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1
GOBBINI, ELISA. « A screen for synthetic phenotypes reveals new Sae2 functions and interactions in the repair of DNA double-strand breaks ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2016. http://hdl.handle.net/10281/102381.
Texte intégralRésumé :
Genome instability is one of the most pervasive characteristics of cancer cells and can be due to DNA repair defects and failure to arrest the cell cycle. Among the many types of DNA damage, the DNA double strand break (DSB) is one of the most severe, because it can cause mutations and chromosomal rearrangements. Generation of DSBs triggers a highly conserved mechanism, known as DNA damage checkpoint, which arrests the cell cycle until DSBs are repaired. DSBs can be repaired by homologous recombination (HR), which requires the DSB ends to be nucleolytically processed (resected) to generate single-strand DNA. In Saccharomyces cerevisiae, initiation of DSB resection requires the conserved MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals) that, together with Sae2 (CtIP in mammals), catalyzes an endonucleolytic cleavage of the 5’ strands. More extensive resection depends on two pathways: one catalyzed by the exonuclease Exo1, and a second requiring the nuclease Dna2 with the helicase Sgs1. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1 (ATM in humans)- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of sae2∆ cells is not known. sae2∆ cells are sensitive to the alkylating agent methyl methanesulfonate (MMS) and camptothecin (CPT), which traps covalent topoisomerase I (Top1)-DNA cleavable complexes and induces DNA replication-dependent cell death. Since this sensitivity has been shown to be due to resection defect, we searched for extragenic suppressors of the sae2∆ sensitivity to CPT and MMS. By performing a genetic screen, we identify three mutant alleles (SGS1-ss, rad53-ss and tel1-ss) that suppress both the DNA damage hypersensitivity and the resection defect of sae2∆ cells.
We show that Sgs1-ss mediated suppression depends on the Dna2 nuclease but not on Exo1. Furthermore, not only Sgs1-ss suppresses the resection defect of sae2∆ cells but it also increases resection efficiency compared to wild type cells. The checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1‐ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced.
rad53-ss and tel1-ss mutant alleles, but also the kinase defective alleles (rad53-kd and tel1-kd), suppress both the DNA damage hypersensitivity and the resection defect of sae2∆ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 cause DNA damage hypersensitivity and defective DSB resection by increasing the amount of Rad9 bound at the DSBs, which in turn inhibits the Sgs1-Dna2 resection machinery.
2
Wardrope, Laura. « Repair of double-strand DNA breaks in Escherichia coli ». Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/13208.
Texte intégralRésumé :
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.
3
Liu, Nan. « Hypersensitivity of ataxia telangiectasia cells to DNA double strand breaks ». Thesis, University of St Andrews, 1994. http://hdl.handle.net/10023/13905.
Texte intégralRésumé :
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.
4
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.
Texte intégralRésumé :
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.
5
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.
Texte intégralRésumé :
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
6
Khalil, Ashraf. « ATM-Dependent ERK Signaling in Response to DNA Double Strand Breaks ». VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd/760.
Texte intégralRésumé :
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.
7
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.
Texte intégralRésumé :
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.
8
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.
Texte intégralRésumé :
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.
9
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.
Texte intégralRésumé :
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.
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North, Matthew Howard. « The formation and repair of DNA double-strand breaks in saccaromyces cerevisiae ». Thesis, University of Sheffield, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.489352.
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Shah, Ketan. « Development of clinical biomarkers of DNA double strand breaks for cancer care ». Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:2e6b2595-fbbc-4fff-80e2-bec8a4d9d15e.
Texte intégralRésumé :
Many anticancer therapies, including radiotherapy, act by damaging the deoxyribosenucleic acid (DNA) that is fundamental to cell function and proliferation. H2AX is a histone protein associated with DNA that is phosphorylated to produce γH2AX in response to DNA double strand breaks (DSBs), the most lethal lesions caused in cancer cells. This thesis examines the translation of γH2AX detection assays to clinical situations in order to provide biomarkers of response that might help to guide the treatment of cancer patients. γH2AX immunohistochemistry was developed in preclinical xenograft models, and validated over a range of radiation doses and over time after irradiation. The method was prepared for translation to archived clinical biopsy and surgical specimens. The DSB Biomarkers Pilot Study was established in order to develop a method for γH2AX quantification in direct tumour cell specimens obtained using the clinical technique of fine needle aspiration (FNA) cytology. Eleven patients undergoing anticancer therapy were recruited to the study, and the method evaluated. The coefficient of variation of the measure was 49%. Non-invasive imaging for γH2AX would allow DNA damage to be quantified in all tumour sites, and on multiple occasions. An antibody-based nuclear medicine imaging agent was re-engineered using Fab fragments of the antibody. The novel agent demonstrated improved pharmacokinetics when compared to the whole antibody agent, but reduced target specificity. The findings further develop the potential to exploit DNA damage biomarker measurements in clinical oncology.
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Yin, Yizhi. « Resection of DNA double strand breaks in the germline of Caenorhabditis elegans ». Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/5883.
Texte intégralRésumé :
Repair of double-strand DNA breaks (DSBs) by the homologous recombination (HR) pathway results in crossovers (COs) required for a successful first meiotic division. DSB resection is the nucleic degradation of DSB ends to expose 3’ single strand DNA (ssDNA), an intermediate required for HR. To investigate genes involved in meiosis, a forward genetic screen was performed to search for novel genes or informative new mutant alleles of known genes. Mre11 is one member of the MRX/N (Mre11-Rad50-Xrs2/Nbs1) complex required for meiotic DSB formation and for resection in budding yeast. In Caenorhabditis elegans, evidence for the MRX/N’s role in DSB resection is limited. We isolated the first separation of function allele in C. elegans , mre 11(iow1), isolated from our forward genetic screen. The mre-11(iow1) mutants are specifically defective in meiotic DSB resection but not in DSB formation. The mre 11(iow1) mutants display chromosomal fragmentation and aggregation in late prophase I. Recombination intermediates and crossover formation is greatly reduced in mre 11(iow1) mutants. Irradiation induced DSBs during meiosis fail to be repaired from the early to middle prophase I in mre 11(iow1) mutants. Our data suggest that some DSBs in mre 11(iow1) mutants are repaired by the non homologous end joining (NHEJ) pathway because removing NHEJ partially suppresses some meiotic defects conferred by mre 11(iow1). In the absence of NHEJ and a functional MRX/N, meiotic DSBs are channeled to an EXO 1 dependent form of recombination repair. Overall, our analysis supports a role for MRE-11 in the resection of DSBs in early to middle meiotic prophase I and in blocking NHEJ.
A reverse genetic screen and a yeast two hybrid screen were performed to search for genes with genetic and/or physical interactions with mre-11. The reverse genetic screen isolated a novel meiotic gene, nhr-2, as a partial suppressor of the meiotic defects conferred by mre-11(iow1). The yeast two hybrid screen identified kin-18 interacting with mre-11. KIN-18 is the C. elegans homolog of mammalian Thousand And One kinase (TAO) kinase. KIN-18/TAO is MAPK kinase kinase whose meiotic role was unknown. We have found that KIN-18 is essential for normal meiotic progression as kin-18 mutants exhibit accelerated meiotic recombination, ectopic germ cell differentiation, and enhanced levels of germline apoptosis. In C.elegans MPK-1 activation in late pachytene is required for physiological apoptosis (nuclei removed by apoptosis serve as nursing cells for oocytes) and oocyte differentiation. The kin-18 mutants also showed absence of MPK-1 activation and aberrant MPK-1 activation that includes ectopic activation in the wrong regions in the germline or more than one time of activation. The progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator, KSR-2, of the canonical MPK-1 signaling. Our data suggest KIN-18 affects meiotic progression by modulating the timing of MPK-1 activation. This regulation ensures the proper timing of recombination and normal apoptosis, which is required for the formation of functional oocytes. Meiosis is a conserved process; revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans will motivate hypothesis for TAO kinase’s role in the germline development in higher eukaryotes.
Meiosis is a crucial for sexually reproducing organisms to maintain ploidy level from one generation to the next. Accurate chromosome segregation in the meiosis requires meiotic recombination between homologous chromosomes. Failure in recombination can lead to abnormal segregation of chromosomes in meiosis, which leads to aneuploidy. Anueploidy is a leading cause of miscarriages and attributes to chromosomal related birth defects. Meiotic recombination starts with programmed DNA double strand breaks (DSBs), followed by repair of these DSBs by homologous recombination (HR) pathway. One key step in HR is resection, a process to covert DSB ends into single strand DNA (ssDNA). To broaden our understanding of meiotic DSB resection, we used a nematode, C. elegans, as a model to investigate genes in DSB resection. We have isolated a specific mutant allele of a meiotic gene, mre-11. Our data suggest meiotic DSB resection in C. elegans requires collaboration of mre-11 and another gene exo-1; efficient resection of DSB ends is important to safeguard repair of DSB by HR against other illegitimate repair pathway. In addition, we identified a gene kin-18 by looking for genes interacting with mre-11. Characterization of kin-18 show meiotic recombination is tightly coordinated with germ cell progression. Our analysis provides significant improvement in the understanding of meiotic recombination in C. elegans. Given the high conservation of the two genes, mre-11 and kin-18, our finding may be applied to other organisms.
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DI, VIRGILIO MICHELA. « The MRN complex and the cellular response to DNA double-strand breaks ». Doctoral thesis, Università degli Studi di Milano, 2006. http://hdl.handle.net/2434/59929.
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The MRN complex and the cellular response to DNA double-strand breaks The Mre11/Rad50/Nbs1 (MRN) complex is a multisubunit nuclease that plays a crucial role in DNA double-strand break (DSB) repair and ATM (Ataxia-Telangiectasia mutated)-mediated checkpoint signaling. The first part of this thesis addresses the controversy over MRN complex involvement in the non-homologous end-joining (NHEJ) DSB repair pathway by the use of a plasmid DSB repair assay in Xenopus laevis cell-free extracts. Mre11-depleted extracts are able to support efficient NHEJ repair of DSBs, regardless of the end-structure. Mre11-depletion does not alter the kinetics of end-joining or the type and frequency of junctions found in repaired products. Finally, Ku70-independent end-joining events are not affected by Mre11 loss. Our data demonstrate that the MRN complex is not required for efficient and accurate NHEJ-mediated repair of DSBs in this vertebrate system. In the second part, I discuss the techniques, molecular tools and preliminary results of the characterization of the molecular mechanisms underlying the involvement of the MRN-ATM pathway in the maintenance of genome integrity during DNA replication.
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D'ALESSANDRO, GIUSEPPINA. « THE ROLE OF RNA AND DNA:RNA HYBRIDS AT DNA DOUBLE-STRAND BREAKS ». Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/562552.
Texte intégralRésumé :
The stability of our genome is constantly challenged by several genotoxic threats. DNA
double-strand breaks (DSBs) are the most dangerous DNA lesions that, if not repaired, can
lead to cancer initiation and progression and/or ageing. These detrimental consequences can
only be avoided if cells promptly recognize the lesions and signal their presence, thus
promoting either efficient repair and transient cell cycle arrest or cell death and cellular
senescence. This is the role of the DNA damage response (DDR) proteins and the newly
identified damage-induced non coding RNAs. We recently discovered that RNA polymerase
II is recruited to DSBs and synthetizes damage-induced non-coding RNAs (dilncRNAs).
DROSHA- and DICER-mediated processing of dilncRNAs generates small RNA species,
named DNA damage response RNA (DDRNAs) (Francia, 2012), that localize to DSBs via
pairing with dilncRNAs and promote DDR signaling (Michelini et al., in press). Similar
small non-coding RNA species discovered in plants are involved in DNA repair by
homologous recombination (HR) (Wei, 2012, Gao, 2014, Wang, 2016). In line with these
results, I report that transcriptional inhibition impairs recruitment of the HR proteins
BRCA1, BRCA2, and RAD51 to DSBs, while partially promoting DNA end resection.
Moreover, I show DNA:RNA hybrids accumulation at DSBs in mammalian cells by both
DRIP analyses and imaging techniques. Damage-induced DNA:RNA hybrids form upon the
hybridization of RNA species, likely dilncRNAs, to the resected DSBs DNA ends generated
during the S/G2 cell cycle phase. I also report that purified recombinant BRCA1 binds
DNA:RNA hybrids in vitro; moreover, DNA:RNA hybrids in vivo contribute to BRCA1
recruitment to DSBs. Consistent with the need to tightly regulate DNA:RNA hybrid levels,
I demonstrate that RNase H2, the major RNase H activity in mammalian nuclei, is recruited
to DSBs through direct interaction with RAD51. In summary, I report for the first time that
DNA:RNA hybrids accumulate at DSBs in mammalian cells in a cell-cycle- and DNA end
resection-depended way. At DSBs, BRCA1 directly recognizes DNA:RNA hybrids and
likely controls their turn-over by mediating the recruitment of RNase H2 via RAD51.
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Lemaître, Charlène. « Nuclear architecture and DNA repair : double-strand breaks repair at the nuclear periphery ». Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ079/document.
Texte intégralRésumé :
L'ADN peut être endommagé par des facteurs environnementaux ou intrinsèques au fonctionnement des cellules. Ces facteurs induisent différents types de lésions dont les cassures double brins (CDBs). Les CDBs sont particulièrement dangereuses pour les cellules et une réparation inefficace ou non précise de ces cassures peut entraîner des mutations ou des translocations qui peuvent être à l'origine de cancer. Afin d'éviter l'instabilité génétique que peuvent induire les CDBs, les cellules ont développé deux principaux mécanismes de réparation: la ligature d'extrémités non homologues (NHEJ pour non homologous end joining) et la recombinaison homologue (HR pour homologous recombination). L’utilisation de l’un ou de l’autre de ces mécanismes est finement régulée et une dérégulation de cet équilibre induit une importante instabilité génomique.Tous ces mécanismes ont lieu dans le noyau des cellules qui, chez les mammifères est fortement hétérogène, comportant différents compartiments et des régions où la chromatine est plus ou moins compacte. Cette hétérogénéité implique que la réparation de l’ADN doit pouvoir être efficace dans différents contextes nucléaires. Au cours de ma thèse, j’ai étudié l’influence de l’architecture nucléaire sur le choix des mécanismes de réparation des CDBs. J’ai montré d’une part que la protéine appartenant au pore nucléaire Nup153 influence l’équilibre entre HR et NHEJ et d’autre part que la position d’une CDB influe sur le choix du mécanisme de réparation.Mes résultats démontrent que l’organisation des gènes dans le noyau est un nouveau paramètre à prendre en compte dans l’étude des mécanismes de réparation de l’ADN et de tumorigénèseDNA is constantly assaulted by various damaging agents, leading to different types of lesions including double-strand breaks (DSBs). DSBs are the most harmful lesions to the cells and their inaccurate or inefficient repair can trigger genomic instability and tumorigenesis. To cope with DSBs, cells evolved several repair pathways, including non-homologous end joining (NHEJ) and homologous recombination (HR). A fine regulation of the balance between these two pathways is necessary to avoid genomic instability.All of these mechanisms happen in the nucleus, which is highly heterogeneous in mammalian cells. Indeed, it encompasses several compartments and regions of various chromatin compaction levels. My PhD project focused on the influence of nuclear architecture on DNA repair pathway choice. I demonstrated on one hand that the nuclear pore protein Nup153 influences the balance between HR and NHEJ and on the other hand that the position of a DSB influences the choice of the repair pathway that will be used.My results demonstrate that gene positioning is a new important parameter in the study of DNA repair and tumorigenesis
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Choudhury, Ananya. « Molecular epidemiology of DNA damage response to double strand breaks in bladder cancer ». Thesis, University of Leeds, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445944.
Texte intégral
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Riches, Lucy C. « Investigating DNA Double Strand Breaks (DSB) in mammalian cells by novel fluorescent reporters ». Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/1363.
Texte intégralRésumé :
An efficient DNA damage response is critical for maintaining the integrity of the mammalian genome, and ensuring the accurate transfer of genetic information between generations. Of particular biological relevance are DNA double strand breaks (DSB), which if repaired incorrectly may contribute to carcinogenesis. Review of contemporary literature has led to the identification of protein interactions and transcriptional events, tightly associated with the mammalian DSB response. Characteristics of selected events have been manipulated, with the notion of developing a reporter system that offers a sensitive and rapid method of detecting DSB in living mammalian cell models. Work presented here provides a quantitative evaluation of DSB generation in various mammalian cell lines, following chemical and irradiation treatment, and highlights the limitations of currently used markers. A series of recombinant proteins comprising peptide interacting domains, which exhibit altered spatio-temporal dynamics in relation with each other following DSB induction, are proposed as potential reporters of damage in mammalian cells. Novel gene constructs have been engineered that encode these peptide interacting domains, sandwiched between fluorescence-resonance-energy transfer (FRET) capable proteins. DSB specific events are predicted to induce peptide interactions that may be tracked in real time, by monitoring alterations in the fluorescent properties of such a recombinant protein. In an alternative approach, the transcriptional up-regulation of RAD52 mRNA following DSB induction was extended to whole cells. Optimisation of a fluorescent molecular beacon probe complementary to mammalian RAD52 mRNA is described, and data obtained in mammalian cells following DSB induction supports the notion that RAD52 is actively transcribed as part of the DSB response.
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Tseng, Tzu-Ling. « Factors influencing the processing of VDE-induced DNA double-strand breaks during meiosis ». Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/8516/.
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Mason, Rebecca Mary Aglaia. « The rejoining of non-homologous DNA double-strand breaks by mammalian cell free extracts ». Thesis, University of Sussex, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336323.
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Traver, Brenna E. « Exogenously-introduced Homing Endonucleases Catalyze Double-stranded DNA Breaks in Aedes aegypti ». Thesis, Virginia Tech, 2009. http://hdl.handle.net/10919/40967.
Texte intégralRésumé :
Aedes aegypti transmits the viruses which cause yellow fever, dengue fever, and dengue hemorrhagic fever. Homing endonucleases are selfish genetic elements which introduce double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this study, we aimed to validate a somatic assay to detect recombinant homing endonuclease (rHE)-induced dsDNA breaks in both cultured cells and adult female Ae. aegypti. While the cell culture-based two plasmid assay used to test rHE ability to induce dsDNA breaks was inconclusive, assays used to test rHEs in Ae. aegypti were successful. Recognition sequences for various rHEs were introduced into Ae. aegypti through germline transformation, and imperfect repair at each of these exogenous sites was evaluated. In mosquitoes containing a single exogenous HE site, imperfect gap repair was detected in 40% and 21% of clones sequenced from mosquitoes exposed to I-PpoI and Iâ SceI, respectively. In mosquitoes containing two exogenous HE sites flanking a marker gene (EGFP), 100% of clones sequenced from mosquitoes exposed to I-PpoI, I-CreI, and I-AniI demonstrated excision of EGFP. No evidence of EGFP excision or imperfect repair at any HE recognition site was detected in mosquitoes not exposed to a rHE. In summary, a somatic genomic footprint assay was developed and validated to detect rHE or other meganuclease-induced site-specific dsDNA breaks in chromosomal DNA in Ae. aegypti.Master of Science in Life Sciences
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Karlsson, Karin. « Role of Non-Homologous End-Joining in Repair of Radiation-Induced DNA Double-Strand Breaks ». Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7219.
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Xie, Hong. « Misrepair of Particulate Chromium(VI)-Induced DNA Double Strand Breaks Leads to Neoplastic Transformation ». Fogler Library, University of Maine, 2007. http://www.library.umaine.edu/theses/pdf/XieH2007.pdf.
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Dobbs, Tracey. « An investigation into the repair of complex double-strand dna breaks using biochemical approach ». Thesis, University of Reading, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.500521.
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du, Plessis David James Philip. « The roles of ABRA1 and ABRO1 in the response to DNA double strand breaks ». Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610459.
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Akagawa, Remi. « UBC13-Mediated Ubiquitin Signaling Promotes Removal of Blocking Adducts from DNA Double-Strand Breaks ». Kyoto University, 2020. http://hdl.handle.net/2433/258998.
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付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラムKyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第22730号
医博第4648号
新制||医||1046(附属図書館)
京都大学大学院医学研究科医学専攻
(主査)教授 遊佐 宏介, 教授 溝脇 尚志, 教授 篠原 隆司
学位規則第4条第1項該当
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Ariyoshi, Kentaro. « Increased chromosome instability and accumulation of DNA double-strand breaks in Werner syndrome cells ». Kyoto University, 2007. http://hdl.handle.net/2433/135762.
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GALBIATI, ALESSANDRO. « NEW APPROACHES TO STUDY DNA DOUBLE-STRAND BREAKS GENOME-WIDE AND IN SINGLE-CELLS ». Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/562490.
Texte intégralRésumé :
Cells have evolved several DNA repair mechanisms to maintain their genetic information unaltered and a DNA damage response pathway (DDR) coordinates DNA repair with several cellular events including a cell-cycle arrest until damaged DNA is repaired. When cells fail to repair DNA lesions, they undergo either apoptosis or cellular senescence. Tools commonly used to detect DNA lesions rely on indirect, antibody-based recognition of proteins associated to DNA breaks. Unfortunately, these tools do not allow direct and precise localization of the breaks, leaving several biological questions unanswered. I validated and optimized BLESS and BLISS, two methods that allow genome-wide single-nucleotide resolution mapping of DNA double strand-breaks (DSBs). Using these techniques, I studied the impact of DSBs on transcription. I characterized a DDR-dependent transcription inhibition around breaks. Differently, I observed that following macrophages LPS-stimulation, a transient wave of DSBs is induced at LPS-specific enhancers and it correlates with their transcription activation, thus suggesting a new mechanism for transcription activation involving controlled DNA damage generation.
BLESS and BLISS cannot be applied to single-cell studies. Thus, I developed a new method, named DI-PLA, for the detection and imaging of DSBs in fixed cells and tissues. I applied DI-PLA to demonstrate that senescence cells and aged tissues accumulate DSBs, which are associated with persistent DDR activation, that is known to fuel cellular senescence.
Finally, I developed a modification of BLESS to discriminate between DSB bearing telomeres and deprotected telomeres which could be applied to further characterize the mechanisms of DDR activation at telomeres.
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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.
Texte intégralRésumé :
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.
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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.
Texte intégralRésumé :
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.
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Stephanou, Nicolas Constantinos. « Mycobacterial non-homologous end-joining : molecular mechanisms and components of a novel DNA double strand break repair pathway / ». Access full-text from WCMC, 2008. http://proquest.umi.com/pqdweb?did=1528973431&sid=21&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Tran, Amy V. « Do BHA and BHT Induce Morphological Changes and DNA Double-Strand Breaks in Schizosaccharomyces pombe ? » Scholarship @ Claremont, 2013. http://scholarship.claremont.edu/scripps_theses/152.
Texte intégralRésumé :
Butylated Hydroxyanisole, BHA, and Butylated Hydroxytoluene, BHT, are commonly used as preservatives for our food as well as additives in many products such as cosmetics, petroleum, and medicine. Although their use has been approved by the Food and Drug Administration (FDA), there have been controversies and debates on whether these phenol derivatives or antioxidants are safe to use. Their accumulative toxicology and side effects need to be thoroughly investigated as we continue to consume them on a daily basis. Data obtained by genomic analysis in Tang lab suggested the involvement of DNA damage checkpoint/repair pathways in the response network to these phenol stress factors. The aims of this thesis are to examine the morphological changes and potential DNA damage induced by exposing cells to BHA and BHT using fission yeast Schizosaccharomyces pombe as a model organism. Fluorescence microscopy was used to assess DNA double-strain breaks (DSBs) by monitoring the nuclear foci formation of Rad22, a DNA repair protein, in the presence of BHA and BHT. Changes in cell morphology were also studied under microscope. Preliminary data showed that cells treated with BHA and BHT exhibited morphological changes. In addition, for the first time in S. pombe cells, Rad22 foci in the nucleus of BHA and BHT treated cells were observed. Further investigation is needed to optimal the experimental condition to continue the study. These results will not only help us to better understand the effect of these phenol derivatives in the cells, but can also establish an experimental system for future studies on the interaction of the cells with stress factors and therapeutic drugs for human-related diseases such as cancer.
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Lewis, Todd Warren. « A novel system for detection of DNA double strand breaks and repair in human cells ». Wright State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=wright1496097422996588.
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Kalliontzi, Eugenia. « Effects of acetylation and deacetylation inhibitors on DNA double strand breaks repair and cell survival ». Thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-366545.
Texte intégralRésumé :
During treatment with ionizing radiation (IR), cells are killed mainly due to ionizing radiation caused acute damage to DNA, such as double-strand breaks, where opposite DNA strands in close proximity are cut. To counteract toxicity of double-strand breaks, cells have evolved DNA damage repair systems such as homologous recombination and non-homologous end joining. An important factor regulating DNA repair after exposure to ionizing radiation is chromatin structure. Interfering with normal functions of chromatin remodelling results in reduced cell survival as seen with histone deacetylase (HDAC) inhibitors such as trichostatin A (TSA) which exhibits antitumoral effects in various cancers. Furthermore, TSA interferes with functions of DNA repair proteins similarly as histone acetyltransferase inhibitors, such as C646, however, the effect of these inhibitors on DNA repair is not fully characterized. In this study, DNA damage repair after exposure to TSA and C646 in combination with ionizing radiation was assessed and the results indicated that both TSA and C646 suppressed the survival and proliferation of cancer cells in a dose-dependent manner. When combined with radiation, clonogenic assay shows that C646 radiosensitized HCT116 cells. As measured with flow cytometry, the drugs had no significant effect on the H3K9ac and H3K9me3 histone modifications over the time course of 24 hours. Futhermore, TSA and C646 inhibited ionizing radiation induced foci (IRIF) formation of 53BP1but had no effects on the ability to repair radiation-induced DSBs.
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CASARI, ERIKA. « Resection of DNA double-strand breaks : novel regulatory mechanisms by checkpoint proteins and chromatin remodelers ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/365496.
Texte intégralRésumé :
L'instabilità genomica è una delle caratteristiche delle cellule tumorali e può essere dovuta a difetti nella riparazione dei danni al DNA. Tra le differenti tipologie di danno al DNA, le rotture della doppia elica di DNA sono lesioni citotossiche che devono essere riparate per garantire stabilità genomica ed evitare la morte cellulare. Le cellule eucariotiche affrontano questi danni attivando una risposta con differenti vie molecolari. Tra esse il meccanismo NHEJ lega direttamente le estremità rotte del DNA. Oppure il meccanismo HR utilizza il cromatidio fratello/il cromosoma omologo come templato per riparare la rottura. HR è avviata dalla degradazione nucleolitica (resection) delle estremità 5' del DSB. La resection è un processo a due fasi: la prima fase, short-range, è catalizzata dal complesso MRX/MRN che, insieme a Sae2/CtIP catalizza un taglio endonucleolitico alle estremità 5’ del DNA rotto. Dopo di che, la seconda fase, long-range, prevede l’intervento delle nucleasi Exo1 e Dna2/Sgs1. Esse sono necessarie per generare code di 3’ssDNA. In seguito a una rottura della doppia elica di DNA, le cellule attivano anche un’altra via chiamata checkpoint da danno al DNA, che coordina la riparazione del danno con la progressione del ciclo cellulare. Tra i principali attori del checkpoint ci sono le chinasi Mec1/ATR e Tel1/ATM. Tel1 riconosce le rotture del doppio filamento di DNA non processate, mentre Mec1 è attivato dal DNA a singolo filamento, prodotto dal processo di resection. Una volta stimolate, queste due chinasi apicali attivano per fosforilazione le chinasi effettrici Rad53/CHK2 e Chk1/CHK1. Questa attivazione richiede anche la proteina conservata Rad9/53BP1 la cui associazione al DNA coinvolge diverse vie. Resta da determinare come la short-range resection sia regolata e contribuisca all'attivazione del checkpoint. In questa tesi, ho contribuito a dimostrare che l’inibizione della long-range resection induce una risposta di checkpoint che dipende dal complesso di checkpoint 9-1-1, che recluta Rad9 al DNA danneggiato. Inoltre, il complesso 9-1-1, indipendentemente da Rad9, limita la short-range resection regolando negativamente la nucleasi di MRX. La riparazione dei danni della doppia elica di DNA coinvolge anche la cromatina. Infatti, i genomi eucariotici sono compattati in una struttura cromatinica che limita l'accesso al DNA agli enzimi dedicati alla riparazione dei danni e solleva la questione di come avvenga la resection in tale contesto. È noto che la cromatina che affianca una rottura della doppia elica di DNA subisce ampie modificazioni da parte di una serie di rimodellatori della cromatina. Pertanto, nella seconda parte di questa tesi, ho studiato il ruolo della proteina di rimodellamento della cromatina Dpb4 nella riparazione dei danni. Nel lievito gemmante, la proteina conservata Dpb4 presenta un dominio istonico ed è condivisa da due complessi proteici: il rimodellatore della cromatina ISW2 e l'oloenzima DNA Pol epsilon. In S. cerevisiae, Dpb4 interagisce con Dls1 nel complesso ISW2 e con Dpb3 nel complesso Pol epsilon. In questa tesi ho dimostrato che Dpb4 promuove la rimozione degli istoni e la resection interagendo con Dls1 per facilitare l'associazione dell'ATPasi Isw2 al DNA danneggiato. Inoltre, promuove l'attivazione del checkpoint interagendo con Dpb3 per facilitare l'associazione di Rad9. Nell'ultima parte della tesi, per comprendere meglio il legame tra il rimodellamento della cromatina e la resection dei danni al DNA, ho contribuito a studiare il ruolo del rimodellatore della cromatina Chd1, frequentemente mutato nel cancro alla prostata. Abbiamo dimostrato che Chd1 partecipa in entrambe le fasi di resection, promuovendo l'associazione di MRX ed Exo1 alle estremità di una rottura del DNA. Inoltre, Chd1 consente la rimozione degli istoni vicino alle estremità del DNA danneggiato promuovendone la riparazione con il meccanismo di HR.Genome instability is one of the hallmarks of cancer cells and can be due to DNA repair defects. Among different types of DNA damage, DNA DSBs are cytotoxic lesions that must be repaired to ensure genomic stability and avoid cell death. Eukaryotic cells deal with DSBs by activating the DNA damage response, that comprises pathways devoted to repair DNA breaks. DSBs can be repaired by NHEJ, which directly ligates the broken DNA ends, or by HR, which uses sister chromatids/homologous chromosomes as a template to repair the DNA break. HR is initiated by nucleolytic degradation (resection) of the 5’-terminated strands at both DSB ends. DSB resection is a two-step process, in which an initial short-range step is catalyzed by Mre11-Rad50-Xrs2/NBS1 (MRX/MRN) complex that, together with Sae2 (CtIP in mammals), catalyzes an endonucleolytic cleavage of the 5’strands. Then, a long-range resection step is carried out by the nucleases Exo1 and Dna2/Sgs1 to generate long 3’ssDNA tails. The DDR comprises also surveillance mechanisms, called DNA damage checkpoint, that couple DSB repair with cell cycle progression. Major checkpoint players include the apical protein kinases Mec1/ATR and Tel1/ATM. Tel1 recognizes unprocessed DSBs, while Mec1 is activated by RPA-coated ssDNA that is generated during the resection process. Once activated, these protein kinases activate by phosphorylation the effector kinases Rad53/CHK2 and Chk1/CHK1. This activation requires the conserved adaptor protein Rad9/53BP1, whose association to chromatin involves multiple pathways. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. In this thesis, I contributed to show that abrogation of long-range resection induces a checkpoint response that depends on the checkpoint complex 9-1-1, which recruits Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated. Repair of DSBs occurs in a chromatin context. In fact, eukaryotic genomes are compacted into chromatin, which restricts the access to DNA of the enzymes devoted to repair DNA DSBs and raises the question as to how DNA end resection occurs in the context of chromatin. For this reason, chromatin near DSBs undergoes extensive modifications by a series of conserved chromatin remodelers that are recruited to DNA DSBs. Thus, given the importance of chromatin remodeling in DSB repair, in the second part of this thesis, I investigated the role of the chromatin remodeling protein Dpb4 in DSB repair. Budding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase epsilon holoenzyme. In S. cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol epsilon complex. I showed that Dpb4 plays two functions in sensing and processing DSBs. It promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. In the last part of my thesis, to better understand the link between chromatin remodeling and DNA end resection, I contributed to examine the role in DSB repair of the S. cerevisiae chromatin remodeler Chd1, whose human counterpart is frequently mutated in prostate cancer. We showed that Chd1 participates in both short- and long- range resection by promoting the association of MRX and Exo1 to the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity.
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Kawale, Ajinkya S. « PROCESSING OF 3′-BLOCKED DNA DOUBLE-STRAND BREAKS BY TYROSYL-DNA PHOSPHODIESTERASE 1, ARTEMIS AND POLYNUCLEOTIDE KINASE/ PHOSPHATASE ». VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5329.
Texte intégralRésumé :
DNA double-strand breaks (DSBs) containing unligatable termini are potent cytotoxic lesions leading to growth arrest or cell death. The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3′-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3′-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. Moreover, Polynucleotide Kinase/ Phosphatase (PNKP) is known to process 3′-phosphates and 5′-hydroxyls during DSB repair. PNKP-deficiency sensitized both HCT116 and HeLa cells to 3′-phosphate ended DSBs formed upon radiation and radiomimetic drug treatment. The increased cytotoxicity in the absence of PNKP was synonymous with persistent, un-rejoined 3′-phosphate-ended DSBs. However, DNA-PK deficiency sensitized PNKP-/- cells to low doses of NCS suggesting that, in the absence of PNKP, alternative enzyme(s) can remove 3′-phosphates in a DNA-PK-dependent manner. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining. In addition, loss of PNKP significantly sensitizes cells to 3′-phosphate-ended DSBs due to a defect in 3′-dephosphorylation.
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Tay, Ian Jun Jie. « A novel DNA damage quantification platform enables high throughput screening for genes that impact DNA double strand breaks ». Thesis, Massachusetts Institute of Technology, 2018. https://hdl.handle.net/1721.1/122836.
Texte intégralRésumé :
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018Cataloged from PDF version of thesis.
Includes bibliographical references.
DNA is the blueprint of life, and the high fidelity transmission of genetic information from parent cells to progeny is essential for an organism's viability. However, our genomes are constantly being damaged by reactive molecules generated from cellular metabolic processes or introduced from the environment. The resulting DNA damage can alter the information encoded in DNA, and can interfere with the accurate transmission of genetic information when cells divide. The accumulation of cells with highly damaged or altered DNA within an organism can cause diseases, such as growth defects, aging and cancer. Fortunately, cells possess the capability to repair damaged DNA. Since DNA repair mechanisms can reverse the deleterious effects of DNA damage, they are important in disease prevention, and in particular play an important role in preventing cancer. DNA repair factors are also important targets for cancer therapies.
Tumor cells frequently harbor defects in DNA repair, rendering them vulnerable to DNA damage. Many cancer therapies exploit this vulnerability by treatment with DNA damaging agents. However, tumor cells can have differential DNA repair capacities based on the expression levels of various DNA repair genes. Thus, some cancer cells are variable in their response to chemotherapy and radiation. It is well established that inhibiting DNA repair can increase the efficacy of treatment. Therefore, it is critical to develop a better understanding of the network of genes that regulate DNA repair mechanisms both to understand susceptibility to cancer, and also in order to improve the outcomes of cancer therapy. DNA repair is a complex process that requires the coordination of many proteins to respond to specific classes of DNA damage. Many of the major proteins that directly participate in DNA repair pathways are well characterized.
However, recent research has indicated that the core DNA repair factors make up only a small fraction of the proteins that respond to DNA damage, suggesting that a large number of novel DNA repair factors have yet to be discovered and characterized. In this work, we leveraged the CometChip, a high-throughput DNA damage quantification assay, to screen thousands of genes for their ability to modulate DNA repair, by knocking them down with shRNAs. We first designed hardware for the CometChip to make it compatible with high-throughput robotics so as to reduce the amount of manual labor needed to execute our screen. We then exploited the ability of our assay to measure DNA damage at an unparalleled rate to screen an shRNA library targeting 2564 oncology-associated genes. We performed gene network analysis on the top candidate genes and found LATS2 to be a novel DNA repair factor. Further investigation revealed that LATS2 is a modulator of the homologous recombination repair pathway.
In addition, we merged our screen data with that from an assay that queries proteins for their ability to bind to DNA double strand breaks. Our results showed that we were able to identify known DNA repair factors via the intersection of the two datasets, and we pinpointed at least one other novel DNA repair gene for further investigation. Taken together, this work represents an advancement in the ability to discover novel DNA repair factors by large-scale parallel measurement of physical DNA damage in cells. Our technology enables high-throughput screening for DNA damage and repair factors faster than ever before, allowing for extensive studies of DNA damage and opening doors to the discovery of new genes and molecules that affect DNA repair.
by Ian Jun Jie Tay.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering
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Hall, Mathew. « Investigation of a mutant Indian muntjac cell line defective in the processing of DNA double strand breaks ». Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338322.
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Zimmermann, Philipp Konstantin [Verfasser], et Christof von [Akademischer Betreuer] Kalle. « Genome‐wide detection of induced DNA double strand breaks / Philipp Konstantin Zimmermann ; Betreuer : Christof von Kalle ». Heidelberg : Universitätsbibliothek Heidelberg, 2016. http://d-nb.info/118073517X/34.
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Hawley, Benjamin Richard. « Investigating the role of Drosha and RNA molecules in the repair of DNA double-strand breaks ». Thesis, University of Leicester, 2018. http://hdl.handle.net/2381/40976.
Texte intégralRésumé :
The error‐free and efficient repair of DNA double‐strand breaks is extremely important for cell survival. Over the past several years, RNA has been increasingly implicated in the DNA damage response, but the mechanism or mechanisms through which it can act remain poorly understood. Various hypotheses have been made in the literature: RNA as a signalling molecule or as a scaffold; RNA long‐range interactions to reorganise or maintain chromatin structure; RNA as a template for repair. In this thesis, the miRNA biogenesis apparatus was demonstrated to be involved in DNA repair outside of their normal roles in miRNA maturation. Drosha was required for efficient repair by the two major repair pathways, suggesting a central role in the DNA damage response. Previous reports looking at the involvement of Drosha in DNA damage had suggested that Drosha processes newly transcribed RNA, the product of which would carry out a role in repair. Here, a comprehensive series of nextgeneration sequencing approaches was unable to validate this. Later experiments instead discovered that pre‐existing RNA molecules could participate in repair. RNA invasion around DNA break sites was observed, and shown to be Drosha‐dependent. These damage‐induced DNA:RNA hybrid structures were important for efficient repair, showing that RNA can be a direct and critical mediator of DNA damage repair.
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Amarh, Vincent. « Visualization of replication-dependent DNA double-strand break repair in Escherichia coli ». Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/29596.
Texte intégralRésumé :
Chromosomal replication is a source of spontaneous DNA double-strand breaks (DSBs). In E. coli, DSBs are repaired by homologous recombination using an undamaged sister template. During repair, the RecA protein polymerizes on single-stranded DNA generated at the site of the DSB and catalyses the search for sequence homologies on the undamaged sister template. This study utilized fluorescence microscopy to investigate the spatial and temporal dynamics of the RecA protein at the site of a replication-dependent DSB generated at the lacZ locus of the E. coli chromosome. The DSB was generated by SbcCD-mediated cleavage of a hairpin DNA structure formed on the lagging strand template of the replication fork by a long palindromic sequence. The tandem insertion of a recA-mCherry gene with the endogenous recA gene at the natural chromosomal locus produced no detectable effect on cell viability in the presence of DSB formation. During repair, the fluorescently-labelled RecA protein formed a transient focus, which was inferred to be the RecA nucleoprotein filament at the site of the replication-dependent DSB. The duration of the RecA focus at the site of the DSB was modestly reduced in a ΔdinI mutant and modestly increased in a ΔuvrD or ΔrecX mutant. Most cells underwent a period of extended cohesion of the sister lacZ loci after disappearance of the RecA focus. Segregation of the sister lacZ loci was followed by cell division, with each daughter cell obtaining a copy of the fluorescently-labelled lacZ locus. The RecA focus at the site of the DSB was observed predominantly between the mid-cell and the 1⁄4 position. In the absence of DSB formation, the lacZ locus exhibited dynamic movement between the mid-cell and the 1⁄4 position until the onset of segregation. Formation of the DSB and initiation of repair occurred at the spatial localization for replication of the lacZ locus while the downstream repair events occurred very close to the mid-cell. Genomic analysis of RecA-DNA interactions by ChIP-seq was used to demonstrate that the RecA focus at the lacZ locus was generated by the repair of the palindrome-induced DSB and not the repair of one-ended DSBs emanating from stalled replication forks at the repressor-bound operator arrays. This study has shown that the repair of a replication-dependent DSB occurs exclusively during the period of cohesion of the sister loci and the repair is efficiently completed prior to segregation of the two sister loci.
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Mitchell, Jody. « DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP-1) function in response to DNA double strand breaks ». Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.519482.
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Attia, Atef Mahmoud Mahmoud. « Induction of DNA double-strand breaks at various stages of the cell cycle using the comet assay ». [S.l.] : [s.n.], 2002. http://webdoc.sub.gwdg.de/diss/2002/attia/attia.pdf.
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Wang, Jinglong. « Insights into the mechanism of DNA double-strand breaks and classic NHEJ by single-molecule magnetic tweezers ». Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7063.
Texte intégralRésumé :
La réparation des coupures de double brin (DSB) de l'ADN par une jonction d'extrémité non homologue (NHEJ) nécessite de multiples protéines pour reconnaître et lier les extrémités de l'ADN, les traiter pour des raisons de compatibilité et les lier ensemble. Nous avons construit de nouveaux substrats d'ADN pour la nano-manipulation à une seule molécule nous permettant de détecter, de sonder et de rompre mécaniquement la synapsis du DSB en temps réel par des composants spécifiques du NHEJ humain. DNA-PKcs et Ku permettent la synapsis des extrémités de l'ADN à une échelle de temps inférieure à la seconde, et l'ajout de PAXX étend cette durée de vie à ~ 2 secondes. Une addition supplémentaire de XRCC4, XLF et Ligase IV a entraîné une synapsis à l'échelle minute et conduit à une réparation robuste des deux brins de l'ADN nanomanipulé. Contrairement à PAXX, un long ARN non codant LINP1 peut également aider la DNA-PK à attacher ensemble les extrémités de l'ADN, ce qui pourrait être plus nécessaire lorsque les extrémités de l'ADN se séparent. De plus, nous interrogeons également le système bactérien Bacillus subtilis NHEJ, qui vient de composer Ku et la ligase D, la bactérie Ku peut également lier l’ADN et introduire la Ligase D renforcer la synapsis et conduire à la ligature. La contribution énergétique des différents composants à la stabilité synaptique est généralement faible, à l’échelle de quelques kCal / mol. Nos résultats combinés définissent les règles d’assemblage des machines NHEJ et révèlent l’importance des interactions faibles, rapidement rompues même sous des forces sous-picoNewton, dans la régulation de ce système chimico-mécanique à plusieurs composants pour l’intégrité du génome. De plus, nous identifions également un nouveau modèle de clivage à double brin d’ADN régulé par Cas9 PAM. En résumé, ce travail de thèse porte sur la génération DSB et le processus détaillé allant de la connexion de l’ADN à la ligatureRepairing DNA double-strand breaks (DSBs) by non-homologous end-joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nano-manipulation allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the sub second timescale, and addition of PAXX extends this lifetime to ~2s. Further addition of XRCC4, XLF and Ligase IV resulted in minute-scale synapsis and led to robust repair of both strands of the nanomanipulated DNA. In contrast with PAXX, a long non-coding RNA LINP1 can also help DNA-PK to tether DNA ends together, which could be more required when DNA ends falling apart in distance. Moreover, we also interrogate the bacteria Bacillus subtilis NHEJ system, which just composed Ku and Ligase D, bacteria Ku also can tether DNA together and introducing the Ligase D strengthen the synapsis and lead to ligation. The energetic contribution of the different components to synaptic stability is typically small, on the scale of a few kCal/mol. Our combined results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity. Moreover, we also identify a novel the DNA double strand cleavage pattern regulated by Cas9 PAM. In sum, this PhD work investigates the DSB generation and the detailed process from DNA end tethering to ligation
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Yuan, Ying. « Modulation of DNA double strand breaks end-joining pathway choice by single stranded oligonucleotides in mammalian cells ». Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30091.
Texte intégralRésumé :
En réponse aux dommages de son génome, le choix par la cellule de la voie de réparation de l'ADN est un crucial par ses conséquences en termes de mutagénèse et de survie. Pour faire face aux cassures double-brin de l'ADN (CDB), les cellules humaines possèdent deux voies principales qui consistent soit à rejoindre les extrémités de la cassure par jonction d'extrémités non-homologues (voie conventionnelle C-NHEJ), soit à reconstituer par recombinaison homologue la séquence clivée en copiant son double non endommagé présent après la réplication (voie RH). La RH nécessite de dégrader l'un des brins d'ADN de part et d'autre de la cassure. Cette dégradation produit de courts fragments d'ADN simple-brin, connus pour aider à signaler le dommage à la cellule. Dans ce travail, nous avons évalué directement l'effet de ces fragments d'ADN simple brin sur la réparation des CDB dans des expériences biochimiques et cellulaires. Nous montrons que de courts fragments d'ADN simple-brin inhibent la C-NHEJ en inactivant sa protéine clef Ku, tout en stimulant une forme minoritaire de jonction des cassures dite NHEJ alternative (A-EJ). Ces travaux permettent de mieux comprendre comment la réparation par la voie peu connue A-EJ peut s'exprimer dans les cellules mais aussi d'envisager des stratégies pour piloter la réponse des cellules cancéreuses aux thérapies induisant des CDBIn response to DNA damage, the choice made by the cells between DNA repair mechanisms is crucial for mutagenic and survival outcomes. In humans, DNA double-strand breaks are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3' single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. In this work, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. Under both conditions, we report that ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process. Our data bring new clues in the understanding of the cellular response to DNA double-strand breaks
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Wang, Lange. « MicroRNAs modulate the response of p53-MDM2 dynamics to DNA double-strand breaks at single-cell level ». Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/micrornas-modulate-the-response-of-p53mdm2-dynamics-to-dna-doublestrand-breaks-at-singlecell-level(0784d06d-bbac-452d-8595-2aa47c98fd47).html.
Texte intégralRésumé :
The tumour suppressor protein p53 plays a pivotal role in the response to various cellular stresses and thus undergoes complicated patterns of regulation, which has been well-studied. However, recent research exploring this topic at single-cell level has revealed that the dynamical behaviour of p53 and its regulator MDM2 in response to DNA damage was distinct from the general trends seen at population level. Moreover, these studies demonstrated that p53-MDM2 dynamics at single-cell level is correlated with cell fate determination. However, the original conclusions were obtained from the simple plasmid-based cell models, which cannot fully recreate the native behaviour of p53-MDM2 in response to stress signals. To better understand this question, we adapted and improved the previous model by introducing a BAC vector containing native p53 gene labelled with a fluorescent protein. This model has also allowed us to investigate whether miRNAs can modulate p53 dynamics and follow cell fate at the single-cell level. By using time-lapse fluorescent microscopy, our results indicated that p53-MDM2 dynamics in response to DNA double-strand breaks was indeed different in single cells when compared with the population-based results obtained in parallel in both models. Furthermore, the dynamics of p53 signalling in individual cells showed the following characteristics: 1) It was variable from cell to cell, but the mean value of some parameters remained fixed; 2) It was able to be fine-tuned by miRNAs; 3) It is involved in shaping cell fate determination with the regulation by miRNAs leading to distinct. This work identified discrepancies in different cell models, suggesting the relative importance of transgenic tools used for single-cell research. In conclusion, our research not only provided a new approach to study the interaction between miRNAs and p53, but also suggested a new dimension of p53 regulatory mechanism and cell fate determination, which further demonstrates the complexity of biological systems that respond to cellular stress in mammalian cells.
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MANFRINI, NICOLA. « Maintenance of genome integrity in gametes : coping with accidental and programmed DNA double-strand breaks during meiosis ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/18999.
Texte intégralRésumé :
DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase. On the other hand, the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed Spo11-dependent meiotic DSBs that are required for meiotic recombination to take place. In Saccharomyces cerevisiae, the Sae2 protein and the Mre11- Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends of meiotic DSBs, which are then resected by so far unknown nucleases.
As several aspects of the control of the response to DSBs during meiosis are still obscure, I focused my research as Ph. D. student on two different aspects of this control: 1) the possible role of Rad53 and inter-relationships with Mek1 in the response to accidental and programmed DSBs during meiosis and 2) the mechanisms responsible for processing Spo11-induced meiotic DSBs.
1. We have provided evidence that exogenous DSBs lead to Rad53 phosphorylation during meiosis, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 phosphorylation prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.
2. By using site directed mutagenesis and gene replacements, we have demonstrated that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, we used different genetic and biochemical tools to demonstrate that the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5’-3’ resection tracts during meiosis by controlling a step subsequent to Spo11 removal. Our findings suggest that, once Spo11 has catalyzed meiosis-specific DSB formation, it is removed from the DSB ends by endonucleolytic cleavage that necessitates CDK-mediated Sae2 phosphorylation and the nuclease activity of MRX. This cleavage is in turn required for resection of the break by either Exo1 or Dna2-Sgs1 activities, thus allowing completion of meiosis-specific DSB processing.
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Stuckey, Samantha Anne. « Gene targeting at and distant from DNA breaks in yeast and human cells ». Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/51721.
Texte intégralRésumé :
Here we developed multiple genetic systems through which genetic modifications driven by DNA breaks caused by the I-SceI nuclease can be assayed in the yeast Saccharomyces cerevisiae and in human cells. Using the delitto perfetto approach for site-directed mutagenesis in yeast, we generated isogenic strains in which we could directly compare the recombination potential of different I-SceI variants. By genetic engineering procedures, we generated constructs in human cells for testing the recombination activity of the same I-SceI variants. Both in yeast and human cells we performed gene correction experiments using oligonucleotides (oligos) following modification and/or optimization of existing gene targeting protocols and development of new ones. We demonstrated that an I-SceI nicking enzyme can stimulate recombination on the chromosome in S. cerevisiae at multiple genomic loci. We also demonstrated in yeast that an I-SceI-driven nick can activate recombination 10 kb distant from the initial site of the chromosomal lesion. Moreover we demonstrated that an I-SceI nick can stimulate recombination at the site of the nick at episomal and chromosomal loci in human cells. We showed that an I-SceI double-strand break (DSB) could trigger recombination up to 2 kb distant from the break at an episomal target locus in human cells, though the same was not observed for the nick. Overall, we demonstrated the capacity for I-SceI nick-induced recombination in yeast and human cells. Importantly, our findings reveal that the nick stimulates gene correction by oligos differently from a DSB lesion, as determined by genetic and molecular analyses in yeast and human cells. This research illustrates the promise of targeted gene correction following generation of a nick.
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Zhang, Hongshan. « A single molecule perspective on DNA double-strand break repair mechanisms ». Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0177.
Texte intégralRésumé :
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
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Little, Kevin C. E. « The patchy human genome : DNA double-strand breaks in human cells can be repaired by the capture of other DNA fragments ». Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85568.
Texte intégralRésumé :
The human genome is composed of very few genes (the DNA which encodes proteins), and the remaining 98% of all our DNA is made up of repetitive sequences or "junk" DNA with little or no known function. Our complexity, and the cause of many genetic diseases such as cancer, is brought about by the differential expression of that genetic material, in part due to shuffling and movement of chromosomes within the cell nucleus. A major threat to the integrity of the genome is the occurrence of a DNA double-strand break (DSB). These DSBs occur frequently in every cell, and if repaired improperly or left unattended can lead to genomic rearrangements and cell death. With the publication of the human genome sequence, we were able to establish two systems to investigate the possibility that DNA fragments can insert into breaks sites during DSB repair in human cells.We find that both foreign and human genomic DNA can insert into extrachromosomal and chromosomal DSBs. Genomic instability syndromes, like those which result from deficiencies in repair proteins, still permit this DSB insertional repair process, however the spectrum of source material provided can differ. The deregulation of replication origins, such as the amplification of sequences flanking viral integration sites, can lead to the spread and further gene amplification of DNA by this insertion mechanism.
Our demonstration that DSB insertional repair takes place in human cells provides a mechanism for reshuffling genomic DNA, and acquiring new sequences. We propose that a selective advantage is conferred upon a cell able to insert DNA at a DSB, providing a complexity of gene content and interaction which could explain the origins and evolutionary role of the vast majority of the human genome.
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Lee, Brian. « A Monte Carlo investigation of radiation damage to chromatin fibers and production of DNA double strand breaks using Geant4-DNA code ». Thesis, Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/53106.
Texte intégralRésumé :
In the presented research we propose to improve on historically accepted radiobiological models via Monte Carlo simulation of radiation tracks passing through a cell nucleus modeled with up-to-date subnuclear structures. This is performed by generating a radiation track database using the Monte Carlo code, Geant4-DNA, that simulates radiation interactions at the nanometer scale of DNA. These tracks are called upon from the database and intersected with a cell nucleus model that incorporates DNA-containing structures. This allows for a Monte Carlo simulation of how DNA double strand breaks are produced by radiation. The results can be used to correlate to many experimentally observed biological endpoints, e.g. chromosome aberrations as well as cell death.