology. "Regulation of DNA replication and the replication initiator, DnaA, in Bacillus subtilis." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/121876.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019Cataloged from PDF version of thesis. "February 2019."
Includes bibliographical references (pages 118-128).
DNA replication is a highly regulated process across all organisms. Improper regulation of DNA replication can be detrimental. I identified an overinitiating, conditional synthetic lethal mutant of Bacillus subtilis. I isolated suppressors of this mutant and uncovered novel genes associated with DNA replication. These suppressors acted both at the steps of initiation and elongation to overcome the detrimental replication initiation of the synthetic lethal [delta]yabA dnaA 1 mutant. One class of suppressors decreased levels of the replicative helicase, DnaC. I showed that decreased levels of helicase are sufficient to limit replication initiation under fast growth conditions. I also explored the regulation of DnaA as a transcription factor. The replication initiation inhibitor, YabA, binds to DnaA and prevents its cooperative binding at the origin. In addition to its role in replication initiation, DnaA also directly regulates expression of several genes. YabA has been shown to inhibit DnaA binding at several promoters but its effect on DnaA-mediated gene expression is unclear. I found that YabA inhibits sda activation by DnaA but does not significantly affect repression of ywlC by DnaA. Lastly, I showed that YabA appears to stimulate autoregulation of dnaA. This preliminary data illustrates a role for YabA regulation in DnaA-mediated gene expression.
by Mary E. Anderson.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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Tavares, de Araujo Felipe. "DNA replication and methylation." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37847.
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One of the main questions of modern biology is how our cells interpret our genetic and epigenetic information. DNA methylation is a covalent modification of the genome that is essential for mammalian development and plays an important role in the control of gene expression, genomic imprinting and X-chromosome inactivation (Bird and Wolffe, 1999; Szyf et al., 2000). Furthermore, changes in DNA methylation and DNA methyltransferase 1 (DNMT1) activity have been widely documented in a number of human cancers (Szyf, 1998a; Szyf et al., 2000).In Escherichia coli, timing and frequency of initiation of DNA replication are controlled by the levels of the bacterial methyltransferase and by the methylation status of its origin of replication (Boye and Lobner-Olesen, 1990; Campbell and Kleckner, 1990). In mammalian cells, however, the importance of methyltransferase activity and of DNA methylation in replication is only now starting to emerge (Araujo et al., 1998; Delgado et al., 1998; DePamphilis, 2000; Knox et al., 2000).
The work described in this thesis focuses mainly on understanding the functional relationship between changes in DNA methylation and DNMT1 activity on mammalian DNA replication. In higher eukaryotes, DNA replication initiates from multiple specific sites throughout the genome (Zannis-Hadjopoulos and Price, 1999). The first part of the thesis describes the identification and characterization of novel in vivo initiation sites of DNA replication within the human dnmt1 locus (Araujo et al., 1999). Subsequently, a study of the temporal relationship between DNA replication and the inheritance of the DNA methylation pattern is presented. We also demonstrate that mammalian origins of replication, similarly to promoters, are differentially methylated (Araujo et al., 1998). We then tested the hypothesis that DNMT1 is a necessary component of the replication machinery. The results presented indicate that inhibition of DNMT1 results in inhibition of DNA replication (Knox et al., 2000). Finally, results are presented, demonstrating that the amino terminal region of DNMT1 is responsible for recognizing hemimethylated CGs, DNMT1's enzymatic target. Taken together, the results presented in this thesis demonstrate that DNMT1 is necessary for proper DNA replication and that DNA methylation may modulate origin function.
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Upton, Amy Louise. "Replication of damaged DNA." Thesis, University of Nottingham, 2009. http://eprints.nottingham.ac.uk/11332/.
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DNA is under constant attack from numerous damaging agents and our cells deal with thousands of lesions every day. With such constant damage it is inevitable that the template will not be completely cleared of lesions before the replication complex arrives. The consequences of the replisome meeting an obstacle will depend upon the nature of the obstacle. I have focussed upon replication in Escherichia coli and the effect of UV-induced lesions, which would block synthesis by the replicative polymerases. It is accepted that a UV lesion in the lagging strand template can be bypassed by the replisome complex, but the consequences of meeting a lesion in the leading strand template remain unclear. A lesion in the leading strand template could block replisome progression and the fork might require extensive processing in order to restart replication. However, it has also been proposed that the replisome could progress past these lesions by re‐priming replication downstream and leaving a gap opposite the lesion. The results of my studies revealed that all modes of synthesis are delayed after UV. I have demonstrated that when synthesis resumed, the majority reflected the combined effects of oriC firing and the initiation of inducible stable DNA replication. These modes of synthesis mask the true extent of the delay in synthesis at existing replication forks. The results also revealed that all synthesis after UV is dependent upon DnaC, suggesting that the replicative helicase and possibly the entire replisome, needs to be reloaded. A functional RecFOR system is required for efficient replication restart, without these proteins replication is capable of resuming but only after a long delay. My data support models proposing that replication forks require extensive processing after meeting a lesion in the leading strand template. Whilst I cannot exclude the possibility that replication forks can progress past some such lesions, my data indicate that they cannot progress past many before stalling. Overall, my results demonstrate the importance of measuring all modes of DNA synthesis when assessing the contribution of any particular protein to recovery after UV irradiation. Thus, although net synthesis in cells lacking RecG appears similar to wild type after UV, the mode of replication is in fact quite different. A dramatic increase in the level of stable DNA replication appears to account for much of the overall synthesis detected and coincides with a major chromosome segregation defect. The importance of stable DNA replication in irradiated recG cells has not previously been considered because the different modes of synthesis were ignored. The significance of this pathology and of the other findings reported in this thesis is discussed in relation to current models of DNA repair and replication restart.
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Borazjani, Gholami Farimah. "Role of replicative primase in lesion bypass during DNA replication." Thesis, University of Sussex, 2017. http://sro.sussex.ac.uk/id/eprint/68762/.
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Maintenance of genome integrity and stability is fundamental for any form of life. This is complicated as DNA is highly reactive and always under attack from a wide range of endogenous and exogenous sources which can lead to different damages in the DNA. To preserve the integrity of DNA replication, cells hav evolved a variety of DNA repair pathways. DNA damage tolerance mechanisms serve as the last line of defence to rescue the stalled replications forks. TLS, error-prone type of DNA damage tolerance, acts to bypass DNA lesions and allows continuation of DNA replication. Surprisingly majority of archaeal species lack canonical TLS polymerases. This poses a question as to how archaea restart stalled replication in the absence of TLS or lesion repair pathways. This thesis establishes that archaeal replicative primase (PriS/L), a member of the archaeo-eukaryotic primase (AEP) superfamily, possessing both primase and polymerase activities, is able to bypass the most common oxidative damages and highly distorting lesions caused by UV radiation. It has been postulated that archaeal replicative polymerases (Pol B and Pol D family Pols) can bind tightly to the deaminated bases uracil and cause replication fork stalling four bases prior to dU. A specific mechanism for resuming replication of uracil containing DNA by PriS/L is suggested in this thesis. In this thesis, we also reported how the enzymatic activities of archaeal PriS/L are regulated. Here, it is demonstrated that in contrast to archaeal replicative polymerases, single-strand binding proteins (RPA) significantly limit the polymerase activity of PriS/L. The remaining results chapter interrogates the possible interactions between PriS/L and RPA. Finally, the attempts to reconstitute an archaeal CMG complex in vitro, with the aim of shedding light on the role of archaeal replicative primase in replication-specific TLS are described.
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Pearson, Christopher Edmund. "DNA cruciforms and mammalian origins of DNA replication." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=28503.
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The objective of the research in this thesis is to investigate, at the molecular level, the sequences and/or structures involved in the initiation of mammalian DNA replication and to investigate the protein interactions with DNA cruciforms which have been implicated in the process of replication initiation. Four plasmids containing monkey (CV-1) early replicating nascent origin enriched sequences (ors), which had been shown previously to replicate autonomously in transfected CV-1, COS-7 and HeLa cells, were used in the establishment of an in vitro DNA replication system that uses HeLa cell extracts. The in vitro replication system is dependent upon the presence of an ors sequence, and HeLa cell extracts. Mapping experiments indicate that there is preferential nucleotide incorporation in the ors sequences, suggesting site-specific initiation, and that replication is bidirectional and semiconservative. Electron microscopy confirmed that in vitro initiation occurs within the ors sequence.Prokaryotic and eukaryotic viral replication origins, mammalian origin enriched sequences (ors) and other mammalian early replicating sequences contain AT-rich sequences and inverted repeats, which have the potential to form bent and cruciform (stem-loop) DNA structures, respectively. Cruciforms have been postulated to form transiently at or near origins to serve as recognition structures for initiator proteins. Using a stable-DNA cruciform as a binding substrate in a band-shift assay, a novel DNA binding activity with specificity for the cruciform-containing DNA and no apparent sequence-specificity was identified in HeLa cell extracts. The activity is protein-dependent and is void of detectable nuclease activity. Cruciform-specific binding was observed to be maximal in early-S phase extracts. A novel cruciform binding protein (CBP) with an apparent molecular weight of 66 kDa was enriched from HeLa cell extracts. Footprinting experiments localized the CBP-DNA cruciform interaction to the four-way junction at the base of the cruciform. CBP appears to interact with the elbow junctions in an asymmetric fashion. Upon CBP binding, structural distortions were observed at the cruciform stems and at a DNA region distal to the junction.
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Schorr, Stephanie. "Replication of Bulky DNA Adducts." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-125267.
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Bennett, Ellen R. (Ellen Ruth). "Activation of papovavirus DNA replication." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=70232.
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To define the viral target sites of cellular permissive factors, simian virus 40-polyomavirus hybrid origins for DNA replication were formed by joining the auxiliary domain of one viral origin to the origin core of the other, and vice versa. The replicative capacity of these constructs were assessed in a number of mouse and monkey cell lines which express the large T antigen of polyomavirus or SV40. The results of this analysis showed that the auxiliary domains of the viral replication origins could substitute for one another in DNA replication, provided that the viral origin core and its cognate large T antigen were present in a permissive cellular milieu. Surprisingly, the large T antigens of the viruses could not substitute for one another, regardless of the species of origin of the host cell, even though the two large T antigens bind to the same sequence motif in vitro. These results suggest that species-specific permissive factors do not interact with the origin auxiliary domains but, rather, with either the origin core, large T antigen, or with both components to effect DNA replication.To determine whether the minimal sequences that constitute a viral enhancer of gene expression are capable of activating DNA replication, a series of recombinant plasmids, composed of elements and subelements (enhansons) of the SV40 enhancer joined to the late border of the polyomavirus origin core domain, were tested for their capacity to replicate in permissive mouse cells synthesizing polyomavirus large T antigen. The results of these experiments demonstrated that a number of reiterated SV40 minimal enhancer sequences are capable of activating polyomavirus DNA replication and that mutations of elements which impair transcriptional activity also disrupt SV40 enhancer-mediated polyomavirus DNA replication. In addition, when the adenovirus E1A gene, a known repressor of gene expression, was examined for its ability to repress the replication of the plasmids described above, it repressed polyomavirus DNA replication in a sequence non-specific manner.
To determine whether activation surfaces of eukaryotic transcription factors participate in activation of DNA replication, a reporter plasmid was made bearing the binding site for a yeast transcriptional activator, GAL4, positioned near the late side of the polyomavirus origin core domain, and tested for its ability to replicate in mouse cells expressing polyomavirus large T antigen and GAL4. The results of these experiments demonstrated that binding of GAL4 next to the polyomavirus core led to enhanced replication of the reporter plasmid. This enhanced replication was dependent on a GAL4 binding site and the presence of amino acid sequences required for transcriptional activation in mammalian cells. Moreover, fusion proteins formed between the GAL4 DNA binding domain and activation surfaces of other viral transactivators also activated polyomavirus DNA replication whereas deletion mutants of fusion proteins impaired in their ability to activate transcription were poor activators of DNA replication. Together, these results implicate transcription factors as well as other components of the transcriptional machinery in DNA replication, and suggest that activation of transcription and DNA replication may occur by a common mechanism.
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Thomas, C. M. "Cauliflower mosaic virus DNA replication." Thesis, Bucks New University, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374828.
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King, Arusha. "Replication of wheat chloroplast DNA." Thesis, University of Hertfordshire, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314575.
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Fennessy, Ross. "Chromatin dynamics during DNA replication." Thesis, University of Dundee, 2014. https://discovery.dundee.ac.uk/en/studentTheses/7898ad5c-ea45-4ce5-a6b7-9b858615368e.
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The primary level of chromatin organisation consists of arrays of nucleosomes that are present across the genetic template. Advances in the post genomics era have made it possible to determine the positions of nucleosomes genome-wide where it has been observed that nucleosomes adopt a distinct organisation with respect to genetic and trans-binding elements. Amongst the best studied of these is the transcription start site where it has been observed that genic nucleosome locations are well maintained with respect to promoters. DNA and chromatin replication are coupled processes whereby chromatin is disrupted ahead of the replication fork and nucleosomes are rapidly assembled on the nascent DNA template. Classically it has been observed that nascent chromatin is more susceptible to digestion, prompting the possibility of an “immature” chromatin organisation post assembly. However it has not been investigated genome-wide if nucleosomes are initially assembled in phase or must be reorganised post assembly to canonical locations. We have developed two methods of isolating nascent DNA fragments representative of nucleosome positions from synchronised and asynchronous populations of the budding yeast S. cerevesiae. High throughput sequencing has revealed that chromatin is assembled and organised rapidly behind the replication fork. The most nascent chromatin isolated displays typical patterns of nucleosome organisation suggesting that reorganisation of nucleosomes on the nascent template is replication coupled. Deletion of specific histone chaperones and chromatin remodelers perturbs this pathway. However, this can be compensated for by a transcription directed reorganisation of nascent chromatin. Our analysis of nascent chromatin has allowed us to investigate the mechanisms that act to direct chromatin organisation in addition to evaluation of models that describe nucleosome organisation genome-wide.
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Monaghan, Alan. "Mechanisms of adenovirus DNA replication." Thesis, University of St Andrews, 1995. http://hdl.handle.net/10023/13935.
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The development of a cell-free system in which adenovirus DNA synthesis can be initiated in vitro by using the viral genome or plasmids containing the origin of replication as template has led to the identification of the sequences important for origin function and the isolation and purification of the proteins required for viral DNA replication. In vitro studies on adenovirus types 2 and 5 have shown that their replication requires the formation of a large nucleoprotein complex. This is composed of three virally encoded proteins: adenovirus DNA polymerase, precursor terminal protein and DNA binding protein, and two cellular proteins nuclear factor I and nuclear factor III. While the presence of DNA helicases in other eukaryotic DNA replication systems have been well characterised, this was not the case for adenovirus DNA replication. Initial attempts to identify DNA helicase activity associated with any of the adenovirus replication proteins were unsuccessful. However, a novel DNA unwinding activity was found associated with the DNA binding protein (Ad.DBP). We examined the interaction of DBP with partial DNA duplexes and demonstrated that it could displace oligonucleotides annealed to single-stranded M13 DNA. In addition, DBP could also unwind small fragments of fully duplex DNA. Unlike a DNA helicase, DBP promoted DNA unwinding was nucleoside-5'-triphosphate and Mg2+ independent and exhibited no directionality. The activity required saturating amounts of DBP and was both efficient and cooperative in nature. The helix-destabilising activity was shown to be situated in the C-terminal domain of the protein. These properties suggest a role for DBP in DNA replication in which DBP destabilises duplex DNA during origin unwinding and replication fork movement. The second part of the thesis dealt with the characterisation of the putative "active site" of the adenovirus DNA polymerase. This experimental approach was prompted by data from earlier studies which indicated that DBP could increase the processitivity of the polymerase as well as its sensitivity to nucleotide analogue inhibitors. The "active site" was labelled with pyridoxal-5'-phosphate (PLP), a substrate binding site directed reagent for DNA polymerases. Treatment of Ad.5 DNA polymerase with PLP followed by reduction of the enzyme-PLP adduct resulted in irreversible inactivation of the polymerase activity while the 3'-5' exonuclease associated with Ad.5 DNA polymerase was minimally affected. Substrate protection studies indicate that PLP inhibition is complex. Neither template-primer nor substrate dNTP alone showed any protective effect from PLP mediated inhibition. However, the presence of both template -primer and complementary dNTP significantly protected against PLP inhibition. Comparative tryptic mapping of labelled enzyme, modified in the presence and absence of substrates by PLP reaction, on a C-18 reverse phase column, indicated the protection of one peptide from pyridoxylation in the presence of substrates. Amino acid sequence analyses found no sequence to be present in this peak.
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Brümmer, Anneke. "Mathematical modelling of DNA replication." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16212.
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Bevor sich eine Zelle teilt muss sie ihr gesamtes genetisches Material verdoppeln. Eukaryotische Genome werden von einer Vielzahl von Replikationsstartpunkten, den sogenannten Origins, aus repliziert, die über das gesamte Genome verteilt sind. In dieser Arbeit wird der zugrundeliegende molekulare Mechanismus quantitativ analysiert, der für die nahezu simultane Initiierung der Origins exakt ein Mal pro Zellzyklus verantwortlich ist. Basierend auf umfangreichen experimentellen Studien, wird zunächst ein molekulares regulatorisches Netzwerk rekonstruiert, welches das Binden von Molekülen an die Origins beschreibt, an denen sich schließlich komplette Replikationskomplexe (RKs) bilden. Die molekularen Reaktionen werden dann in ein Differentialgleichungssystem übersetzt. Um dieses mathematische Modell zu parametrisieren, werden gemessene Proteinkonzentrationen als Anfangswerte verwendet, während kinetische Parametersätze in einen Optimierungsverfahren erzeugt werden, in welchem die Dauer, in der sich eine Mindestanzahl von RKs gebildet hat, minimiert wird. Das Modell identifiziert einen Konflikt zwischen einer schnellen Initiierung der Origins und einer effizienten Verhinderung der DNA Rereplikation. Modellanalysen deuten darauf hin, dass eine zeitlich verzögerte Origininitiierung verursacht durch die multiple Phosphorylierung der Proteine Sic1 und Sld2 durch Cyclin-abhängige Kinasen, G1-Cdk bzw. S-Cdk, essentiell für die Lösung dieses Konfliktes ist. Insbesondere verschafft die Mehrfach-Phosphorylierung von Sld2 durch S-Cdk eine zeitliche Verzögerung, die robust gegenüber Veränderungen in der S-Cdk Aktivierungskinetik ist und außerdem eine nahezu simultane Aktivierung der Origins ermöglicht. Die berechnete Verteilung der Fertigstellungszeiten der RKs, oder die Verteilung der Originaktivierungszeiten, wird auch genutzt, um die Konsequenzen bestimmter Mutationen im Assemblierungsprozess auf das Kopieren des genetischen Materials in der S Phase des Zellzyklus zu simulieren.Before a cell divides it has to duplicate its entire genetic material. Eukaryotic genomes are replicated from multiple replication origins across the genome. This work is focused on the quantitative analysis of the underlying molecular mechanism that allows these origins to initiate DNA replication almost simultaneously and exactly once per cell cycle. Based on a vast amount of experimental findings, a molecular regulatory network is constructed that describes the assembly of the molecules at the replication origins that finally form complete replication complexes. Using mass–action kinetics, the molecular reactions are translated into a system of differential equations. To parameterize the mathematical model, the initial protein concentrations are taken from experimental data, while kinetic parameter sets are determined using an optimization approach, in particular a minimization of the duration, in which a minimum number of replication complexes has formed. The model identifies a conflict between the rapid initiation of replication origins and the efficient inhibition of DNA rereplication. Analyses of the model suggest that a time delay before the initiation of DNA replication provided by the multiple phosphorylations of the proteins Sic1 and Sld2 by cyclin-dependent kinases in G1 and S phase, G1-Cdk and S-Cdk, respectively, may be essential to solve this conflict. In particular, multisite phosphorylation of Sld2 by S-Cdk creates a time delay that is robust to changes in the S-Cdk activation kinetics and additionally allows the near-simultaneous activation of multiple replication origins. The calculated distribution of the assembly times of replication complexes, that is also the distribution of origin activation times, is then used to simulate the consequences of certain mutations in the assembly process on the copying of the genetic material in S phase of the cell cycle.
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Mackenney, Victoria Jane. "Human DNA ligase I in DNA replication and repair." Thesis, King's College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267515.
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Minchell, Nicola E. "DNA topological stress during DNA replication in Saccharomyces cerevisiae." Thesis, University of Sussex, 2019. http://sro.sussex.ac.uk/id/eprint/81222/.
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DNA topological stress impedes normal DNA replication. If topological stress is allowed to build up in front of the replication fork, the fork rotates to overcome the stress, leading to formation of DNA pre-catenanes. The formation of DNA pre-catenanes is therefore a marker of DNA topological stress. In this study, I have examined how transcription linked DNA topological stress impacts on fork rotation and on endogenous DNA damage. Transcription, similar to replication, affects the topology of the DNA; and collision between the two machineries is likely to lead to high levels of DNA topological stress. I found that the frequency of fork rotation during DNA replication, increases with the number of genes present on a plasmid. Interestingly, I also found that this increase in pre-catenation is dependent on the cohesin complex. Cohesin and transcription are known to be linked, as transcription leads to the translocation of cohesin along budding yeast DNA away from its loading sites. Cohesin plays a major role in establishing chromosomal structure, influencing gene expression and genetic inheritance. In this work, I have analysed the relationship between cohesin and the generation of topological stress and found that topological stress associated with cohesin can lead to DNA replication stress and DNA damage.
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Isoz, Isabelle. "Role of yeast DNA polymerase epsilon during DNA replication." Doctoral thesis, Umeå : Umeå University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1932.
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Tao, Liang 1960. "DNA replication in human transformed cells." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37846.
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Cell transformation and malignancy may modulate some regulatory parameters of DNA replication, resulting in altered features of initiation of DNA replication in these cells. The objectives of this research were to examine the possibility for tumor-specific DNA replication origins and activation of replication origins in human transformed cells.Conventional PCR was used to detect chromosomal activities of several known and putative replication origins in four human cell lines (HeLa, NSF, WI38 and SK-MG-1). Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 were approximately twice as active in HeLa cells as in NSF cells.
To test whether the differential origin activities at certain loci in HeLa and NSF cells were due to shifts in origin usage among multiple initiation sites, DNA replication initiation sites and initiation frequencies over 12.5 kb of the human c-myc locus, including 4.6 kb of new 5 ' sequence, were determined by competitive PCR. In this study, one predominant site was located at ∼0.5 kb upstream of the exon 1, and a second new major site was identified in exon 2. The relative usage of origins over the same region of the c-myc locus in the two types of cells was similar, but activities of all origins in HeLa cells were approximately twice those in NSF cells.
In order to better determine if cell type (Hela vs. NSF) or transformation were most likely responsible for altered origin activities, we studied initiation of replication in an isogenic cell pair, WI38 and SV40-transformed WI38 (WI38 VA13 2RA). We found, once again, that the activities of all origins at the c-myc locus were approximately twice as high in immortalized and transformed WI38(SV40) cells as in WI38 cells. This also suggests that the increased activities of origins at the c-myc locus in HeLa cells are caused by the processes leading to malignancy. Although there was significantly increased activity of promoter P2 (7.5--8.0-fold), it had no preferential influence on origin activities at the major sites. Finally, the potential associations between the DNA replication origin activity and transcription activity through higher order regulation by nuclear structure have been discussed.
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Hameister, Heike. "Mathematical models for DNA replication machinery." Thesis, University of Aberdeen, 2012. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=186178.
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DNA replication and associated processes take place in all living organisms with the same constitutions. The knowledge of the duplication process, chromatin building and repair mechanisms has increased explosively over the last years, but the complex interplay of different proteins and their mechanisms are not conceived properly. During DNA replication, the DNA has to be unpacked, duplicated and finally repacked into chromatin. These steps require different proteins, e.g. new histone proteins on demand to secure an error-free and undelayed DNA replication. This thesis includes different mathematical models for DNA replication, repair and chromatin formation, which are based on experimental results. Three models of chromatin formation provide a simplified description of histone gene expression and protein synthesis during G1/S/G2 phase and include the contribution of different regulatory elements. Furthermore, all models present two different mechanisms of regulation to test possible scenarios of newly synthesised histones and free DNA binding sites. The basic model presents a single histone gene, which codes for a single histone protein. The stem-loop binding protein (SLBP) acts as a master regulator, which is only present during S phase. Different analyses of early S-phase, over- and underexpressed replication and the down-regulation of SLBP proof the model under extreme conditions. This basic model serves as a template for further scenarios with several genes and different histone families. For this, a second model is realised to simulate imbalances in the histone mRNA synthesis and translation. Additionally, a third model tests a gene knock-out and mRNA silencing. The initial histone model is able to qualitatively reproduce experimental observations and shows basic regulatory principles. The adaptation with several genes and different histone families presents qualitatively different system responses for the discussed regulatory mechanisms and illustrates the ability to compensate the effect of mRNA silencing.
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Hawkins, Michelle. "DNA replication origins in Haloferax volcanii." Thesis, University of Nottingham, 2009. http://eprints.nottingham.ac.uk/10853/.
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DNA replication is fundamental to the proliferation of life. Sites of DNA replication initiation are called replication origins. Bacteria replicate from a single origin whereas eukaryotes utilise multiple origins for each chromosome. The archaeal domain includes species which replicate using multiple origins of replication in addition to those which use a single origin. Archaeal DNA replication proteins are similar to eukaryotic replication machinery. Most characterised archaeal origins are adjacent to an orc gene which encodes a homologue of the Orc1 subunit of the eukaryotic initiator protein complex. Replication origins of the halophilic archaeon Haloferax volcanii were identified using a combination of genetic, biochemical and bioinformatic approaches. H. volcanii has a multireplicon genome consisting of a circular main chromosome and three mini-chromosomes: pHV1, pHV3 and pHV4. The major chromosome contains multiple origins of replication and is the first example of multiple origins on a single replicon in the Euryarchaeota. Each characterised origin is adjacent to an orc gene and contains repeated sequence motifs surrounding an A/T-rich duplex unwinding element. The archaeal recombinase, RadA, is homologous to eukaryotic and bacterial Rad51/RecA. It is widely held that deletion of radA results in elimination of homologous recombination. In this study the discovery of a radA-independent recombination pathway specific to replication origins is described. This dynamic mechanism was identified by observing chromosomal integration of plasmids containing H. volcanii replication origins in a radA deletion strain. The eukaryotic RAD25 gene is involved in nucleotide excision repair and transcription. H. volcanii has four RAD25 homologues, one on pHV4 and three near the oriC-2 locus on the main chromosome. A role for the assistance of oriC-2 firing is proposed based on autonomously replicating plasmid assays. Deletion of all four RAD25 homologues did not increase DNA damage sensitivity but resulted in a minor growth defect.
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Blow, J. J. "The control of eukaryotic DNA replication." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233674.
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One of the major limitations on research into the control of eukaryotic DNA replication has been the lack of any cell-free system that initiates DNA replication in vitro. The first part of the disseration describes the establishment of a eukaryotic system, derived from the activated eggs of the South African clawed toad, Xenopus laevis, that efficiently initiates and completes DNA replication in vitro. Using a variety of biochemical techniques I show that DNA added to the extract in the form of sperm nuclei is efficiently replicated over a period of 4 - 6 hours. Replication of nuclear DNA represents a single round of semiconservative, semidiscon-tinuous replication. The extract will also replicate naked DNA incubated in it, regardless of sequence, though less efficiently than nuclear templates. This is probably related to the unusual ability of the egg extract to assemble apparently normal interphase nuclei from any DNA molecule incubated in it Evidence is presented that initiation, rather than chain elongation, is the rate-limiting step for replication in vitro. In this and in other ways the cell-free system behaves as though it were an early embryo blocked in a single cell cycle. The second part of the dissertation describes experiments that examine the control of DNA replication in the extract The first set of experiments suggest that on replication, DNA is marked in some way so that it can no longer act as a substrate for further initiation. This provides a mechanism by which the template DNA is replicated precisely once per incubation in vitro (or per cell cycle in vivo). The second set of experiments investigate the relationship between nuclear assembly and the initiation of DNA replication in vitro. A novel method for quantifying DNA replication in intact nuclei using the nucleotide analogue biotin-11-dUTP is described. This technique reveals that although they are in the common cytoplasm of the egg extract, different nuclei start to replicate at different times. Entry into S-phase is characterised by a burst of many synchronous or near-synchronous initiations within individual nuclei. This means that nuclei act as independent and integrated units of replication in the cell-free system, and suggests a fundamental role for nuclear assembly in controlling DNA replication in vitro.
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Bowmaker, Mark Richard. "Replication of the mouse mitochondrial DNA." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614689.
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Green, Brian M. "The consequences of DNA re-replication." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3251930.
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You, Zhongsheng. "Sensing and responding to DNA replication /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2002. http://wwwlib.umi.com/cr/ucsd/fullcit?p3055806.
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Cerutti, A. "ONCOGENE-INDUCED ALTERED DNA REPLICATION DYNAMICS." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/234135.
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Oncogene Induced Senescence (OIS) is a tumor suppressive barrier that blocks cell cycle permanently. OIS results from a robust DNA damage response (DDR) activation due to oncogene-induced hyper-proliferation. By performing a whole genome analysis of DNA replication dynamics occurring upon oncogene activation, I discovered that oncogene activation alters DNA replication by increasing replication fork speed and fork stalling, while decreasing the frequency of replication initiation. This is accompanied by a prompt DDR activation. As cells approach senescence the frequency of initiation increases, the level of fork stalling and fork speed decreases.
Oncogene activation leads to DNA replication stress mainly at fragile sites and since telomeres resemble fragile sites, I then demonstrated that oncogene activation impairs telomere replication, by increasing fork stalling at telomeres. This is accompanied by increased fragile telomeres, stochastic telomeric attrition and persistent telomeric DDR. These results revealed a novel link between oncogene activation and telomere dysfunction, refining the model underlying OIS establishment.
Oncogene activation increases Reactive Oxygen Species (ROS). Beyond their toxicity, ROS are essential second messengers mediating mitogenic signalling. We discovered that oncogene-induced ROS is mediated by the NADPH oxidase NOX4. Upon oncogene activation, NOX4 pharmacological inhibition blocks ROS production, resulting in fork speed reduction and differential regulation of local replication origin initiation. These results revealed a fundamental role of NOX4 and ROS in mediating oncogene-induced hyperproliferation.
Polycomb repressive complexes (PRCs) repress genes involved in development, proliferation and differentiation by EZH2-mediated H3K27 trimethylation. Recent independent studies revealed a more direct PRCs role on S phase progression and DNA replication. We show that EZH2 KO leads to impairment of cell cycle progression, with cells blocked at G1/S transition. Furthermore EZH2 KO impairs DNA replication, by reducing fork speed, increasing the frequency of initiation and fork stalling, demonstrating that PRCs deficiency leads to replication stress.
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Muñoz, Castellano Casilda. "Understanding DNA replication of Plasmodium falciparum." Electronic Thesis or Diss., Université de Montpellier (2022-....), 2023. http://www.theses.fr/2023UMONT016.
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Le parasite Plasmodium falciparum, l’agent responsable du paludisme, a un cycle de vie complexe et emploie des stratégies de multiplication atypiques, adaptées à l'expansion rapide de la population pendant la colonisation de l'hôte et la transmission, qui ne sont pas entièrement comprises. Afin de comprendre l'initiation de la réplication de l'ADN de ce pathogène humain, mon projet s'est concentré sur deux aspects principaux de ce processus : l'identification et la caractérisation des origines de la réplication et de la machinerie de réplication.Dans la première partie de ce projet, j'ai combiné trois approches différentes pour cartographier les origines de réplication chez P. falciparum : ChIP-seq, le séquençage des brins naissants d'ADN (SNS-Seq) et la méthode NanoForkSpeed (NFS). J'ai d'abord réalisé les expériences ChIP-seq sur deux sous-unités du complexe de reconnaissance des origines de réplication (PfORC1 et PfORC2) au début de la schizogonie afin d'obtenir une cartographie de tous les sites d'initiation potentiels. Ensuite, j'ai identifié les sites de réplication actifs en utilisant deux stratégies : SNS-seq et la cartographie de l'incorporation de l'analogue de la thymidine BrdU dans l'ADN en réplication, en utilisant le séquençage nanopore et l'algorithme NFS. En combinant les données issues de ces différentes méthodes, j'ai obtenu un ensemble robuste d'origines de réplication qui présentent des caractéristiques similaires à celles des origines des mammifères, comme la distribution non aléatoire dans des zones d'initiation ou ‘clusters’ et l'association avec des séquences formant des G-quadruplex. Les origines de réplication montrent également des caractéristiques uniques. Elles sont associées à des gènes fortement transcrits, mais dépourvus de sites de début de transcription (TSS). En outre, les résultats montrent que la vitesse de la fourche de réplication est uniforme sur l'ensemble du génome, à l’exception d’une diminution significative au niveau des centromères et des télomères. Des informations obtenues sur molécules uniques, utilisant des lectures contenant de multiples événements d'initiation qui n'auraient pu provenir que de cellules individuelles, ont révélé une relation entre la vitesse à laquelle les fourches de réplication se déplacent et la distance par rapport à l'origine la plus proche. Cette approche à multiples facettes a fourni la première analyse complète du paysage génétique des origines de réplication chez P. falciparum.La deuxième partie de mon projet s'est concentrée sur la caractérisation du complexe réplicatif de P. falciparum en isolant des protéines sur l'ADN naissant, qui font potentiellement partie de la machinerie du réplisome. Dans l'ensemble, ce travail contribue aux études en plein essor, sur la réplication de l’ADN des parasites Plasmodium falciparum, dans l'hôte humain, et fournira une base solide pour les futures recherches dans ce domainePlasmodium falciparum parasites have a complex life cycle and employ atypical multiplication strategies, tailored for fast population expansion during host colonisation and transmission, that are not fully understood. In an effort to unravel the initiation of DNA replication of these human pathogens my project focused on two main aspects of this process: (i) identifying and characterising the origins of replication and (ii) identifying and characterising the replication machinery.Regarding the first part, I combined three different approaches to map the origins of replication in P. falciparum. These were ChIP-seq, SNS-Seq and NFS. I first performed ChIP-seq on two subunits of the origin recognition complex (of PfORC1 and PfORC2) at the beginning of schizogony to obtain a cartography of all potential replication initiation sites. Next, I mapped sites of active replication using two strategies: sequencing DNA nascent strands (SNS-seq); and mapping the incorporation of the thymidine analogue BrdU into replicating DNA, using nanopore sequencing combined with NanoForkSpeed (NFS). By combining data from these different methods, I have obtained a robust set of origins of replication that display some characteristics similar to those of mammalian origins, such as the non-random distribution in initiation zones or clusters and the association with G-quadruplex forming sequences. Strikingly, they also display unique characteristics, since they are associated with highly transcribed genes but depleted from transcription start sites (TSS). Additionally, the results showed a uniform fork speed across the genome with a significant decrease in centromeres and telomeres. Single molecule information, using reads containing multiple initiation events which could have only come from individual cells, revealed a relationship between the pace at which replication forks travel and the distance to the nearest origin. This multifaceted approach provided the first comprehensive analysis of the genetic landscape of the origins of replication in P. falciparum.The second part of my project focused on the characterization of the replicative complex of P. falciparum by isolating proteins bound to nascent DNA at active replication forks.Overall, this work contributes to the growing field of studying how Plasmodium falciparum parasites replicate their genome within the human host, and I am confident that it will serve as a solid foundation for further investigations
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Komori, Hirofumi. "Structural studies on DNA-binding proteins : DNA replication initiator and DNA photolyase." 京都大学 (Kyoto University), 2002. http://hdl.handle.net/2433/150005.
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Brüning, Jan-Gert. "Underpinning replication of protein-bound DNA by the accessory replicative helicase Rep." Thesis, University of York, 2015. http://etheses.whiterose.ac.uk/8220/.
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Accurate DNA replication must occur prior to every cell division. However, replication forks often stall at sites of DNA damage and protein-DNA complexes. If not removed, these blocks can threaten the viability of both daughter cells by preventing the completion of genome duplication or by targeting of blocked forks by recombination enzymes that can result in gross chromosomal rearrangements and genome instability. The importance of minimising fork blockage has resulted in cells evolving repair systems to remove lesions from DNA whilst accessory replicative helicases can underpin replication fork movement through hard-to-replicate sites including protein-DNA complexes. This thesis investigates the Escherichia coli accessory replicative helicase Rep. It is shown that efficient recruitment of Rep to the replisome via an interaction with the replicative helicase DnaB is dependent on the extreme Rep C terminus. This work also indicates that the DnaB C terminus is necessary for this interaction. Secondly, this work determines the function of the 2B subdomain, a conserved feature of Superfamily 1A (SF1A) helicases. Characterisation of a Rep mutant lacking this domain (RepΔ2B) showed greatly reduced levels of protein displacement from DNA, indicating a central role of the 2B subdomain in the removal of nucleoprotein blocks. Complementation of this mutation by a 2B subdomain of the homologous helicase UvrD supports the idea that the accessory replicative helicase function of Rep is dependent on a 2B subdomain. These data also demonstrate that the function of 2B subdomains is conserved among other SF1A helicases. Previous work had also shown that the 2B subdomain of SF1A helicases is flexible. Mutations in the hinge that connect the 2B subdomain to the rest of the helicase resulted in activation of DNA helicase activity and increased levels of nucleoprotein removal from single-stranded (ss) and double-stranded (ds) DNA. These data shed new light on how translocation along DNA is coupled to protein displacement during helicase catalysis, a conserved function of many helicases. A model is proposed where ATP hydrolysis is closely linked to conformational changes of the 2B subdomain of Rep, facilitating protein displacement by Rep.
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Martin, Eleyna. "Initiation of DNA replication in Bacillus subtilis : structural studies of the DnaA-DnaD interaction." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/53443/.
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Replication of genetic information is a vital process across all domains of life. Bacillus subtilis is considered the gram-positive model bacterium for studying DNA replication (Escherichia coli has been studied extensively as the gram-negative model) and is most representative of the ancestral phylum of prokaryotes. DNA replication has three distinct stages; initiation, elongation and termination. Replication initiation is the focus of this research and this process occurs at a single origin conserved throughout bacteria, termed oriC. B. subtilis primosomal machinery is formed of replication initiator proteins DnaA, DnaD and DnaB, the helicase loader DnaI, replicative helicase DnaC and primase DnaG. The role of the initiator proteins is to melt the DNA double helix and enable loading of the hexameric ring helicase onto each strand of DNA for bidirectional replication. Initiation is the first stage in DNA replication and despite its importance the molecular mechanisms of replication initiation remain largely unclear. The work presented in this thesis has focussed on the essential interaction between replication initiator proteins DnaA and DnaD, with an aim to characterise their binding interface and reveal molecular details of their mechanisms of interaction during DNA replication initiation. The direct interaction between isolated DnaA domain I and DnaD DDBH2 domain was detected by NMR spectroscopy which was subsequently used to identify the specific residues involved and characterise the nature of the binding interface. The kinetics of the interaction were investigated by SPR and computational techniques were used to model the DnaA-DnaD complex. This structural characterisation of the DnaA-DnaD interaction provides greater understanding of the molecular mechanisms of DnaA and DnaD during DNA replication initiation.
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Feu, i. Coll Sònia. "Replication stress: mechanisms and molecules involved in DNA replication progression and reinitiation." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/669609.
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This thesis investigates the mechanisms that are activated in response to replication stress induced with hydroxyurea, an agent that causes deoxyribonucleotides depletion and arrest cells in S phase, in the non-transformed hTERT-RPE cells. The results obtained in the non- transformed cells are compared with the ones obtained with HCT116 colorectal cancer cells.
Previous results indicated that, in response to an acute replication stress in hTERT-RPE, replication forks were able to restart without generating genomic instability, while in response to severe replication stress APC/CCdh1 was activated in S phase, which prevented the reinitiation of replication due to the inactivation of new origins.
In this thesis we verified that, in response to an acute replication stress in hTERT-RPE cells, replication forks undergo fork remodelling, although CMG complexes maintain their integrity and can be reused to restart replication once replication stress is eliminated.
In response to a prolonged replication stress, the lack of APC/CCdh1 activation in S phase in HCT116 tumour cells allows the resumption of replication by the activation of new origins, despite the acquisition of genomic instability during this process.
The role of some proteins, RAD51 and OZF, in replication fork progression or restart during replication stress conditions is also examined. During a mild replication stress, RAD51 and OZF are important for replication fork progression in hTERT-RPE cells. At the same time, they are necessary for the correct progression of replication forks in HCT116 cells, which present a higher basal replication stress, even under unperturbed conditions. In response to an acute replication stress, RAD51 is also important for an efficient replication fork restart and progression in hTERT-RPE cells.
Understanding the mechanisms that are working on non-transformed cells in replication stress, and comparing them with tumour cells, can help us to understand the alterations that tumour cells have acquired in order to bypass these regulations and to discover new molecules relevant to deal with replication stress.En aquesta tesi s’investiga sobre els mecanismes que s’activen en resposta a un estrès de replicació induït amb hidroxiurea, un agent que causa una depleció de desoxiribonucleòtids a la cèl·lula generant una aturada de la replicació de l’ADN, en les cèl·lules no transformades hTERT-RPE i es comparen els resultats amb els obtinguts en les cèl·lules de càncer colorectal HCT116. Resultats previs indicaven que, en resposta a un estrès de replicació agut en les cèl·lules hTERT-RPE, les forquetes de replicació eren capaces de reiniciar sense generar inestabilitat genòmica, mentre que en resposta a un estrès sever s’activava l’APC/CCdh1 en la fase S del cicle cel·lular, el qual impedia el reinici de la replicació degut a la inactivació de nous orígens de replicació. En aquesta tesi s’ha comprovat que, en resposta a un estrès de replicació agut, les forquetes de replicació de les cèl·lules hTERT-RPE pateixen dos tipus de remodelació, tot i que els complexes CMG mantenen la seva integritat i poden ser reutilitzats per reiniciar la replicació una vegada l’agent causant de l’estrès sigui eliminat. En resposta a un estrès de replicació prolongat, la falta d’activació de l’APC/CCdh1 en la fase S de les cèl·lules tumorals HCT116 permet el reinici de la replicació per l’activació de nous orígens, malgrat l’adquisició d’inestabilitat genòmica. També s’indaga sobre la funció d’algunes proteïnes, RAD51 i OZF, en la progressió o reinici de les forquetes de replicació en condicions d’estrès de replicació. Durant un estrès de replicació suau, RAD51 i OZF són importants per la progressió de la forqueta en les cèl·lules hTERT-RPE. A la vegada, resulten necessàries per la correcta progressió de la forqueta de replicació en condicions no pertorbades de les cèl·lules tumorals HCT116, les quals presenten un estrès de replicació basal més alt que les cèl·lules hTERT-RPE. Finalment, RAD51 també resulta important pel correcte reinici i progressió de la forqueta de replicació en les cèl·lules hTERT-RPE després d’un estrès de replicació agut.
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Schorr, Stephanie [Verfasser]. "Replication of Bulky DNA Adducts / Stephanie Schorr." München : Verlag Dr. Hut, 2010. http://d-nb.info/100997274X/34.
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Brown, Stephanie Marie. "Replication of damaged DNA in mammalian cells." Thesis, University of Sussex, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445620.
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Beckman, Jeffrey William. "Studying DNA replication fidelity using nucleotide analogues." Diss., Connect to online resource, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3239428.
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Heichinger, Christian. "Characterisation of fission yeast DNA replication origins." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1444737/.
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In many eukaryotic organisms the chromosomal origins of DNA replication (ORIs) are not characterised by a clearly defined consensus sequence. In this thesis using the fission yeast, for the first time I have carried out a genome-wide analysis to identify such ORIs during the mitotic and meiotic cell cycles. The data can be summarised as follows: a total of 401 ORIs were identified which were used 29 percent of the time during mitotic S-phase and were spaced every 31 kilobases (kb) on average. The same ORIs were used during pre-meiotic S-phase although with lower efficiency in most chromosomal regions. A further 503 potential ORIs were used less efficiently at eight percent of the time during mitotic S-phase. This totals 904 ORIs which were distributed at an average inter-origin distance of 14 kilobases (kb) throughout 12.5 megabases (Mb) of the three chromosomes of fission yeast. These data support the idea of a continuum of ORI activity. The 401 efficient ORI loci contained A+T-rich regions located between genes, and these intergenic regions were typically larger than average. ORIs were not defined by a strict sequence consensus but the presence of AT-hook binding sequences. When the initiation factors Cdc18 and Cdt1 were over-expressed, regions of DNA containing particularly efficient ORIs with exceptionally large AT-hook binding domains became over-amplified, suggesting that interactions between these factors and efficient ORIs may be important for the mechanism ensuring that an ORI only fires once in each S-phase.
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Loveland, Anna Barbara. "Single-Molecule Studies of Eukaryotic DNA Replication." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10076.
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DNA replication is a fundamental cellular process. However, the structure and dynamics of the eukaryotic DNA replication machinery remain poorly understood. A soluble extract system prepared from Xenopus eggs recapitulates eukaryotic DNA replication outside of a cell on a variety of DNA templates. This system has been used to reveal many aspects of DNA replication using a variety of ensemble biochemical techniques. Single-molecule fluorescence imaging is a powerful tool to dissect biochemical mechanisms. By immobilizing or confining a substrate, its interaction with individual, soluble, fluorescently-labeled reactants can be imaged over time and without the need for synchrony. These molecular movies reveal binding parameters of the reactant and any population heterogeneity. Moreover, if the experiments are imaged in wide-field format, the location or motion of the labeled species along the substrate can be followed with nanometer accuracy. This dissertation describes the use and development of novel single-molecule fluorescence imaging techniques to study eukaryotic DNA replication. A biophysical characterization of a replication fork protein, PCNA, revealed both helical and non-helical sliding modes along DNA. Previous experiments demonstrate that the egg extracts efficiently replicate surface-immobilized linear DNA. This finding suggested replication of DNA could be followed as motion of the replication fork along the extended DNA. However, individual proteins bound at the replication fork could not be visualized in the wide-field due to the background from the high concentration of the fluorescent protein needed to compete with the extract’s endogenous protein. To overcome this concentration barrier, I have developed a wide-field technique that enables sensitive detection of single molecules at micromolar concentrations of the labeled protein of interest. The acronym for this method, PhADE, denotes three essential steps: (1) Localized PhotoActivation of fluorescence at the immobilized substrate, (2) Diffusion of unbound fluorescent molecules to reduce the background and (3) Excitation and imaging of the substrate-bound molecules. PhADE imaging of flap endonuclease I (Fen1) during replication revealed the time-evolved pattern of replication initiation, elongation and termination and the kinetics of Fen1 exchange during Okazaki fragment maturation. In the future, PhADE will enable the elucidation of the dynamic events at the eukaryotic DNA replication fork. PhADE will also be broadly applicable to the investigation of other complex biochemical process and low affinity interactions. It will be especially useful to those researchers wishing to correlate motion with binding events.
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Trautinger, Brigitte W. "Interplay between DNA replication, transcription and repair." Thesis, University of Nottingham, 2002. http://eprints.nottingham.ac.uk/14281/.
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The Ruv ABC and RecBCD protein complexes together can collapse and repair arrested replication forks. With their help a fork structure can be re-established on which replication can be restarted. ruv and recB mutants are therefore quite sensitive to UV light. Their survival is greatly decreased in the absence of the signalling molecules (p)ppGpp and increased when excess (p)ppGpp is present. (p)ppGpp are the effector molecules of the stringent response, regulating adaptation to starvation and other stressful environmental changes. Absence of (p)ppGpp can be compensated for by mutations in RNA polymerase that are called stringent mutations. Some of those, called rpo *, also - like excess (p)ppGpp - increase the survival of UV irradiated ruv and recB cells. A model proposed by McGlynn and Lloyd (Cell, Vol. 101, pp35-45, March 31, 2000) suggests that this is achieved by modulation of RNA polymerase, which decreases the incidence of replication fork blocks. In this work twenty-seven rpo * mutants were isolated, sequenced and mapped on the 3D structure of Thermus aquatic us RNA polymerase. I have found mutants in the ~ and ~' subunits of RNA polymerase. They lie mostly on the inner surface of the protein, well placed to make contact with the DNA substrate or the RNA product. A large number of rifampicin resistant mutations among rpo* mutations is explained by an overlap between the so-called Rif pocket and the "rpo* pocket". rpo * mutations, like stringent mutations, lead to a decrease in cell size, suppress filamentation and increase viability. For in vitro studies I purified wild type and two mutant RNA polymerases with help of a his-tagged a subunit. The experiments confirmed that rpo* mutant RNA polymerases form less stable open complexes than wild type, just like previously investigated stringent RNA polymerases. In addition I have shown here that (p)ppGpp leads to the destabilisation of RNA polymerase complexes stalled by nucleotide starvation or UV-induced lesions, though there is as yet no indication that rpo * mutations act in the same way.
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Smith, Colin. "DNA repair and replication in Streptomyces coelicolor." Thesis, University of Liverpool, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329440.
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Wasson, Gillian Rachel. "Patterns of DNA replication in human cells." Thesis, University of Ulster, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.232846.
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Parkes, Vincent. "Replication of DNA by isolated wheat chloroplasts." Thesis, University of Hertfordshire, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329036.
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Jones, M. C. "Replication of DNA by isolated wheat chloroplasts." Thesis, University of Hertfordshire, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356358.
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Maloney, Michael F. (Michael Finnan). "Mechanism of Mcm10 function during DNA replication." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/119912.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
All life needs to replicate its genome completely and do so with limited errors. In eukaryotic cells, DNA replication is accomplished by a multi-stage process involving numerous protein assemblies. The core component of this process is the replicative DNA helicase, Mcm2-7. Mcm2-7 complexes are loaded around origins of DNA replication during G1 phase. As cells transition from G1 to S phase, the Mcm2-7 helicases are activated resulting in the recruitment of DNA polymerases and accessory proteins to begin DNA synthesis. Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Mcm2-7 helicase-activation requires binding of the helicase-activating proteins, Cdc45 and GINS (forming the CMG complex). DNA unwinding cannot proceed, however, until an additional protein, Mcm10, associates with the CMG. Mcm10 continues to travel with the replication fork after stimulating initial DNA unwinding but a function for Mcm10 during DNA replication elongation had not been established. Using a combination of molecular genetics and reconstituted biochemical assays, this thesis will outline the function of Mcm10 throughout DNA replication. I show that Mcm10 binds a conserved motif located between the OB-fold and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. In addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding is identified. These findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication.
by Michael F. Maloney.
Ph. D.
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Cluett, Tricia Joy. "The mechanism of mammalian mitochondrial DNA replication." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611167.
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Marsh, Victoria Louise. "DNA replication and chromatin in Sulfolobus solfataricus." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613746.
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Sarosh, Alvina. "DNA replication and segregation in Staphylococcus aureus." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/16476.
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Antibiotic resistant strains of S. aureus are responsible for hospital-acquired infections around the world, and also cause serious infections in the wider community, thereby posing a serious threat to human health. Resistance genes may be chromosomally encoded and/or carried by one or more plasmids. Efficient partitioning of replicated chromosomes and plasmids ensures their faithful inheritance. The S. aureus chromosome carries a putative partitioning gene, parB, and predicted parS sites at which ParB might act. Analysis of a S. aureus parB mutant revealed an increased frequency of anucleate cell production under some conditions. ParB was also shown to bind specifically to three parS sites. The partitioning system of the S. aureus multiresistance plasmid pSK41 comprises two genes in an operon, parM and parR, and a centromere site, parC. ParM interacts with ParR bound to parC repeats to form a partitioning complex. Binding of ParR to parC also mediates transcriptional autoregulation of the operon. Results described here indicate that the minimal parC region required for function is larger than anticipated. The pSK41 orf86 gene is located upstream of the replication initiation gene, rep, but its function was a mystery. Orf86 was shown to negatively regulate rep expression. It repressed transcription from the rep promoter, and reduced the copy number of pSK41 mini-replicons. Transcription of orf86 is dependent on transcripts from the RNAI promoter, which therefore mediates the production of two RNA species that regulate rep expression; a small antisense transcript RNAI that inhibits Rep translation, and orf86 mRNA that is translated into Orf86, which inhibits rep transcription. Based on its newly described role in copy number control, orf86 was renamed cop. Detailed knowledge about DNA replication and segregation mechanisms arising from these studies may contribute to the development of strategies to combat the threat of antibiotic resistant S. aureus.
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Wilson, Rosemary Helen Clare. "Organisation of the initiation of DNA replication." Thesis, University of York, 2013. http://etheses.whiterose.ac.uk/4802/.
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Multiple lines of evidence show that DNA replication and the proteins involved with its preparation reside at the nuclear matrix (NM). Some of these, such as cyclin E, are recruited to the NM during differentiation, implying that NM attachment may help to fix cell-type specific replication programmes, and potentially therefore restrict plasticity. However, our understanding of the interplay between the NM, the cell cycle and cell type is still limited. The aim of this PhD is to develop our understanding of the NM in relation to the preparation for DNA replication. The function of the replication-promoting and cancer-associated NM protein CIZ1 was investigated using cells derived from a CIZ1-/- mouse. Dysregulated cyclin and CDKi expression and immobilisation suggest that CIZ1 has a widespread role as a cell cycle regulator with an exchange function that is consistent with previous analysis of its interaction with cyclin E and A. More importantly, in the absence of CIZ1, the DNA damage response is activated as cells receive growth inhibition signals, leading to the formation of foci outgrowths (and lymphoid malignancies in the animal). Together with published data, this suggests a tumour suppressor role for CIZ1. This also implies that different forms of CIZ1 have tumour suppressor or promoting roles and that the balance between these may be critically important for proper integration of growth control signals. CIZ1 has also previously been implicated in chromatin loop organisation during G1-phase. My analysis of NM attached chromatin loops during cell cycle re-entry from quiescence shows that average chromatin loop size is smaller in quiescence but recovers in size in a very early, growth factor-independent step during G1. Moreover, chromatin loops were also found to become larger and more unstable following controlled transformation, correlating with changes in CIZ1 expression. This suggests weaker attachment of DNA in cancer cells and mirrors the lack of attachment of cyclin E and CIZ1. These data support the hypothesis that loss of NM attachment is a feature of cancer cells and were collected using novel methods developed and validated during this project that allow rapid, non-subjective computer measurement of chromatin loops.
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Stracker, Travis Hileman. "DNA virus interactions with host cell DNA replication and repair pathways /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2002. http://wwwlib.umi.com/cr/ucsd/fullcit?p3070999.
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Morley, Stewart Anthony. "Interactions Between the Organellar Pol1A, Pol1B, and Twinkle DNA Replication Proteins and Their Role in Plant Organelle DNA Replication." BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/8128.
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Plants maintain organelle genomes that are descended from ancient microbes. Ages ago, these ancient microbes were engulfed by larger cells, beginning a process of co-evolution we now call the endo-symbiotic theory. Over time, DNA from the engulfed microbe was transferred to the genome of the larger engulfing cell, eventually losing the ability to be free-living, and establishing a permanent residency in the larger cell. Similarly, the larger cell came to rely so much on the microbe it had engulfed, that it too lost its ability to survive without it. Thus, mitochondria and plastids were born. Nearly all multicellular eukaryotes possess mitochondria; however, different evolutionary pressures have created drastically different genomes in plants versus animals. For one, animals have very compact, efficient mitochondrial genomes, with about 97% of the DNA coding for genes. These genomes are very consistent in size across different animal species. Plants, on the other hand, have mitochondrial genomes 10 to more than 100 times as large as animal mitochondrial genomes. Plants also use a variety of mechanisms to replicate and maintain their DNA. Central to these mechanisms are nuclear-encoded, organelle targeted replication proteins. To date, there are two DNA polymerases that have been identified in plant mitochondria and chloroplasts, Pol1A and Pol1B. There is also a DNA helicase-primase that localizes to mitochondria and chloroplasts called Twinkle, which has similarities to the gp4 protein from T7 phage. In this dissertation, we discuss the roles of the polymerases and the effects of mutating the Pol1A and Pol1B genes respectively. We show that organelle genome copy number decreases slightly and over time but with little effect on plant development. We also detail the interactions between Twinkle and Pol1A or Pol1B. Plants possess the same organellar proteins found in animal mitochondria, which are homologs to T7 phage DNA replication proteins. We show that similar to animals and some phage, plants utilize the same proteins in similar interactions to form the basis of a DNA replisome. However, we also show that plants mutated for Twinkle protein show no discernable growth defects, suggesting there are alternative replication mechanisms available to plant mitochondria that are not accessible in animals.
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Russell, Iain Alasdair, and n/a. "Involvement of p53 and Rad51 in adenovirus replication." University of Otago. Dunedin School of Medicine, 2007. http://adt.otago.ac.nz./public/adt-NZDU20070521.094929.
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As an Adenovirus infects a host cell a multitude of molecular interactions occur, some driven by the virus and some driven by the cell it is infecting. Many of these areas of Adenovirus biology have been intensely studied over the last half century, however, many questions remain unanswered. The aim of this study was to investigate, more closely, a long studied molecular interaction, namely the role of the tumour suppressor p53 in the Adenovirus life cycle, and also to investigate the related, but much less studied, interaction between Adenoviruses and the host cell DNA repair machinery.
Controversy surrounds the role of p53 in the Adenovirus life cycle, with current dogma favouring the view that p53 is inactivated, as it presumably presents an obstacle to a productive infection. In Chapter 3, a standardised infection protocol was developed to examine this area of Adenovirus biology more closely. This was followed with an array of cell viability and western blotting analyses that not only showed p53 was not an antagonist of the Adenovirus life cycle, but in some cases p53 acted as a protagonist. Isogenic cell lines were used to reinforce this point. Following this, data were provided that virus DNA replication was linked to the ability of an Adenovirus to kill cells. Furthermore, p53 was shown by immunofluorescence to be present in infected cells at a time that corresponded with virus DNA replication, albeit at low levels. By adding p53 back into cells, it was shown that the number of Adenovirus progeny could be stimulated to levels produced in genetically wild type TP53 cells. A selection of promoter/reporter assays and infection/transfection assays then showed how p53 might be aiding the virus life cycle. These data showed that low levels of p53 cooperated with the Adenovirus transactivator, E1A, to promote late gene expression, and this translated into a modest increase in virus late antigens in infected cells. Taken together these data show that, contrary to current dogma, p53 generally aids an Adenovirus infection and it may do this through promoting virus late gene expression.
Recent data have emerged suggesting Adenoviruses must disable the host DNA double-strand break machinery to achieve a productive infection. As this area of Adenovirus biology is in its infancy, and as p53 has recently been identified as an integral component of these DNA repair processes, the contributions of the host cell repair machinery to Adenovirus biology were examined in Chapters 4 and 5. In Chapter 4, western blotting showed that upon Adenovirus infection, a key component of the homologous recombination repair machinery, Rad51, was markedly up-regulated. This up-regulation occurred independently of other key repair proteins, and was found to be a generalised feature of an Adenovirus infection. Surprisingly, p53 did not appear to be involved in this up-regulation, and neither were several other nodal host regulatory proteins. The up-regulation was then linked to Adenovirus DNA replication using a temperature-sensitive mutant Adenovirus, ts125. In Chapter 5, functional analysis of this up-regulated protein showed that Rad51 colocalised with Adenovirus replication centres. This colocalisation coincided with a time when virus DNA replication was occurring. Furthermore, transient over-expression of Rad51 drastically increased the amount of virus progeny produced. This effect was reproduced in two very different cell types and with a selection of attenuated mutant viruses. Finally, several models were proposed that might account for this newfound effect of Rad51 on the Adenovirus life cycle.
The data presented in this thesis shows that Adenovirus not only interacts with key molecular machinery within the host cell, but also manipulates this machinery to its own end. These data add additional layers of complexity to current knowledge of the virus/host cell relationship, and thus reveal new avenues of research for future work.
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Panciatici, Claire. "DNA replication in budding yeast : link between chromatin conformation and kinetics of replication." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS473/document.
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L’information génétique contenue dans le noyau de la cellule doit être dupliquée fidèlement afin d’être transmise aux cellules filles pendant la division cellulaire. Pour organiser leur division, les cellules suivent un cycle reproductible composé de quatre étapes appelé cycle cellulaire. La préparation et l’exécution du programme de réplication de l’ADN ont lieu pendant des phases spécifiques du cycle grâce à l’intervention de multiples partenaires protéiques et de régulateurs structuraux. En particulier, la réplication de l’ADN s’effectue sur une matrice complexe constituée d’ADN associé à des protéines appelée chromatine. Cette dernière influence et est influencée par la réplication de l’ADN. Le travail présenté ici a pour objectif de faire le lien entre la conformation de la chromatine et la cinétique de réplication de l’ADN. Pour ce faire, nous combinons plusieurs techniques. La cytométrie de flux nous permet de suivre la quantité d’ADN présent dans une population de cellules et, à l’aide d’une méthode développée dans notre laboratoire, d’extraire le programme de réplication moyen d’une population de cellules. La technique de SAXS fournit des informations sur l’organisation locale des protéines et de l’ADN in vivo. Nos données peuvent être interprétées comme un cristal liquide avec un ordre nématique et une faible longueur de corrélation, ce qui suggère que la chromatine de la levure est majoritairement dépourvue d’une organisation en fibre de 30nm in vivo. Par ailleurs, par la méthode de peignage d’ADN, nous reproduisons les résultats précédemment obtenus montrant que la distance entre zones répliquées est d’environ ~60kb qui correspond à la distance entre des origines de réplication identifiées. Cependant, d’après l’étude du comportement dynamique de l’initiation, nous proposons que les initiations sont plus fréquentes que ce qui a été mesuré précédemment et correspondent à la distance entre les protéines MCM disposées sur le génomeGenetic information carried in the cell nucleus must be faithfully duplicated to be transmitted to daughter cells during cell division. In order to orchestrate their division, cells go through a reproducible 4 stages cycle called «cell cycle». The preparation and execution of the DNA replication program is restricted to specific phases and implies many proteic and structural regulators. In particular, DNA replication occurs on a complex template of DNA associated with proteins. The latter is both influencing and influenced by DNA replication. This work aims at investigating the link between chromatin conformation and the kinetics of DNA replication. In order to do so, we combine several techniques. Using flow cytometry, we follow the evolution of a cell population with regards to their DNA content and, with a method developed in our laboratory, decipher the population averaged temporal program of DNA replication. SAXS data provide information on the local organisation of protein and DNA in vivo. Our data can be interpreted as a liquid crystal with a nematic order and a short correlation length, which suggest that yeast chromatin in vivo is predominantly devoid of 30 nm fibres organisation. On the other hand, we performed DNA combing to study the replication program in single cells. We reproduce previously obtained result showing that distance between replicated tracks is of ~60kb which corresponds to the distance between known origins of replication. However, studying the behaviour of initiation, we propose that the initiation events are more frequent than previously measured and correspond to distances between MCMs proteins loaded on the genome