Дисертації з теми "DNA strand"
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1
Lo, Allen Tak Yiu. "Protein dynamics on the lagging strand during DNA synthesis." Thesis, School of Chemistry, 2012. https://ro.uow.edu.au/theses/3684.
Анотація:
DNA replication is one of the vital processes in the cell; it duplicates chromosomal DNA before a cell divides. In all organisms, DNA synthesis on the leading-strand template occurs continuously, whereas on the lagging strand a different mechanism is required. Due to the anti-parallel structure of double-stranded DNA, lagging-strand synthesis requires repeated RNA priming by a specialist primase and synthesis of short Okazaki fragments. How proteins carry out this dynamic process is still unknown. For Escherichia coli DNA replication, a lagging-strand three-point switch was proposed in 1999 to explain priming by DnaG primase while it is associated with the DnaB6 helicase, and its subsequent hand-off from the primer to the χ subunit of DNA polymerase III holenzyme to enable primer utilization for Okazaki fragment synthesis. The main aims of this project were to study the interactions involved in this switch to understand better how the proteins coordinate their roles during lagging-strand DNA synthesis.
2
Tingey, Andrew Philip. "Strand passage in DNA gyrase." Thesis, University of Leicester, 1996. http://hdl.handle.net/2381/35173.
Анотація:
DNA gyrase, a type II topoisomerase, catalyses the introduction of negative supercoils into closed-circular DNA, using the energy from ATP hydrolysis. The reaction mechanism involves the breakage of one DNA double strand (the DNA gate) and the passing of another DNA strand (the passage helix) through that break and finally the re-sealing of the DNA gate. The strand-passage reaction was studied by the use of novel DNA substrates and by site-directed mutagenesis of one of the gyrase proteins. The DNA substrates were used to attempt to define the DNA segments used by the enzyme as the DNA gate and passage helix in a catenation reaction. This was achieved by using oligonucleotides to form partial duplex regions in single-stranded DNA. A high-affinity gyrase cleavage site from the plasmid pBR322 was cloned into M13mpl8 and generated both the single and double-stranded circular forms of the molecule (MAT1). It was shown that gyrase could form a specific DNA gate in a short duplex region in single-stranded MAT1 when quinolone drugs were present. This DNA gate was much smaller than that normally utilised by the enzyme. The catenation and decatenation reactions were examined in detail with normal duplex substrates; reactions using a non-hydrolysable ATP analogue gave different results to those previously reported for the eukaryotic homologue of gyrase, indicating a possible mechanistic difference between the enzymes. Conditions under which the partial duplex substrates would be catenated were not found. Site-directed mutagenesis was used to alter arginine residues thought to interact with the passage helix during the reaction cycle. Assays of the mutant protein revealed that supercoiling activity was markedly reduced, but that partial activities of gyrase, such as the ATPase and DNA cleavage reactions, were close to wild-type levels.
3
Ho, F. M. "Strand exchange for duplex DNA detection." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604106.
Анотація:
The phenomenon of strand exchange between an unlabelled double-stranded target oligonucleotide and a single-stranded, fluorophore labelled probe oliognucleotide was investigated. This behaviour was characterised using fluorescence resonance energy transfer (FRET). The individual fluorescence characteristics of the fluorophores the minor-groove binder Hoechst 33258 and the dye Oregon Green 488 were studied, as well as their properties in combination as a FRET pair. These dyes allowed the use of FRET for the study of duplex DNA without the need for covalently attaching two labels on the component strands. Two strategies were studied for the detection of a duplex target. Firstly, detection could be by monitoring the FRET process as a function of time, monitoring the fluorescence intensities at both the donor and acceptor emission peak wavelengths. Single base pair discrimination was achieved, with very high reproducibility, especially if ratiometric analysis of the emission signals was employed. The mechanism of this process was examined using mathematical modelling, and comparisons made with the experimental results. Secondly, an in-gel detection technique was investigated for the detection of the target duplex within a complex mixture. The target sequence was successfully detected from within the enzyme digestion products of plasmids extracted from cloned E. coli cells. This was performed directly from a polyacrylamide electrophoresis gel without the need for blotting, and was possible with or without polymerase chain reaction amplification. Multiplexing was also demonstrated using this in-gel strategy, giving simultaneous detection of two targets of different base sequences and lengths. Finally, the synthesis of an acceptor fluorophore labelled dendrimer was proposed. This opened up the prospect of exploiting the properties of the dendrimer to enhance the FRET signal upon strand exchange.
4
Washbrook, Elinor. "Alternate strand DNA triple helix formation." Thesis, University of Southampton, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242223.
5
Lansita, Janice A. (Janice Ann) 1975. "Physicochemical characterization of immortal strand DNA." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/18038.
Анотація:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.Includes bibliographical references.
Adult tissue differentiation involves the generation of distinct cell types from adult stem cells (ASCs). Current understanding of tissue differentiation mechanisms is based on studies of protein and RNAs that asymmetrically segregate between daughter cells during embryogenesis. Whether or not other types of biomolecules segregate asymmetrically has not been widely studied. In 1975, John Cairns proposed that ASCs preferentially segregate the oldest parental template DNA strands to themselves and pass on newly replicated DNA strands to their differentiating progeny in order to protect the stem cell from inheriting DNA replication mutations. This laboratory has shown non-random chromosome segregation in murine fetal fibroblasts that model asymmetric self-renewal like ASCs. In these cells, chromosomes that contain the oldest DNA strands co-segregate to the cycling daughter stem-like cells, while chromosomes with more recently replicated DNA segregate to the non-stem cell daughters. Previously, cytological methods were reported to elucidate non-random segregation in these cells. This dissertation research provides additional confirmation of the mechanism using physicochemical methods. Specifically, buoyant density-shift experiments in equilibrium CsCl density gradients were used to detect co-segregated "immortal DNA strands" based on incorporation of the thymidine base analogue bromodeoxyuridine. In addition, DNA from cells undergoing non-random mitotic chromosome segregation was analyzed for unique DNA base modifications and global structural modifications (by HPLC and melting temperature analyses). To date, these studies show no significant differences compared to control randomly segregated DNA. Components of the mitotic chromosome separation
(cont.) apparatus that might play a role in the co-segregation mechanism were also evaluated. Two homologous proteins, essential for proper chromosome segregation and cytokinesis, Aurora A kinase and Aurora B kinase, were highly reduced in expression in cells retaining immortal DNA strands and may indicate a role for them in the immortal strand mechanism. These studies independently confirm the immortal strand mechanism and provide methods for its detection in other cell lines. In addition, observed changes in chromosome segregation proteins that are potential candidates for involvement in the mechanism have revealed a new area of investigation in the laboratory. These findings are relevant to understanding normal tissue development, cancer, and aging.
y Janice A. Lansita.
Ph.D.
6
Absalon, Michael Joseph. "DNA double-strand cleavage mediated by bleomycin." Thesis, Massachusetts Institute of Technology, 1994. http://hdl.handle.net/1721.1/11927.
7
Morant, Nick. "Novel thermostable DNA polymerases for isothermal DNA amplification." Thesis, University of Bath, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.667735.
Анотація:
DNA polymerases play a fundamental role in the transmission and maintenance of genetic information and have become an important in vitro diagnostic and analytical tool. The Loop-mediated isothermal DNA amplification (LAMP) method has major applications for disease and pathogen detection and utilises the unique strand-displacement activity of a small group of thermostable DNA polymerases. The Large (Klenow-like) Fragment of Geobacillus stearothermophilus DNA polymerase I (B.st LF Pol I) currently serves as the enzyme of choice for the majority of these isothermal reactions, with few alternatives commercially available. An increasing need for point-of-care nucleic acid diagnostics is now shifting detection methods away from traditional laboratory based chemistries, such as the polymerase chain reaction (PCR), in favour of faster, and often simpler, isothermal methods. It was recognised that in order to facilitate these rapid isothermal reactions there was a requirement for alternative thermostable, strand-displacing DNA polymerases and this was the basis of this thesis. This thesis reports the successful identification of polymerases from Family A, chosen for their inherent strand-displacement activity, which is essential for the removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis in vivo. Twelve thermophilic organisms, with growth temperature ranges between 50oC and 80oC, were identified and the genomic DNA extracted. Where DNA sequences were unavailable, a gene-walking technique revealed the polA sequences, enabling the Large Fragment Pol I to be cloned and the recombinant protein over-expressed in Escherichia coli. A three-stage column chromatography purification permitted the characterisation of ten newly identified Pol I enzymes suitable for use in LAMP. Thermodesulfatator indicus (T.in) Pol I proved to be the most interesting enzyme isolated. Demonstrating strong strand-displacement activity and thermostability to 98oC, T.in Pol I is uniquely suitable to a newly termed heat-denaturing LAMP (HD-LAMP) reaction offering many potential advantages over the existing LAMP protocol. The current understanding of strand-displacement activity of Pol I is poorly understood. This thesis recognised the need to identify the exact regions and motifs responsible for this activity of the enzyme, enabling potential enhancements to be made. Enzyme engineering using site-directed mutagenesis and the formation of chimeras confirmed the importance of specific subdomains in strand-separation activity. With this knowledge, a unique Thermus aquaticus (T.aq) Pol I mutant demonstrated sufficient strand-displacement activity to permit its use in LAMP for the first time. The fusion of Cren7, a double-stranded DNA binding protein, to Pol I for use in LAMP is also reported. Although the fusion construct was found to reduce amplification speed, enhancements were observed in the presence of increased salt concentrations and it is suggested here as a means for future enzyme development.
8
Tatavarthi, Haritha. "Action of Tyrosyl DNA Phosphodiesterase on 3'-Phosphoglycolate Terminated DNA Strand Breaks." VCU Scholars Compass, 2006. http://hdl.handle.net/10156/1799.
9
Razavy, Haide. "Single-strand DNA ends in recombination in vivo." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/mq22661.pdf.
10
Fan, Saijun. "DNA strand breaks induced by gamma-ray irradiation." Thesis, University of Leicester, 1992. http://hdl.handle.net/2381/33667.
Анотація:
Part I: Plasmid DNA System The effects of a range of buffers and additives on the radiation damage in frozen aqueous plasmid DNA have been studied. In studies of various buffers, the results show that phosphate buffer system sensitise radiation DNA damage, EDTA and Tris present protections against DNA damage, in comparison with pure water system. In studies of other additives, radioprotection by NaI and LiCl increase with increasing concentrations, whilst radiosensitivity of DNA with Na2SO4 and NaClO4 increase with increasing their concentrations. DMSO shows a radioprotection. A range concentrations of spermidine and spermine are used to probe the radioprotection of DNA by polyamines. The results suggest that the protection efficiencies of polyamines increase with increasing their concentrations, moreover, spermine has a greater effect than spermidine. Part II: Cell system 10 mM concentration of spermine shows a radioprotection against DNA DSB and cell death. Metronidazole acts as a sensitiser in the induction of DSB and cell killing. However, spermine-linked metronidazole (AM1229) acts as radioprotectors against DSB under the condition of free-oxygen, and as sensitiser in induction of cell killing under the condition of atmospheric oxygen. The yields of DSBs are compared between cells irradiated at 77K and 0 C. The results show that there is a reduction of DSB in cells exposed at 77K, approximately 35% less than that in cells exposed at 0 C. It may suggest that ca. 65% DNA DSBs formed from direct effect, 35% from indirect effects. There is a difference of DSB yield in cells exposed to gamma-rays in the presence of hypotonic (0.05M) and hypertonic (1.5M) NaCl solutions. The results show that there is 20% increase in hypotonic solution, 8% reduction in hypertonic solution. However, these influences disappear when the cells are irradiated at 77K. The results suggest that the water concentration within cells has an effect on the radiation damage to DNA. There is no evidence to show that an adaptive response of DNA DSB is induced in cell pre-exposed to low doses and subsequently to high doses. The results might suggest that there is no a simple link between repair of DNA DSB and the induction of adaptive response which is found in chromosomal aberration.
11
Mahalingam, Kalpana. "Involution codes with application to DNA strand design." [Tampa, Fla.] : University of South Florida, 2004. http://purl.fcla.edu/fcla/etd/SFE0000409.
12
Krietsch, Jana. "PARP-1 activation regulates the DNA damage response to DNA double-strand breaks." Thesis, Université Laval, 2014. http://www.theses.ulaval.ca/2014/30722/30722.pdf.
Анотація:
Les cassures double-brin de l'ADN, lorsque incorrectement réparées, peuvent avoir des conséquences fatales telles que des délétions et des réarrangements chromosomiques, favorisant la carcinogenèse. La poly(ADP-ribosyl)ation réalisée par la protéine poly(ADP-ribose) polymérase-1 (PARP-1) est l'une des premières modifications post-traductionnelles qui se produisent en réponse aux dommages à l'ADN. La PARP-1 utilise la nicotinamide pour générer un polymère chargé négativement, nommé poly(ADP-ribose) polymère (PAR), lequel est attaché en majorité à la PARP-1 elle-même ainsi qu'à d'autres protéines cibles. Le PAR a récemment été reconnu comme un signal de recrutement pour certaines protéines de réparation aux sites de dommages à l'ADN, mais un débat est en cours quant au rôle précis de la PARP-1 et du PAR dans la réponse aux dommages de l'ADN. Au cours de mon projet de doctorat, nous avons pu confirmer que les protéines qui se retrouvent en complexe avec le PAR immédiatement après les dommages à l'ADN sont principalement des facteurs de réparation. Étonnamment, les complexes protéiques associés au PAR pendant la période de récupération suite aux dommages sont enrichis en facteurs de liaison à l'ARN. Toutefois, la protéine liant l'ARN la plus abondante que nous avons détectée dans l'interactome du PAR, soit NONO, ne suit pas cette dernière cinétique puisqu'elle est fortement enrichie immédiatement après les dommages à l'ADN. Notre étude subséquente de NONO dans la réponse aux cassures double-brin de l'ADN a étonnamment révélé une implication directe de celle-ci par le mécanismede réparation de jonction des extrémités non-homologues. En plus, nous avons constaté que NONO se lie fortement et spécifiquement au PAR via son motif 1 de la reconnaissance de l'ARN, soulignant la compétition entre les PAR et l'ARN pour le même site de liaison. Fait intéressant, le recrutement in vivo de NONO aux sites de dommages de l'ADN dépend entièrement du PAR et nécessite le motif 1 de la reconnaissance de l'ARN. En conclusion, nos résultats établissent NONO comme une nouvelle protéine impliquée dans la réponse aux cassures double-brin de l'ADN et plus généralement démontrent un autre niveau de complexité supplémentaire dans l'interdépendance de la biologie de l'ARN et la réparation de l'ADN.DNA double-strand breaks are potentially lethal lesions, which if not repaired correctly, can have harmful consequences such as carcinogenesis promoted by chromosome deletions and rearrangements. Poly(ADP-ribosyl)ation carried out by poly(ADP-ribose) polymerase 1 (PARP-1) is one of the first posttranslational modifications occurring in response to DNA damage. In brief, PARP-1 uses nicotinamide to generate a negatively charged polymer called poly(ADP-ribose) polymer (PAR), that can be attached to acceptor proteins, which is to a large extent PARP-1 itself. PAR has recently been recognized as a recruitment signal for key DNA repair proteins to sites of DNA damage but the precise role of PARP-1 and its catalytic product PAR in the DNA damage response are still a matter of ongoing debate. Throughout my doctoral work, we confirmed that the proteins in complex with PAR promptly after DNA damage are mostly DNA repair proteins, whereas during the period of recovery from DNA damage, the PAR interactome is highly enriched with RNA processing factors. Interestingly, one of the most abundant RNA-binding proteins detected in the PAR interactome, namely NONO, did not follow these kinetics as it was highly enriched immediately after DNA damage in the DNA repair protein complexes centered on PAR. Our subsequent investigation of NONO in the DNA damage response to double-strand breaks strikingly revealed a direct implication for NONO in repair by nonhomologous end joining (NHEJ). Moreover, we found that NONO strongly and specifically binds to PAR through its RNA-recognition motif 1 (RRM1), highlighting competition between PAR and RNA for the same binding site. Remarkably, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR and requires the RRM1 motif. In conclusion, our results establish NONO as a new protein implicated in the DNA damage response to double-strand break and in broader terms add another layer of complexity to the cross-talk between RNA-biology and DNA repair.
13
Zabolotnaya, Ekaterina. "DNA double-strand break repair studied by atomic force microscopy." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275890.
Анотація:
DNA double-strand breaks (DSBs), where both strands of the DNA duplex are simultaneously fractured, are considered the most lethal type of DNA damage. The conserved Mre11-Rad50 DNA repair complex enables the catalytic activities of the Mre11 nuclease and the Rad50 ATPase to function together to coordinate the recognition and processing of DSBs prior to the recruitment of long-range end-resection machinery required to trigger the DSB repair by the homologous recombination (HR) pathway. Fast-scan atomic force microscopy (AFM) in fluid conditions was primarily used to explore the architectural arrangement, DNA binding and processing machinery of the Mre11-Rad50 complex from the thermophilic crenarchaeote Sulfolobus acidocaldarius. The structural analysis identified four distinct architectural arrangements and demonstrates the key role of the Rad50 zinc hooks in the oligomerisation of the complex. AFM imaging showed a dynamic and Velcro-like interplay between Mre11-Rad50 protein complexes and the DNA double-helix using the Rad50 coiled-coils in a novel mode of DNA binding. The complex appears to use the Rad50 zinc hook region to bind to and track along dsDNA for broken DNA-terminals. Furthermore, the present study shows that this archaeal complex can drive extensive ATP-dependent unwinding of DNA templates. It is the first time that such unwinding has been observed in a single molecule study. These observations reveal novel activities leading to the proposal of a new model for Mre11-Rad50 action during DSB repair. AFM was also used to visualise the structure and activity of the HerA-NurA protein complex, which has been predicted to combine the activity of the NurA nuclease and hexameric HerA-translocase to generate long single-stranded DNA overhangs essential for DSB repair by HR in archaea. The present data verify and clarify the presumed biological role of this complex. Overall, the present study provides new insights into the initial steps of DNA DSB repair by the HR pathway and, most importantly, the detection of the broken ends.
14
Couto, Claudia Anne-Marie. "Investigating DNA double-strand break repair in Dictyostelium discoideum." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558280.
Анотація:
DNA double-strand breaks (DSBs) are toxic lesions that can be repaired by numerous mechanistically distinct pathways. However, regulation of DSB repair pathway utilisation is also essential for maintaining genomic integrity, and eukaryotes have evolved several mechanisms to achieve this. This includes post-translational modifications of DSB repair proteins to modulate their activity at DNA DSB ends. One example of a post-translational modification with a role in DNA repair is Poly-ADP ribosylation (PARylation), which in mammals is mediated by Poly-ADP ribose Polymerase 1 and 2 (PARP1 and 2). The best characterised roles of PARP1 and 2 is in single-strand break repair (SSBR) and base-excision repair (BER). However, their roles in DSB repair remain unclear, and to address this, the eukaryotic model Dictyostelium discoideum has been utilised. Several putative repair PARPs are conserved in the Dictyostelium genome, and the induction of P ARylation following DNA SSBs and base-damage has been observed. Using a genetic approach, multiple Dictyostelium PARPs have been implicated in SSBR/BER, as is the case in mammals. PARylation is also induced following DNA DSBs, with the major contributor being Adprt1a. This work has demonstrated the regulatory role of Adprt1a in DSB repair by promoting non-homologous end joining (NHEJ), at the expense of homologous recombination (HR). This may be achieved by the enhanced association of the Ku70/Ku80 heterodimer, the DSB sensor that initiates NHEJ, with chromatin following DNA damage in a PAR-dependent manner. Consistent with this, a PAR binding zinc-finger, the PBZ domain, has been identified in Dictyostelium Ku70, which is involved in the enhanced chromatin-association of Ku post-DSB induction. This work therefore highlights a mechanism by which Adprtl a-induced PARylation post-DSBs promotes NHEJ via Ku recruitment. The enhanced Ku association at damaged chromatin reduces engagement of other DSB repair pathways, placing Adprt 1 a at the crossroads of DSB repair pathway choice.
15
Wardrope, Laura. "Repair of double-strand DNA breaks in Escherichia coli." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/13208.
Анотація:
Double-strand DNA breaks (DSBs) occur during normal cell metabolism and are lethal unless repaired. E. coli repairs DSBs using a pathway that involves homologous recombination. The mechanisms involved in this process were investigated by manipulating the EcoKI restriction-modification system of E. coli so that the restriction activity cleaves chromosomes to produce DSBs. The viability of recombination and repair mutants was measured following the induction of DSBs. The results show that RecG and RuvABC facilitate the survival of DSBs. Surprisingly, RuvABC was able to promote survival even when recombination could not be initiated. Pulsed field gel electrophoresis (PFGE) was carried out on the genomic DNA of mutants exposed to DSBs. This allowed Holliday junctions (HJs) linking the chromosomes of strains lacking RuvABC to be detected. Most significantly, the PFGE phenotype of a recG mutant mirrored that of the wild-type, suggesting that the RecG protein is not involved in the resolution of HJs. The outcome of HJ resolution to form crossover or non-crossover products was also investigated in mutants exposed to DSBs by measuring the effect on viability of inactivating the XerCD/dif system that is involved in chromosome dimer resolution. The deleterious effect of xerC mutations on recG and ruvAC mutants was approximately 10-fold greater than on wild-type. These results prompted an interesting discussion as to how the functions of the products of these genes interact in the cell. Finally, the theory that the product of the essential yqgF gene is an alternative HJ resolvase was investigated. yqgF was placed under the control of an inducible promoter and the effect of depleting YqgF levels on survival of DSBs was measured. No evidence to suggest that YqgF can resolve HJs was found.
16
Amato, Nicholas J. "Impact of DNA Structure and Aeropyrum pernix Single-Strand DNA Binding Protein on Oxidative Damage to DNA." University of Toledo / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1372296254.
17
VILLA, MATTEO. "Regulation of DNA-end resection at DNA double strand breaks and stalled replication forks." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2018. http://hdl.handle.net/10281/198950.
Анотація:
L’instabilità genomica è una delle principali caratteristiche delle cellule tumorali e può essere generata da danni al DNA o da stress replicativi. Le rotture della doppia elica di DNA, Double Strand Breaks-DSBs, sono tra i danni più pericolosi che le cellule devono affrontare. In risposta ai DSBs, le cellule attivano un meccanismo molto conservato noto come checkpoint da danno al DNA, il cui effetto primario è quello di bloccare il ciclo cellulare fino a quando la rottura non è stata riparata. L’attivazione del checkpoint è dovuta alle chinasi apicali Tel1 e Mec1 che fosforilano e attivano le chinasi effettrici Rad53 e Chk1. I DSBs possono essere riparati mediante la ricombinazione omologa che inizia con la degradazione nucleolitica-resection- dell’estremità della rottura catalizzata dal complesso MRX e da Sae2. In seguito, le nucleasi Exo1 e Dna2, insieme all’elicasi Sgs1, catalizzano la formazione di lunghi tratti di DNA a singolo filamento. La resection è controllata negativamente dal complesso Ku, che inibisce Exo1, e dalla proteina di checkpoint Rad9, il cui meccanismo di regolazione non è noto. In lievito, l’assenza di Sae2 genera un difetto di resection che è responsabile dell’attivazione persistente del checkpoint dipendente da Tel1 e da Rad53. Per via di questo difetto, mutanti sae2 sono sensibili ad agenti genotossici che inducono DSBs. Tuttavia, la causa del difetto di resection e come l’attivazione incontrollata del checkpoint contribuiscano al fenotipo di sensibilità non è ancora noto. Per questo abbiamo cercato altri meccanismi che regolano l’inizio della resection, identificando mutazioni extrageniche in grado di sopprimere le sensibilità di cellule sae2. Abbiamo quindi isolato tre alleli SGS1-G1298R, rad53-Y88H e tel1-N2021D, in grado di sopprimere non solo le sensibilità ma anche il difetto di resection di mutanti sae2. La soppressione mediata da Sgs1-G1298R dipende da Dna2 e non da Exo1. Inoltre, l’azione di Sgs1-G1298R non solo sopprime il difetto di resection di cellule sae2 ma aumenta anche l’efficienza del processo rispetto ad un ceppo selvatico, a causa della resistenza all’inibizione mediata da Rad9. Infatti, Rad9 regola negativamente il reclutamento di Sgs1 alle estremità della lesione. Quando l’azione inibitoria di Rad9 viene meno, la richiesta del complesso MRX e di Sae2 nell’inizio della resection è ridotta. Rad53-Y88H e Tel1-N2021 sono varianti con perdita di funzione in grado di sopprimere le sensibilità di cellule sae2, in maniera dipendente da Sgs1-Dna2. Inoltre, anche l’assenza dell’attività chinasica di Rad53 e Tel1 permette di ottenere lo stesso fenotipo di soppressione che, tuttavia, non è dovuto al ruolo delle stesse nel blocco del ciclo cellulare. Infatti, queste mutazioni diminuiscono la quantità di Rad9 legato al DSB. Ciò facilita l’azione dell’elicasi Sgs1 e della nucleasi Dna2, sopprimendo così il difetto di resection di cellule sae2. Tali dati portano ad ipotizzare che l’attivazione persistente del checkpoint Tel1 e Rad53 dipendente causi un aumento del reclutamento dell’inibitore Rad9 nell’intorno della lesione che, a sua volta, è responsabile del difetto di resection e delle sensibilità di cellule sae2. Gli stress replicativi inducono il blocco della forca di replicazione e il processo di resection può essere un valido meccanismo per risolverlo. A questo proposito, abbiamo dimostrato che l’assenza dell’inibizione mediata da Rad9 compromette la risposta agli stress replicativi di cellule difettive nell’attività chinasica di Mec1, attraverso la degradazione delle forche bloccate in maniera dipendente da Sgs1 e Dna2. Tale funzione protettiva di Rad9 è indipendente dalla sua funzione nel checkpoint ma dipende principalmente dall’interazione di Rad9 con la proteina Dpb11. Per questo, abbiamo ipotizzato che Rad9 sia in grado di regolare la resection non solo al DSB ma anche alle forche di replicazione bloccate.Genome instability is an hallmark of cancer cells and can be due to DNA damage or replication stress. DNA double strand breaks (DSBs) are the most dangerous type of damage that cells have to manage. In response to DSBs, cells activate an highly conserved mechanism known as DNA damage checkpoint (DDC), whose primary effect is to halt the cell cycle until the damage is repaired. DDC is activated by the apical kinases Tel1/ATM and Mec1/ATR, which phosphorylate and activate the effector kinases Rad53/CHK2 and Chk1/CHK1. The Homologous Recombination (HR)-mediated repair of a DSB starts with the nucleolytic degradation (resection) of the 5’ ends to create long ssDNA tails. In Saccharomyces cerevisiae, resection starts with an endonucleolytic cleavage catalyzed by the MRX complex together with Sae2. More extensive resection relies on two parallel pathways that involve the nucleases Exo1 and Dna2, together with the helicase Sgs1. Resection must be tightly controlled to avoid excessive ssDNA creation. The Ku complex and the checkpoint protein Rad9 negatively regulate resection. While Ku inhibits Exo1, Rad9 restrains nucleolytic degradation by an unknown mechanism. The absence of Sae2 impairs DSB resection and causes prolonged MRX binding at DSB that leads to persistent Tel1 and Rad53-dependent DNA damage checkpoint. SAE2 deleted strains are sensitive to DSBs inducing agents, like camptothecin (CPT). This sensitivity has been associated to the resection defect of sae2∆ cells, but what causes this resection defect and if the enhanced checkpoint signaling contributes to the DNA damage sensitivity of sae2∆ cells is unknown. For these reasons, we tried to identify other possible mechanisms regulating MRX/Sae2 requirement in DSB resection by searching extragenic mutations that suppressed the sensitivity to DNA damaging agents of sae2Δ cells. We identified three mutant alleles (SGS1-G1298R, rad53-Y88H and tel1-N2021D) that suppress both the DNA damage hypersensitivity and the resection defect of sae2∆ cells. We show that Sgs1-G1298R-mediated suppression depends on Dna2 but not on Exo1. Furthermore, not only Sgs1-G1298R suppresses the resection defect of sae2∆ cells but also increases resection efficiency even in a wild type context by escaping Rad9-mediated inhibition. In fact, Rad9 negatively regulates the binding/persistence of Sgs1 at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1-G1298R mutant variant, the requirement for MRX/Sae2 in DSBs resection is reduced. Rad53-Y88H and Tel1-N2021 are loss of function mutant variants that suppress sae2∆ cells sensitivity in a Sgs1-Dna2 dependent manner. Furthermore, abolishing Rad53 and Tel1 kinase activity results in a similar suppression phenotype which does not involve the escape from the checkpoint mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function in DSBs resection by decreasing the amount of Rad9 bound at DSBs. This increases the Sgs1-Dna2 activity that, in turn, can compensate for the lack of Sae2. We propose that persistent Tel1 and Rad53 checkpoint signaling in sae2∆ cells causes DNA damage hypersensitivity and defective DSB resection by increasing the amount of Rad9 that, in turn, inhibits Sgs1-Dna2. Replication stress can induce fork stalling and controlled resection can be a relevant mechanism to allow repair/restart of stalled replication forks. We show that loss of the inhibition that Rad9 exerts on resection exacerbates the sensitivity to replication stress of Mec1 defective yeast cells by exposing stalled replication forks to Dna2-dependent degradation. This Rad9 protective function is independent of checkpoint activation and relies mainly on Rad9-Dpb11 interaction. We propose that Rad9 not only regulates the action of Sgs1-Dna2 at DSBs but also at stalled replication forks, supporting cell viability when the S-phase checkpoint is not fully functional.
18
Tentner, Andrea R. (Andrea Ruth). "Quantitative measurement and modeling of the DNA damage signaling network : DNA double-strand breaks." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/61234.
Анотація:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009."September 2009." Cataloged from PDF version of thesis.
Includes bibliographical references (p. 218-229).
DNA double-strand breaks (DSB) are one of the major mediators of chemotherapy-induced cytotoxicity in tumors. Cells that experience DNA damage can initiate a DNA damage-mediated cell-cycle arrest, attempt to repair the damage and, if successful, resume the cell-cycle (arrest/repair/resume). Cells can also initiate an active cell-death program known as apoptosis. However, it is not known what "formula" a cell uses to integrate protein signaling molecule activities to determine which of these paths it will take, or what protein signaling-molecules are essential to the execution of that decision. A better understanding of how these cellular decisions are made and mediated on a molecular level is essential to the improvement of existing combination and targeted chemotherapies, and to the development of novel targeted and personalized therapies. Our goal has been to gain an understanding of how cells responding to DSB integrate protein signaling-molecule activities across distinct signaling networks to make and execute binary cell-fate decisions, under conditions relevant to tumor physiology and treatment. We created a quantitative signal-response dataset, measuring signals that widely sample the response of signaling networks activated by the induction of DSB, and the associated cellular phenotypic responses, that together reflect the dynamic cellular responses that follow the induction of DSB. We made use of mathematical modeling approaches to systematically discover signal-response relationships within the DSB-responsive protein signaling network. The structure and content of the signal-response dataset is described, and the use of mathematical modeling approaches to analyze the dataset and discover specific signal-response relationships is illustrated. As a specific example, we selected a particularly strong set of identified signal-response correlations between ERK1/2 activity and S phase cell-cycle phenotype, identified in the mathematical data analysis, to posit a causal relationship between ERK1/2 and S phase cell cycle phenotype. We translated this posited causal relationship into an experimental hypothesis and experimentally test this hypothesis. We describe the validation of an experimental hypothesis based upon model-derived signal response relationships, and demonstrate a dual role for ERK1/2 in mediating cell-cycle arrest and apoptosis following DNA damage. Directions for the extension of the signal-response dataset and mathematical modeling approaches are outlined.
by Andrea R. Tentner.
Ph.D.
19
MARSELLA, ANTONIO. "Functions and regulation of the MRX complex at DNA double strand breaks." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/310478.
Анотація:
Le rotture del doppio filamento del DNA (DSB) sono tra le lesioni del DNA le più gravi. Se non adeguatamente riparati, i DSB potrebbero portare alla perdita di informazioni genetiche e all'instabilità del genoma, che è uno dei tratti distintivi delle cellule tumorali.
Le cellule eucariotiche riparano i DSB mediante il non-homologous end joining (NHEJ), che ricongiunge direttamente le estremità rotte del DNA e la ricombinazione omologa (HR), che utilizza la sequenza di DNA omologa per riparare il DSB. L'HR richiede una degradazione nucleolitica delle estremità, in un processo chiamato resection. In Saccharomyces cerevisiae, il complesso MRX (Mre11, Rad50 e Xrs2), aiutato da Sae2, avvia il processamento delle estremità del DSB eseguendo un taglio sulle estremità 5'. Questo taglio, catalizzato dalla subunità Mre11, consente l'accesso alle nucleasi Exo1 e Dna2. Nel NHEJ, le due estremità devono essere collegate per consentire la loro corretta riparazione. Questa funzione, chiamata end tethering, dipende dalla subunità Rad50, che lega e idrolizza l'ATP. Una transizione da uno stato legato all'ATP a uno stato di taglio post-idrolisi regola le attività di associazione e processamento del DNA di MRX. Il complesso MRX è essenziale anche nell'attivazione del checkpoint perché recluta la chinasi del checkpoint Tel1 al DSB.
In questa tesi, abbiamo studiato le funzioni e la regolazione del complesso MRX nella riparazione dei DSB. Abbiamo trovato degli alleli mre11 che sopprimono l'ipersensibilità delle cellule sae2Δ agli agenti genotossici. Le mutazioni nell'N-terminale di Mre11 sopprimono il difetto di resection delle cellule sae2Δ riducendo l'associazione di MRX e Tel1 al DSB. La ridotta persistenza di Tel1 potenzia l'attività di resection di Dna2 diminuendo l'associazione di Rad9 al DSB. Al contrario, le mutazioni di mre11 localizzate nel C-terminale non necessitano di Sae2 nel tethering ma non nella resection, possibilmente destabilizzando la conformazione aperta di Mre11 - Rad50. Questi risultati mostrano l'esistenza di domini Mre11 strutturalmente distinti che supportano la resistenza agli agenti genotossici mediando diversi processi.
L'attivazione di Tel1 in vitro da parte di MRX richiede il legame dell'ATP a Rad50. In questa tesi, descriviamo due alleli, mre11-S499P e rad50-A78T, che influenzano l'attivazione di Tel1 senza compromettere le funzioni MRX nella riparazione DSB. Queste due varianti riducono l'interazione Tel1-MRX portando a una bassa associazione Tel1 ai DSB che ne riduce l'attivazione. Le simulazioni di dinamica molecolare mostrano che il sub-complesso MR wild-type legato all'ATP rimane in una conformazione 'chiusa', mentre la presenza di ADP porta alla destabilizzazione del dimero Rad50 e dell'associazione Mre11-Rad50, entrambi gli eventi sono richiesti per la transizione conformazionale MR ad uno stato aperto. Al contrario, MRA78T provoca un'apertura del complesso anche se legato all'ATP, indicando che il difetto di attivazione di Tel1 causato da MRA78T risulta dalla destabilizzazione dello stato conformazionale legato all'ATP.
La mancanza di Sae2 aumenta la persistenza di MRX ai DSB e all'attivazione dei checkpoint. In questa tesi, dimostriamo anche che la proteina telomerica Rif2, che stimola l'idrolisi dell'ATP da parte di Rad50, inibisce l'attività dell'endonucleasi Mre11 ed è responsabile dell’aumento di MRX ai DSB nelle cellule sae2Δ. Abbiamo identificato un residuo di Rad50 che è importante per l'interazione Rad50-Rif2 e l'inibizione mediata da Rif2 della nucleasi Mre11. Questo residuo altera l'interazione Mre11-Rad50. Proponiamo che Sae2 stimoli l'attività endonucleasica di MRX stabilizzando lo stato di taglio, mentre Rif2 lo inibisce antagonizzando il legame di Sae2 e stabilizzando una conformazione di MR che non è adatta al taglio.DNA double strand breaks (DSBs) are among the most severe DNA lesions. If not properly repaired, DSBs could lead to loss of genetic information and genome instability, which is one of the hallmarks of cancer cells. Eukaryotic cells repair DSBs by non-homologous end joining (NHEJ), which directly re-ligates the DNA broken ends, and homologous recombination (HR), which uses the intact homologous DNA sequence as a template to repair the DSB. HR requires a nucleolytic degradation of the broken DNA ends, in a process called resection. In Saccharomyces cerevisiae, the MRX (Mre11, Rad50 and Xrs2) complex, aided by Sae2, initiates resection of the DSB ends by performing an endonucleolytic cleavage on the 5’-ended strands. This cleavage, catalyzed by the Mre11 subunit, allows the access of Exo1 and Dna2 nucleases that elongate the ssDNA ends. In NHEJ, the two broken ends need to be physically connected to allow their correct religation. This function, called end tethering, depends on the Rad50 subunit, which binds and hydrolyses ATP. A transitions between an ATP-bound state to a post-hydrolysis cutting state regulates MRX DNA binding and processing activities. The MRX complex is also essential in DNA damage checkpoint activation because it recruits the checkpoint kinase Tel1 at the break site. In this thesis, we studied functions and regulation of the MRX complex in DSB repair. We found mre11 alleles that suppress the hypersensitivity of sae2Δ cells to genotoxic agents. The mutations in the Mre11 N-terminus suppress the resection defect of sae2Δ cells by lowering MRX and Tel1 association to DSBs. The diminished Tel1 persistence potentiates Dna2 resection activity by decreasing Rad9 association to DSBs. By contrast, the mre11 mutations localized at the C-terminus bypass Sae2 function in end-tethering but not in DSB resection, possibly by destabilizing the Mre11–Rad50 open conformation. These findings unmask the existence of structurally distinct Mre11 domains that support resistance to genotoxic agents by mediating different processes. In vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role for the MR subcomplex in Tel1 activation. In this thesis, we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1–MRX interaction leading to low Tel1 association at DSBs that reduces Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly ‘closed’ conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11–Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP- bound conformational state. The lack of Sae2 increases MRX persistence at DSBs and checkpoint activation. In this thesis, we also show that the telomeric protein Rif2, which stimulates ATP hydrolysis by Rad50, inhibits the Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identified a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2-mediated inhibition of Mre11 nuclease. This residue is located nearby a Rad50 surface that binds Sae2 and is important to stabilize the Mre11-Rad50 interaction in the cutting state. We propose that Sae2 stimulates MRX endonuclease activity by stabilizing the cutting state, whereas Rif2 inhibits it by antagonizing Sae2 binding to Rad50 and stabilizing a MR conformation that is not competent for DNA cleavage. The results described in this PhD thesis contribute to the understanding of the molecular mechanisms supporting functions and regulation of the MRX complex at DSBs.
20
Lempidaki, Styliani. "Study of DNA double strand break repair in Dictyostelium discoideum." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:3d0035a5-6f17-435d-990f-22ec24ec441e.
Анотація:
The homologous recombination (HR) pathway contributes to genome integrity by mediating double strand break (DSB) repair using a homologous DNA sequence as a template. In mammals Rad51 and Brca2 are molecules central to this process. Little is known about HR repair in Dictyostelium. However, research previously conducted on DSB repair using this organism has shown that DSB repair pathways are highly conserved when compared to humans. This encouraged study of HR in this organism. In this study, through a bioinformatics search I have identified putative orthologues of most human HR proteins and most interestingly of BRCA2, which cannot be found in other lower eukaryotes used as models for DSB repair, such as the budding yeast S.cerevisiae. Brcp, the Dictyostelium BRCA2 ortholog, shows similar domain structure when compared to BRCA2-related proteins identified in other organisms. To verify the implication of HR proteins in DSB repair, I developed a method to monitor recruitment of DNA repair proteins on chromatin upon DSB induction. Findings of this study suggest that both Brcp and Rad51 get recruited to chromatin upon DSB induction and are therefore implicated in DSB repair in Dictyostelium. To further study Brcp function and based on findings suggesting that disruption of brcp might be lethal, I developed a novel system for specific and conditional depletion of endogenous Dictyostelium proteins. Utilizing this system, I conducted phenotypic studies in a strain depleted of Brcp to examine its role in DNA repair. Overall this study shows that the HR pathway in Dictyostelium shows great similarity to vertebrates, making Dictyostelium an appealing model for the study of DSB repair and specifically HR.
21
Dickman, Rebekah. "Thermodynamic Effects of 5' and 3' Single Strand Dangling Ends on Short Duplex DNA." PDXScholar, 2010. https://pdxscholar.library.pdx.edu/open_access_etds/94.
Анотація:
Differential scanning calorimetry (DSC) melting analysis was performed on 27 short double stranded DNA duplexes containing 15 to 25 base pairs and short single stranded overhangs from one to 10 bases, on both ends. Molecules have two 5' dangling ends or one 5' and one 3' dangling end. For these molecules the duplex region was incrementally reduced from 25 to 15 base pairs with increased length of the dangling ends from one to 10 bases. A third set of molecules contained 21 base pair duplexes with a four base dangling end on either the 5' or 3' end. Blunt ended duplexes from 15 to 25 base pairs were also examined and served as control duplexes. DSC melting curves were measured in solution containing 85 mM, 300 mM or 1.0 M Na+. From these measurements, thermodynamic parameters for 5' and 3' dangling ends as a function of end length were evaluated. Results showed the 5' ends were slightly stabilizing, and this stability was essentially constant with end length, while the 3' ends were generally destabilizing with increasing length of the end. This finding of lower stability for the 3' ends is consistent with results of published studies that have found 5' dangling ends to be more than or equally as stabilizing as 3' dangling ends. Our finding that 3' dangling ends are actually destabilizing for duplex DNA contrasts with published results. The 3' ends also display a stronger dependence on the [Na+]. In the lower Na+ environment the 3' ends are more destabilizing than at the higher salt environments. Analysis of the thermodynamic parameters of the dangling-ended duplexes as a function [Na+] indicated the 3' dangling end molecules behave differently compared to 5' dangling ended and blunt ended duplexes. The net counterion release per phosphate upon melting the molecules having one 5' and one 3' end was approximately 15% smaller as a function of end length compared to the duplex having two 5' ends. Further analysis of the DSC evaluated thermodynamic transition parameter, ΔHcal, and its relationship to the measured transition temperatures of the DNA molecules, provided an estimate on the excess heat capacity differences, ΔCp, between duplex and melted single strands for the dangling-ended molecules. The analysis revealed the molecules with one 5' and one 3' dangling end had very different ΔCp values compared to the blunt-ended molecule; while the molecules with two 5' ends have ΔCp that are essentially the same as the blunt-ended duplex. These observations are interpreted as differences in the interactions with Na+, solvent and the terminal base pairs of the duplex for the 5' versus 3' dangling ends.
22
Liu, Nan. "Hypersensitivity of ataxia telangiectasia cells to DNA double strand breaks." Thesis, University of St Andrews, 1994. http://hdl.handle.net/10023/13905.
Анотація:
Cells of ataxia telangiectasia (AT) individuals are hypersensitive to a variety of DNA damaging agents such as ionizing radiation and bleomycin, presumed to be due to an intrinsic defect in repair of DNA damage. The nature of the DNA lesion(s) to which AT cells are abnormally sensitive, and the defect in DNA repair are presently unclear. The major part of this project aimed at investigating the sensitivity of AT cells to DNA double-strand breaks (dsb) generated by restriction endonucleases (RE), thereby verifying the hypothesis that AT cells are deficient in the processing of dsb. AT lymphoblastoid cell lines (AT-PA and AT-KM) used in this study were initially characterized and found to be approximately 3 times more sensitive to ionizing radiation in the induction of micronuclei (Mn) and chromosomal aberrations (CA) compared with a normal lymphoblastoid cell line (N-SW). Other cellular characteristics were observed in AT-PA cells following-irradiation such as normal induction and rejoining of dsb and reduced inhibition of DNA synthesis. By using SLO poration, RE were introduced into the AT and normal cell lines and the yield of CA resulting from RE-induced dsb were subsequently investigated. The frequencies of CA induced by Pvu II were 2 - 4 fold higher in AT-PA than in N-SW cells at both 5 h and 24 h sampling times. The enhanced frequency of CA in AT cells treated with Pvu II was principally a result of an increase of chromatid aberrations, rather than chromosome aberrations at 24 h. higher frequencies of chromatid exchanges appeared in AT-PA than in N-SW cells. The results suggest that AT cells are characterized by a defect in dsb processing that converts a higher number of dsb into CA than in the normal cell line. With respect to the different end-structures of RE-induced dsb, cohesive-ended dsb generated by BamH I and Pst I were found to induce lower frequencies of CA than blunt-ended dsb generated by Pvu II and EcoR V in both the AT cell lines and the normal cell line. The results support the previous observations that cohesive-ended dsb are less clastogenic than blunt-ended dsb (Bryant 1984). Although inducing lower frequencies of CA than Pvu II and EcoR V, BamH I and Pst I induced higher number of CA in both AT-PA and AT-KM cells when compared with N-SW cells, again indicating a defect in processing cohesive-ended dsb exists in AT cells. A potent DNA repair inhibitor, Ara A, was found to potentiate the production of CA by RE in AT and normal cells. The enhancement ratios (by ara A) for CA induced by Pvu II and Pst 1 were higher in N-SW cells than in AT-PA and AT-KM cells. Ara A appeared to have no effect on the frequencies of CA induced by BamH I in any of the cell lines tested. Based on these findings, a mechanism for the rejoining of RE-induced dsb in which DNA repair synthesis may be involved is proposed, and it is postulated that dsb in AT cells are subjected to greater end degradation. Inhibition of DNA synthesis was observed in normal cells after treatment with Pvu II and EcoR V, while EcoR I and BamH I had only minor effect. AT-PA cells were found to be resistant to RE-induced inhibition of DNA synthesis, as in the case of ionizing radiation. This result suggests that RE-induced blunt-ended dsb mimic radiation-induced lesions in supressing DNA synthesis in normal cells and that AT cells respond to RE-induced dsb in a similar way to damage induced by ionizing radiation. Finally, when a nuclear extract from N-SW cells was introduced into Pvu Il-treated AT-PA cells, it was able to confer a normal frequency of CA. In contrast, neither whole cell nor nuclear extracts from normal cells influenced the production of CA induced by y-rays. These findings provide evidence for the presence of factor(s) in normal nuclear extract which complements the defect in processing of RE-induced dsb in AT cells.
23
Choudhury, Sibgat Ahmed. "Role of TRM2RNC1 endo-exonuclease in DNA double strand break repair." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103373.
Анотація:
DNA double strand breaks (DSB) are the most toxic of all types of DNA lesions. In Saccharomyces cerevisiae, DNA DSBs are predominantly repaired by the homologous recombination repair (HRR) pathway. The initial step of HRR requires extensive processing of DNA ends from the 5' to 3' direction by specific endo-exonuclease(s) (EE) at the DSB sites, but no endo-exonuclease(s) has yet been conclusively determined for such processing of DSBs. S. cerevisiae TRM2/RNC1 is a candidate endo-exonuclease that was previously implicated for its role in the HRR pathway and was also shown to have methyl transferase activity primarily located at its c-terminus.In this dissertation, we provided compelling biochemical and genetic evidence that linked TRM2/RNC1 to the DNA end processing role in HRR. Trm2/Rnc1p purified with a small calmodulin binding peptide (CBP) tag displayed single strand (ss) specific endonuclease and double strand (ds) specific 5' to 3' exonuclease activity characteristic of endo-exonucleases involved in HRR. Intriguingly, purified Trm2/Rnc1p deleted for its C-terminal methyl transferase domain retained its nuclease activity but not the methyl transferase activity indicating that the C-terminal part responsible for its methyl transferase function is not required for its nuclease activity.
Our genetic and functional studies with S. cerevisiae trm2/rnc1 single mutants alone or in combination with other DNA DSB repair mutants after treatment with the DNA damaging drug methyl methane sulfonate (MMS) or IR that is believed to produce DSBs, or with specific induction of DNA DSBs at the MAT locus by HO-endonuclease demonstrated an epistatic relationship of TRM2/RNC1 with the major recombination factor RAD52. These studies suggested that TRM2/RNC1 probably acts at an earlier step than RAD52 in the HRR pathway. The genetic evidence also indicated a possible functional redundancy with the bona fide endo-exonuclease EXO1 in the processing of DNA ends at the DSB sites.
In a recent report, the immuno-purified mouse homologue of TRM2/RNC1 exhibited similar enzymatic properties as the endo-exonucleases involved in HRR. A small molecular inhibitor pentamidine specifically inhibited the nuclease activity of the mouse EE and sensitized various cancer cells to DNA damaging agents commonly used in cancer chemotherapy. We specifically suppressed expression of the mouse EE using small interfering RNA (siRNA) that conferred sensitivity of B16F10 melanoma cells to a variety of DNA damaging drugs often used in cancer treatment. This further validated our earlier claim of the endo-exonuclease as a potential therapeutic target in treating cancer.
24
Song, Daqing. "Homologous Strand Exchange and DNA Helicase Activities in Plant Mitochondria." Diss., CLICK HERE for online access, 2005. http://contentdm.lib.byu.edu/ETD/image/etd931.pdf.
25
Kegel, Andreas. "Silencing and DNA double-strand break repair in budding yeast /." Stockholm : Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-1059.
26
Harer, Christine Joan. "DNA double strand break rejoining by NHEJ and interfacing components." Thesis, University of Sussex, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437460.
27
Sawlekar, Rucha. "Programming dynamic nonlinear biomolecular devices using DNA strand displacement reactions." Thesis, University of Warwick, 2016. http://wrap.warwick.ac.uk/91757/.
Анотація:
Recent advances in DNA computing have greatly facilitated the design of biomolecular circuitry based on toehold-mediated DNA strand displacement (DSD) reactions. The synthesis of biomolecular circuits for controlling molecular-scale processes is an important goal of synthetic biology with a wide range of in vitro and in vivo applications. In this thesis, new results are presented on how chemical reaction networks (CRNs) can be used as a programming language to implement commonly used linear and nonlinear system theoretic operators that can be further utilised in combination to form complex biomolecular circuits. Within the same framework, the design of an important class of nonlinear feedback controller, i.e. a quasi sliding mode (QSM) feedback controller, is proposed. The closed loop response of the nonlinear QSM controller is shown to outperform a traditional linear proportional+integrator (PI) controller by facilitating much faster tracking response dynamics without introducing overshoots in the transient response. The resulting controller is highly modular and is less affected by retroactivity effects than standard linear designs. An important issue to consider in this design process for synthetic circuits is the effect of biological and experimental uncertainties on the functionality and reliability of the overall circuit. In the case of biomolecular feedback control circuits, such uncertainties could lead to a range of adverse effects, including achieving wrong concentration levels, sluggish performance and even instability. In this thesis, the robustness properties of two biomolecular feedback controllers; PI and QSM, subject to uncertainties in the experimentally implemented rates of their underlying chemical reactions, and to variations in accumulative time delays in the process to be controlled, are analysed. The simulation results show that the proposed QSM controller is significantly more robust against investigated uncertainties, highlighting its potential as a practically implementable biomolecular feedback controller for future synthetic biology applications. Finally, the thesis presents new results on the design of biomolecular feedback controllers using the set of chemical reactions underlying covalent modification cycles.
28
Azeroglu, Benura. "DNA synthesis during double-strand break repair in Escherichia coli." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/16213.
Анотація:
Efficient and accurate repair of DNA double strand breaks (DSBs) is required to maintain genomic stability in both eukaryotes and prokaryotes. In Escherichia coli, DSBs are repaired by homologous recombination (HR). During this process, DNA synthesis needs to be primed and templated from an intact homologous sequence to restore any information that may have been lost on the broken DNA molecule. Two critical late stages of the pathway are repair DNA synthesis and the processing of Holliday junctions (HJs). However, our knowledge of the detailed mechanisms of these steps is still limited. Our laboratory has developed a system that permits the induction of a site-specific DSB in the bacterial chromosome. This break forms in a replication dependent manner on one of the sister chromosomes, leaving the second sister chromosome intact for repair by HR. Unlike previously available systems, the repairable nature of these breaks has made it possible to physically investigate the different stages of DNA double-strand break repair (DSBR) in a chromosomal context. In this thesis, I have addressed some fundamental questions relating to repair DNA synthesis and processing of HJs by using a combination of mutants defective in specific biochemical reactions and an assay that I have developed to detect repair DNA synthesis, using a polar termination sequence (terB). First, by using terB sites located at different locations around the break point, it was shown that the DnaB-dependent repair forks are established in a coordinated manner, meaning that the collision of the repair forks occurs between two repair DNA synthesis initiation sites. Second, DSBR was shown to require the PriB protein known to transduce the DNA synthesis initiation signal from PriA protein to DnaT. Conversely, the PriC protein (known as an alternative to PriB in some reactions) was not required in this process. PriB was also shown to be required to establish DnaB-dependent repair synthesis using the terB assay. Third, the establishment and termination of repair DNA synthesis by collision of converging repair forks were shown to occur independently of HJ resolution. This conclusion results from the comparison of the viability of single and double mutants, deficient in either the establishment of DNA synthesis, HJ resolution or in both reactions, subjected to DSBs and from the study of the DNA intermediates that accumulated in these mutants as detected by two-dimensional gel electrophoresis. Fourth, the role of RecG protein during DSB repair was investigated. Solexa sequencing analyses showed that recG null mutant cells undergoing DSBs accumulate more DNA around the break point (Mawer and Leach, unpublished data). This phenomenon was further investigated by two different approaches. Using terB sites in different locations around the break point and ChIP-Seq analyses to investigate the distribution of RecA in a recG null mutant demonstrating that the establishment of repair forks depends on the presence of RecG. Further studies using PriA helicase-dead mutant showed that the interplay between RecG and PriA proteins is essential for the establishment of correctly oriented repair forks during DSBR. As a whole, this work provides evidence on the coordinated nature of the establishment and termination of DNA synthesis during DSBR and how this requires a correct interplay between PriA-PriB and RecG. A new adapted model of homologous recombination is presented.
29
Mawer, Julia Sofia Pamela. "Intermediates of DNA double strand break repair in Escherichia coli." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6258.
Анотація:
A DNA double-strand break (DSB) is a severe form of DNA damage. In fastgrowing cells, DSBs are commonly repaired by homologous recombination (HR) and in E. coli they are exclusively repaired by this mechanism. Failure to accurately repair DSBs can lead to genomic instability. Characterising the DNA intermediates formed during DSB repair by HR is key to understanding this process. A system for inducing a site-specific DSB in the E. coli chromosome has previously been described (Eykelenboom et al., 2008). Here, this system has been used to determine the nature of the intermediates of the repair. It was shown that in a Rec+ background the repair process is rapid and efficient. By contrast, in a ruvAB mutant, which is defective for the Holliday junction (HJ) migration and cleavage complex, RuvABC, HJs are accumulated on both sides of the breakpoint. Replication forks also accumulate at defined positions from the DSB, indicating that unresolved HJs are a barrier to efficient replication that is associated with the repair. This suggests that the resolution of HJs needs to occur prior to the establishment of DNA synthesis. Despite the accumulation of HJs in a ruvAB mutant, cell survival occurs when DSBs are induced for short periods, suggesting that HJs can be resolved in a RuvAB-independent manner. In contrast, the RecG helicase is essential for survival. In a recG mutant, replication forks but not HJs are detected in the region of DSB repair. In a ruvAB recG mutant, intermediates in this region are lost. These observations are consistent with a role of RecG in the stabilisation and maturation of D-loops and not the resolution of Holliday junctions. Nevertheless, an additional role for RecG in later stages of repair cannot yet be excluded. This work provides a solid framework for the further study of DSB repair in E. coli.
30
Dever, Seth. "The Role of BRCA1 in DNA Double-strand Break Repair." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1741.
Анотація:
Mutations in the breast cancer susceptibility 1 (BRCA1) gene are linked to breast as well as ovarian cancers. However, most cancer-causing mutations within the BRCA1 gene have been found in the N’ and C’ terminal regions of the BRCA1 protein, both believed to be important for DNA double-strand break (DSB) repair. The BRCA1 C’ terminal (BRCT) repeats have been implicated in phospho-serine protein binding whereas the N’ terminal RING domain interacts with the BARD1 protein to form a hetero-dimeric complex with E3 ubiquitin ligase activity. The BRCA1 BRCT domain binds CtIP, BACH1, and RAP80, all of which have been directly implicated in homologous recombination repair (HRR). Lysine 1702 (K1702) of BRCA1 resides within the phospho-serine binding pocket of the first BRCT repeat of BRCA1. To determine the effect of manipulating the ability of BRCA1 to bind CtIP and other phospho-proteins binding to the BRCA1 BRCT domain on DSB repair, and specifically HRR, we introduced a K1702M mutation into BRCA1 known to impair BRCT binding to a pSer-X-X-Phe peptide representing BACH1. Surprisingly, instead of impairing HRR, we found that BRCA1 K1702M resulted in hyper-recombination with > 3-fold higher levels of HRR compared to wild-type BRCA1 using an HRR assay based on GFP expression in BRCA1-defective HCC1937 cells. This hyper-recombinogenic phenotype coincided with cell-cycle arrest in S/G2 suggesting that the potential lack of binding of critical proteins to the BRCA1 BRCT domain results in abnormal HRR by priming cells to undergo more HRR which is enhanced during the S and G2 phases of the cell-cycle. In line with the increased HRR seen with the HRR/GFP assay, HCC1937 cells expressing BRCA1 K1702M showed increased levels of RAD51 foci and nuclear staining suggesting that HRR was highly elevated. Interestingly, the hyper-recombinogenic phenotype of BRCA1 K1702M could be reduced to normal levels with a second mutation (I26A) in BRCA1 that affects BRCA1 and CtIP ubiquitination. These results reveal a hierarchal regulation of HRR with ubiquitination having a dominate role in DSB repair by BRCA1 and suggests that targeted disruption of BRCT-CtIP binding increases HRR that is in turn controlled by ubiquitination. In addition, we provide evidence that BRCA1 serine 1387 phosphorylation within the SQ cluster region of BRCA1 is involved in the cell survival and DNA damage response to IR. The BRCA1 S1387A mutant only partially increased the radiosurvival of HCC1937 cells compared to cells expressing wild-type BRCA1 and immunocytochemical analysis revealed wild-type BRCA1 was located in the nucleus whereas the S1387A mutant was cytoplasmic in response to IR. We also show that BRCA1 SQ cluster serine phosphorylation in addition to serine 1387 is involved in HRR. Altogether, these findings reveal the importance of various regions of BRCA1 in DSB repair and may lead to multiple strategies of modulating BRCA1 function in response to DNA damage.
31
Wang, Xin. "PTIP promotes DNA double-strand break repair through homologous recombination." Kyoto University, 2010. http://hdl.handle.net/2433/120541.
32
Damit, Michael James. "Condensin recruitment to the DNA double-strand break in meiosis." Tallahassee, Fla. : Florida State University, 2008. http://purl.fcla.edu/fsu/lib/digcoll/undergraduate/honors-theses/341780.
Анотація:
Thesis (Honors paper)--Florida State University, 2008.Advisor: Dr. Hong-Guo Yu, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Includes bibliographical references.
33
Wechsler, Thomas. "Characterization of new interaction partners of the DNA double-strand break repair protein DNA-PKcs." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-42460.
34
Coogan, Christian P. "Reduced fidelity of E. coli leading strand DNA replication opposite ENU-induced thymine adducts in the transcribed strand /." Available to subscribers only, 2005. http://proquest.umi.com/pqdweb?did=1079660821&sid=4&Fmt=2&clientId=1509&RQT=309&VName=PQD.
35
Hiller, Natalie. "H2A.Z-dependent cellular responses to a persistent DNA double-strand break." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-124885.
36
Zhang, Hongshan. "A single molecule perspective on DNA double-strand break repair mechanisms." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0177.
Анотація:
Les cassures double brin de l'ADN altèrent l'intégrité physique du chromosome et constituent l'un des types les plus sévères de dommages à l'ADN. Pour préserver l'intégrité du génome contre les effets potentiellement néfastes des cassures double brin de l'ADN, les cellules humaines ont développé plusieurs mécanismes de réparation, dont la réparation par recombinaison de l'ADN et la jonction d'extrémités non-homologues (NHEJ), catalysés par des enzymes spécifiques. Pendant ma thèse, nous avons caractérisé la dynamique de certaines des interactions protéines/ADN impliquées dans ces mécanismes au niveau de la molécule unique. Dans ce but, nous avons combiné des pinces optiques et de la micro-fluidique avec de la microscopie de fluorescence à champ large afin de manipuler une ou deux molécules d'ADN individuelles et d'observer directement les protéines de la réparation marquées par fluorescence agissant sur l'ADN. Nous avons concentré notre analyse sur trois protéines/complexes essentiels impliqués dans la réparation de l'ADN: (i) la protéine humaine d’appariement de brin RAD52, (ii) les protéines humaines XRCC4, XLF et le complexe XRCC4/Ligase IV de la NHEJ et (iii) le complexe humain MRE11/RAD50/NBS1DNA double-strand breaks disrupt the physical continuity of the chromosome and are one of the most severe types of DNA damage. To preserve genome integrity against the potentially deleterious effects of DNA double-strand breaks, human cells have evolved several repair mechanisms including DNA recombinational repair and Non-Homologous End Joining (NHEJ), each catalyzed by specific enzymes. In this thesis we aimed at unraveling the dynamics of protein/DNA transactions involved in DNA double-strand break repair mechanisms at single molecule level. To do this, we combined optical tweezers and microfluidics with wide-field fluorescence microscopy, which allowed us to manipulate individual DNA molecules while directly visualize fluorescently-labeled DNA repair proteins acting on them. We focused the study on three crucial proteins/complexes involved in DNA repair: (i) the human DNA annealing protein RAD52, (ii) the non-homologous end joining human proteins XRCC4 and XLF and the complex XRCC4/Ligase IV, and (iii) the human MRE11/RAD50/NBS1 complex
37
Hudson, Jessica. "The conservation of DNA double strand repair proteind in Dictyostelium discoideum." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442763.
38
Shaheen, Fadhel Sulaiman. "Targeting the DNA double strand break repair machinery in prostate cancer." Thesis, University of Newcastle Upon Tyne, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.500921.
Анотація:
Prostate cancer is the most common cancer in males in western societies. In spite of the successful first line treatment using surgery, radiation therapy, antiandrogen treatment or combination therapy the disease progresses towards a hormone refractory state where the only effective treatment is chemotherapy which prolongs overall survival, however it is not curative. The resistance that hormone refractory disease displays highlights the importance of developing new targeted therapies which may be curative or at least may improve the patient's quality of life and overall survival. Current chemotherapeutic regimens used in prostate cancer treatment mostly contain agents that induce DNA damage and specifically double strand breaks, such as Doxorubicin, mitoxantrone and etoposide.
39
Dean, Philip John. "Double strand break repair and DNA damage signalling pathways in Arabidopsis." Thesis, University of Leeds, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487719.
Анотація:
The external environment and internal cellular processes generate DNA double strand breaks (DSBs), a particularly toxic form of DNA damage that can result in chromosome fragmentation, replication failure, mutagenesis and cell death. Cells have evolved effective mechanisms to preserve the mtegrity of the genome including DNA damage signalling, cell cycle checkpoint activation and DNA repair.
40
Renkawitz, Jörg. "Monitoring homology search during DNA double-strand break repair in vivo." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-169454.
41
Golding, Sarah E. "DNA double-strand break repair and signalling in human glioma cells." Thesis, University of the West of England, Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431304.
42
Blunt, Tracy. "The identification of genes involved in DNA double strand break repair." Thesis, University of Sussex, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320363.
43
Monteiro, Emanuela. "Dynamics of p53 signalling in response to single-strand DNA damage." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/dynamics-of-p53-signalling-in-response-to-singlestrand-dna-damage(ddb35d2e-98ac-4e17-a620-c8a047878477).html.
Анотація:
Every day cells are exposed to different stresses that may cause DNA damage. UV radiation and certain anti-cancer drugs mainly cause single-strand DNA breaks, leading to the activation of the ATR pathway and consequently of the tumour suppressor p53. Actually, p53 is a central node in the DNA damage response pathways. Studies on this protein have mainly been made at the population level using classic biochemistry approaches. In recent years, single-cell microscopy analysis, using plasmid-based expression systems, have revealed that p53 shows a series of nuclear translocations in response to double-strand DNA damage, a behaviour that can be masked at the population level. In the present work, a BAC transgenesis system containing the p53 gene tagged to dsRedXP was improved and transiently transfected into MCF7 cells. By using this system it was possible to see that, in contrast with what has been proposed in the past, cells mainly displayed multiple p53 translocations (peaks) in response to UV damage. However, there was an increase in the number of cells showing a single, albeit wider, p53 peak as the UV dose increased, suggesting a longer p53 occupancy within the nucleus. As UV dose increased, the cumulative levels of p53 in the nucleus also appeared to increase, as possibly a higher level of repair was needed. Interestingly, with this work it was possible to see that even though the time of appearance of the first p53 nuclear translocations following UV stress was very variable, the timing of consecutive p53-dsRedXP nuclear translocations as well as nuclear import and export rates of this protein were very stable, independently of level of stress. Through population-based qRT-PCR assays it was also possible to see that the activation of p21, Mdm2 and Wip1, all p53 transcriptional targets, appeared to occur within the same time as the average first p53 nuclear translocation following UV damage. Cells also appeared to present a basal p53 response against damage, which was similar to the response seen in cells exposed to a low dose of UV. Different cancer cell lines were also shown to present a different wiring of the p53 network, leading to different profiles of mRNA response. Overall, with this work it was possible to show that the choice of transgenesis system to study the single-cell behaviour of p53 is of utmost importance. p53 shows a very heterogeneous behaviour in cells exposed to UV stress, even within a defined dose, thus showing that its response to damage and activity is not only dependent on the type and level of stress but possibly on the location of the damage and other factors inherent to each cell and its local environment.
44
Mills, Kevin D. (Kevin David) 1972. "Silencing, heterochromatin, and DNA double strand break repair in Saccharomyces cerevisiae." Thesis, Massachusetts Institute of Technology, 1999. http://hdl.handle.net/1721.1/84768.
45
Roy, Rajat. "Functional analysis of the DNA double-strand break repair protein Ku." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619963.
46
Rakhimova, Alina. "Role of histones in DNA double-strand break repair in Dictyostelium." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:f6ae6f6a-9f83-4326-89e3-719ea9c43cc0.
Анотація:
Correct repair of DNA double-strand breaks is crucial for maintenance of genome integrity. Despite data showing the importance of histones variants and histone post-translational modifications in the cellular response to DNA damage, there is still a lack of knowledge concerning the role of histone H3 and its variants as well as histone ADP-ribosylation in such processes. In this work Dictyostelium discoideum was employed as genetically tractable model organism to address the role of histone H3 variants and histone ADP-ribosylation in DNA double-strand break (DSB) repair. Vegetative cells lacking two out of three histone H3 variants - H3b and H3c, were shown not to be sensitive to DNA DSB. No evidence for altered DSB repair was found as phosphorylation of histone H2AX (a marker of DSB) and one of the pathways of DSB repair, non-homologous end joining, were not altered. Altogether, this work demonstrates that H3b and c variants are not required for overall DNA DSB repair in Dictyostelium. Among the core histones histone H2B was discovered to be the major acceptor of ADP-ribosylation by major ADP-ribosyl transferase involved in DSB repair, Adprt1a, in vitro. ADP-ribosylation in vitro was shown to occur on glutamate E18 with E19 being a potential regulator of this modification. Using an epitope-tagged overexpressed H2B, in vivo H2B ADP-ribosylation in response to DSBs was observed in Dictyostelium for the first time. Decreased ADP-ribosylation of epitope-tagged H2B mutated in both E18 and E19 residues was demonstrated. Overall, this work demonstrates the presence of the ADP-ribosylation of H2B in Dictyostelium in response to DSBs and identifies the major site of this modification.
47
Ma, Yue. "Double-strand breaks (DSBs) and structure transition on genome-sized DNA." Thesis, https://doors.doshisha.ac.jp/opac/opac_link/bibid/BB13097333/?lang=0, 2018. https://doors.doshisha.ac.jp/opac/opac_link/bibid/BB13097333/?lang=0.
Анотація:
DNA中の二本鎖切断(DSB)に対するアスコルビン酸(AA)およびDMSOの保護効果を、蛍光顕微鏡による巨大DNA(T4 DNA; 166kbp)の単分子観察によって評価した。凍結／解凍の状態に対して3つの異なる形態の放射源、可視光、γ線、および超音波の環境下にさらした。1‐プロパノールと2‐プロパノールの間で異なる効果が表れた。ゲノムDNA分子の高次構造の変化は、1−プロパノールを用いると、長軸長が濃度60％で最小を示し、次にアルコール含有量の増加と共に増加する傾向があることを見出した。一方、2−プロパノールを用いると、長軸長はアルコール含有量の増加と共にほぼ単調な減少を示した。The protective effect of ascorbic acid (AA) and DMSO against double-strand breaks (DSBs) in DNA was evaluated by single-molecule observation of giant DNA (T4 DNA; 166kbp) through fluorescence microscopy. Samples were exposed to three different forms of radiation: visible light, γ-ray, and ultrasound or freeze/thawing. The change of the higher-order structure of genomic DNA molecules in the presence of alcohols by use of single DNA observation with fluorescence microscopy, by focusing our attention to unveil the different effect between 1-propanol and 2-propanol.
博士(工学)
Doctor of Philosophy in Engineering
同志社大学
Doshisha University
48
Khalil, Ashraf. "ATM-Dependent ERK Signaling in Response to DNA Double Strand Breaks." VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd/760.
Анотація:
Ionizing radiation (IR) triggers many signaling pathways stemming from DNA damage, and, independently, from extra-nuclear events. To generate radio-mimetic DNA double-strand breaks (DSBs) without and minimizing the effects on extra-nuclear radiation targets, human (p53+) glioma and carcinoma cells containing bromodeoxyuridine (BrdU)- substituted DNA were treated with Hoechst 33258 followed by long wave-length UV (UV-A) (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose-dependent generation of DSBs equivalent to 0.2 - 20 Gy of IR, as detected by pulse-field gel electrophoresis, accompanied by dose-dependent H2AX phosphorylation at ser-139 and ATM phosphorylation at ser-1981, indicating ATM activation. Furthermore, BrdU photolysis increased phosphorylation of Chk2 (at thr-68) and p53 (at ser-15). p53 phosphorylation was reduced by the ATM inhibitor caffeine, and H2AX phosphorylation was greatly reduced in AT cells, confirming that phosphorylation was primarily ATM-dependent. We also examined the effects of BrdU photolysis on the major cellular signaling ERK pathways. Interestingly, low-dose (≤ 2 Gy-equivalents) BrdU photolysis stimulated ERK1/2 phosphorylation whereas higher doses (≥ 5 Gy eq.) resulted in Em1/2 dephosphorylation. ERK1/2 phosphorylation was ATM-dependent, whereas dephosphorylation was ATM-independent and DSBs dose-dependent. Thus ERK1/2 appear to be both positively and negatively regulated by ATM depending on the severity of the insult to DNA. In summary, few DSBs trigger ATM-dependent ERK1/2 pro-survival signals whereas more DSBs result in ERK1/2 dephosphorylation consistent with a switch from pro-survival to anti-survival signaling that might affect DSBs repair.
49
Sinha, Manisha. "Recombinational Repair of a Chromosomal DNA Double Strand Break: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/412.
Анотація:
Repairing a chromosomal DNA double strand break is essential for survival and maintenance of genomic integrity of a eukaryotic organism. The eukaryotic cell has therefore evolved intricate mechanisms to counteract all sorts of genomic insults in the context of chromatin structure. Modulating chromatin structure has been crucial and integral in regulating a number of conserved repair processes along with other fundamental genomic processes like replication and transcription.
The work in this dissertation has focused on understanding the role of chromatin remodeling enzymes in the repair of a chromosomal DNA double strand break by homologous recombination. This has been approached by recapitulating the biochemical formation of recombination intermediates on chromatin in vitro. In this study, we have demonstrated that the mere packaging of DNA into nucleosomal structure does not present a barrier for successful capture of homologous DNA sequences, a central step of the biochemical pathway of recombinational repair. It is only the assembly of heterochromatin-like more complex nucleo-protein structure that presents additional constraints to this key step. And, this additional constraint can be overcome by the activities of ATP-dependent chromatin remodeling enzymes. These findings have great implications for our perception of the mechanism of the recombinational repair process of a chromosomal DNA double strand break within the eukaryotic genome.
50
Phipps, Jamie. "Cohesin and maintenance of genome integrity at DNA double-strand breaks." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL005.
Анотація:
Il est essentiel que les extrémités du DSB soient maintenues ensemble pour une réparation rapide. Chez Saccharomyces cerevisiae, deux voies mal comprises interviennent dans l'attache finale du DSB. L'un utilise le complexe Mre11-Rad50-Xrs2 (MRX) pour relier physiquement les extrémités DSB. Un autre nécessite la conversion des extrémités DSB en ADN simple brin (ssDNA) par Exo1, mais les protéines de pontage sont inconnues. Nous découvrons que la cohésine, son chargeur et Smc5/6 agissent avec Exo1 pour attacher les extrémités du DSB. Remarquablement, la cohésine spécifiquement altérée lors de l'oligomérisation ne parvient pas à attacher les extrémités du DSB, révélant une nouvelle fonction pour l'oligomérisation de la cohésine. En plus de l'importance connue de la cohésion des chromatides sœurs, des expériences microfluidiques basées sur la microscopie dévoilent un nouveau rôle de la cohésine dans la réparation en garantissant l'attache des extrémités du DSB. Globalement, nos résultats démontrent que l'oligomérisation de la cohésine empêche la séparation des extrémités du DSB et favorise la réparation du DSB, révélant ainsi un nouveau mode d'action et un nouveau rôle pour la cohésine dans la sauvegarde de l'intégrité du génomeDNA double-strand breaks (DSB) must be repaired to ensure genome stability. Crucially, DSB ends must be kept together for timely repair. In Saccharomyces cerevisiae, two poorly understood pathways mediate DSB end-tethering. One employs the Mre11-Rad50-Xrs2 (MRX) complex to physically bridge DSB ends. Another requires the conversion of DSB ends into single-strand DNA (ssDNA) by Exo1, but the bridging proteins are unknown. We uncover that cohesin, its loader and Smc5/6 act with Exo1 to tether DSB ends. Remarkably, cohesin specifically impaired in oligomerization fails to tether DSB ends, revealing a new function for cohesin oligomerization. In addition to the known importance of sister chromatid cohesion, microscopy-based microfluidic experiments unveil a new role for cohesin in repair by ensuring DSB end-tethering. Altogether, our findings demonstrate that oligomerization of cohesin prevents DSB end separation and promotes DSB repair, revealing a novel mode of action and role for cohesin in safeguarding genome integrity