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The repair of DNA double-strand breaks (DSBs) is required for the survival of all organisms. In bacteria, DNA DSBs can occur during normal housekeeping processes such as DNA replication or by exogenous damage due to chemicals or radiation. DSBs will compromise the integrity of the genome if left un-repaired, and can be fatal to an organism. Repair of DSBs by homologous recombination (HR) replicates missing chromosomal regions before joining of the separated DNA ends. In Escherichia coli the HR repair steps are; pre-synapsis, synapsis and post-synapsis. In the pre-synaptic stage a DSB is processed into a 3′ single-strand overhang, the substrate required for strand invasion in the synapsis stage and the eventual repair of the DSB. At present there are three identified pre-synapsis systems involved in recombination in bacteria; represented by the AdnAB, AddAB and the RecBCD protein complexes. Each system functions in a similar manner but differ in the physical composition of the machinery. This project investigated the pre-synaptic system of Bacteroides fragilis NCTC9343. Genes encoding putative pre-synapsis proteins were initially identified through analysis of the NCTC9343 genome. The function of these proteins was investigated in vivo by rescue of a repair-deficient strain of E. coli. This demonstrated that Bacteroides fragilis encodes a two component system, where both genes products are required to work in concert for pre-synaptic processing of DSBs. The identified genes were BF2192 and BF2191, and have been renamed addA and addB, respectively. To further examine the role of the AddAB proteins in DSB repair, a Bacteroides fragilis strain with a deletion of addAB was constructed and shown to be extremely sensitive to DNA damaging agents. The AddAB complex was purified and found to be an ATP-dependant helicase and exonuclease that acted on double-stranded DNA ends. In conclusion, this project has identified the proteins involved in pre-synaptic processing of DSBs in B. fragilis NCTC9343, consisting of AddAB homologues, and shown their protective role in repair of DNA damage.

Efficient and accurate repair of DNA double strand breaks (DSBs) is required to maintain genomic stability in both eukaryotes and prokaryotes. In Escherichia coli, DSBs are repaired by homologous recombination (HR). During this process, DNA synthesis needs to be primed and templated from an intact homologous sequence to restore any information that may have been lost on the broken DNA molecule. Two critical late stages of the pathway are repair DNA synthesis and the processing of Holliday junctions (HJs). However, our knowledge of the detailed mechanisms of these steps is still limited. Our laboratory has developed a system that permits the induction of a site-specific DSB in the bacterial chromosome. This break forms in a replication dependent manner on one of the sister chromosomes, leaving the second sister chromosome intact for repair by HR. Unlike previously available systems, the repairable nature of these breaks has made it possible to physically investigate the different stages of DNA double-strand break repair (DSBR) in a chromosomal context. In this thesis, I have addressed some fundamental questions relating to repair DNA synthesis and processing of HJs by using a combination of mutants defective in specific biochemical reactions and an assay that I have developed to detect repair DNA synthesis, using a polar termination sequence (terB). First, by using terB sites located at different locations around the break point, it was shown that the DnaB-dependent repair forks are established in a coordinated manner, meaning that the collision of the repair forks occurs between two repair DNA synthesis initiation sites. Second, DSBR was shown to require the PriB protein known to transduce the DNA synthesis initiation signal from PriA protein to DnaT. Conversely, the PriC protein (known as an alternative to PriB in some reactions) was not required in this process. PriB was also shown to be required to establish DnaB-dependent repair synthesis using the terB assay. Third, the establishment and termination of repair DNA synthesis by collision of converging repair forks were shown to occur independently of HJ resolution. This conclusion results from the comparison of the viability of single and double mutants, deficient in either the establishment of DNA synthesis, HJ resolution or in both reactions, subjected to DSBs and from the study of the DNA intermediates that accumulated in these mutants as detected by two-dimensional gel electrophoresis. Fourth, the role of RecG protein during DSB repair was investigated. Solexa sequencing analyses showed that recG null mutant cells undergoing DSBs accumulate more DNA around the break point (Mawer and Leach, unpublished data). This phenomenon was further investigated by two different approaches. Using terB sites in different locations around the break point and ChIP-Seq analyses to investigate the distribution of RecA in a recG null mutant demonstrating that the establishment of repair forks depends on the presence of RecG. Further studies using PriA helicase-dead mutant showed that the interplay between RecG and PriA proteins is essential for the establishment of correctly oriented repair forks during DSBR. As a whole, this work provides evidence on the coordinated nature of the establishment and termination of DNA synthesis during DSBR and how this requires a correct interplay between PriA-PriB and RecG. A new adapted model of homologous recombination is presented.

A DNA double-strand break (DSB) is a severe form of DNA damage. In fastgrowing cells, DSBs are commonly repaired by homologous recombination (HR) and in E. coli they are exclusively repaired by this mechanism. Failure to accurately repair DSBs can lead to genomic instability. Characterising the DNA intermediates formed during DSB repair by HR is key to understanding this process. A system for inducing a site-specific DSB in the E. coli chromosome has previously been described (Eykelenboom et al., 2008). Here, this system has been used to determine the nature of the intermediates of the repair. It was shown that in a Rec+ background the repair process is rapid and efficient. By contrast, in a ruvAB mutant, which is defective for the Holliday junction (HJ) migration and cleavage complex, RuvABC, HJs are accumulated on both sides of the breakpoint. Replication forks also accumulate at defined positions from the DSB, indicating that unresolved HJs are a barrier to efficient replication that is associated with the repair. This suggests that the resolution of HJs needs to occur prior to the establishment of DNA synthesis. Despite the accumulation of HJs in a ruvAB mutant, cell survival occurs when DSBs are induced for short periods, suggesting that HJs can be resolved in a RuvAB-independent manner. In contrast, the RecG helicase is essential for survival. In a recG mutant, replication forks but not HJs are detected in the region of DSB repair. In a ruvAB recG mutant, intermediates in this region are lost. These observations are consistent with a role of RecG in the stabilisation and maturation of D-loops and not the resolution of Holliday junctions. Nevertheless, an additional role for RecG in later stages of repair cannot yet be excluded. This work provides a solid framework for the further study of DSB repair in E. coli.

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.

<p>The genetic information necessary for the survival and propagation of a species is contained within a physical structure, DNA. However, this molecule is sensitive to damage arising from both exogenous and endogenous sources. DNA damage can prevent metabolic processes such as replication and transcription; thus, systems to bypass or repair DNA lesions are essential. One type of lesion in particular - the double strand break (DSB) - is extremely dangerous as inappropriate repair of DSBs can lead to deletions, mutations and rearrangements. Homologous recombination (HR) uses a template with sequence homology to the region near the DSB to restore the damaged molecule. However, this high-fidelity pathway can contribute to genome instability when recombination occurs between diverged substrates. To further our understanding of the regulation of HR during vegetative growth, we have used the budding yeast Saccharomyces cerevisiae as a model system and a plasmid-based assay to model repair of a DSB. In the first part of this work, the molecular structures of noncrossover (NCO) and crossover (CO) products of recombination were examined. While the majority of NCOs had regions of heteroduplex DNA (hDNA) on one side of the gap in the repaired allele and no change to the donor allele, most COs had two tracts of hDNA. They were present on opposite sides of the gap, one in each allele. Our results suggest that the majority of NCOs are generated through synthesis-dependent strand annealing (SDSA), and COs are the result of constrained cleavage of a Holliday junction (HJ) intermediate. To clarify the mechanisms regulating NCO production, the effects of three DNA helicases - Mph1, Sgs1 and Srs2 - on the structures of NCO events were examined. All three helicases promote NCO formation by SDSA, but Sgs1 and Srs2 also assist in NCO formation arising from an HJ-containing intermediate, consistent with HJ-dissolution. To study how CO products are generated, we have investigated the contribution of the following candidate HJ resolvases to the structures of CO events: Mus81, Yen1 and Rad1. The results suggest that Rad1 is important to normal CO formation in this assay, but Mus81 and Yen1 are largely dispensable. Together, this work advances our knowledge of how the NCO versus CO outcome is determined during HR, expanding our understanding of how mitotic recombination is regulated.</p> / Dissertation

Acute myeloid leukaemia is a heterogeneous disease characterized by uncontrolled proliferation of neoplastic haematopoietic precursor cells, which leads to the disruption of normal haematopoiesis and bone marrow failure. Impaired haematopoiesis is often associated with balanced chromosomal translocations that involve the nucleoporin Nup98 fused to around 30 different partner genes, such as the homeobox genes HOXA9 and PMX1. Nup98-associated AML is characterized by poor prognosis and poor treatment outcome for the patients. The aim of the study was to elucidate the mechanisms underlying chemotherapy-resistance. Previous experiments showed that the expression of Nup98 fusion proteins leads to changes in nuclear organization. Based on these observations, we hypothesize that the expression of Nup98 fusion proteins affect DNA double-strand break (DSB) repair. Our work shows that the expression of Nup98-HoxA9 and Nup98-HHEX in U2OS cells does not induce any DSBs. Further, we examined the repair phenotype of exogenously induced DSBs. Experiments carried out using etoposide (ETO) or neocarzinostatin (NCS) revealed that Nup98 fusion proteins affect non-homologous end joining (NHEJ). The second major DSB repair pathway, homologous recombination (HR), remains unaffected by Nup98 fusion proteins. The repair phenotype showed that at most timepoints analyzed, cells expressing Nup98 fusion proteins present less DSBs that control cells. We further performed single cell gel electrophoresis assays, also called COMET assay. This assay determines the amount of broken DNA at the single cell level. COMET assays showed that cells expressing Nup98-HoxA9 get equally damaged as control cells. Taken together, these results show that Nup98-HoxA9 induces faster DNA repair by affecting NHEJ. Additional experiments, pointed toward a role of p53 in the effect of Nup98 fusion proteins on DSB repair. Monitoring the repair phenotype in a wild-type and p53 depletion background, revealed that the effect of Nup98-HoxA9 on NHEJ is partially p53 dependent. A further search for the potentially implicated factor in the accelerated NHEJ remained inconclusive so far. In conclusion, Nup98-HoxA9 induces accelerated NHEJ in a partially p53-dependent manner. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

During IgH class switch recombination (CSR) in mature B lymphocytes, activation-induced cytidine deaminase (AID) initiates DNA double strand breaks (DSBs) within switch (S) regions flanking different sets of the IgH locus (IgH) constant \((C_H)\) region exons. End-Joining of DSBs in the upstream donor S region (Sm) to DSBs in a downstream acceptor S region \((S_{acc})\) replaces the initial set of \(C_H\) exons, Cm, with a set of downstream \(C_H\) exons, leading to Ig class switching from IgM to another IgH class (e.g., IgG, IgE, or IgA). In addition to joining to DSBs within another S region, AID-induced DSBs within a given S region are often rejoined or joined to other DSBs in the same S region to form internal switch deletions (ISDs). ISDs were frequently observed in Sm but rarely in \(S_{acc}s\), suggesting that AID targeting to \(S_{acc}s\) requires prior recruitment to Sm. To test this hypothesis, we assessed CSR and ISDs in B cells lacking Sm and found that AID frequently targets downstream \(S_{acc}s\) independently of Sm. These studies also led us to propose an alternative pathway of "downstream" IgE class switching that involves joining of DSBs within the downstream \(S\gamma1\) and \(S\epsilon\) regions as a first step before joining of \(S\mu\) to the hybrid downstream S region. To further elucidate the CSR mechanism, we addressed the long-standing question of whether S region DSBs during CSR involves a direction-specific mechanism similar to joining of RAG1/2 endonuclease-generated DSBs during V(D)J recombination. We used an unbiased high throughput method to isolate junctions between I-SceI meganuclease-generated DSBs at a target site that replaces the IgH \(S\gamma1\) region and other genomic DSBs of endogenous origin. Remarkably, we found that the I-SceI-generated DSBs were joined to both upstream DSBs in \(S\mu\) and downstream DSBs in \(S\epsilon\) predominantly in orientations associated with joining during productive CSR. This process required the DSB response factor 53BP1 to maintain the orientation-dependence, but not the overall levels, of joining between these widely separated IgH breaks. We propose that CSR exploits a mechanism involving 53BP1 to enhance directional joining of DSBs within IgH in an orientation that leads to productive CSR.

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and check-point activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.