No description available.

Nucleotide synthesis is a conserved and highly regulated response to DNA damage, required for the efficient repair of DNA double strand breaks (DSB) by homologous recombination (HR). This is essential to prevent loss of heterozygosity (LOH) and maintain genome stability. The aim of this study was to identify new genes important for HR through roles in damage-induced nucleotide synthesis. A screen was performed to identify S. pombe gene deletion strains whose DSB sensitivity was suppressed by deleting the ribonucleotide reductase (RNR) inhibitor spd1⁺ to promote nucleotide synthesis. The screen identified a number of genes including ddb1⁺, cdt2⁺, rad3⁺ and csn1⁺ which have known roles in nucleotide synthesis. Distinct roles were identified for the DNA damage checkpoint in suppressing LOH. rad3⁺, rad26⁺, rad17⁺ and the rad9⁺, rad1⁺ and hus1⁺ genes encoding the 9-1-1 complex were required for DNA damage-induced nucleotide synthesis through Cdt2 induction to promote Spd1 degradation. The HR repair defect of rad3⁺ and rad26⁺ deletion strains was partially suppressed by spd1⁺ deletion. However, the HR repair defect of rad17⁺, rad9⁺, rad1⁺ and hus1⁺ deletion strains was not suppressed. An additional role was confirmed for Rad17 and the 9-1-1 complex in preventing LOH by promoting DSB resection. A role was identified for the Gcn5 histone acetyl transferase (HAT) protein module, consisting of Gcn5, Ngg1, Ada2 and Sgf29, in suppressing DSB sensitivity by promoting nucleotide synthesis. This was independent of Cdt2 or RNR protein levels. The Gcn5 HAT module was also found to regulate DSB repair pathway choice consistent with previous observations. Deletion of gcn5⁺, ngg1⁺ or ada2⁺ decreased HR and increased non-homologous end joining. Surprisingly, deletion of spd1⁺ in a gcn5∆, ngg1∆ or ada2∆ background also promoted HR. This predicts a role for nucleotide pools in regulating DSB repair pathway choice. Eleven other candidates showed repeatable suppression of DSB sensitivity following spd1⁺ deletion. However many of these candidates did not show reduced nucleotide levels. This suggests deleting spd1⁺ may also suppress DSB sensitivity by a different mechanism.

<p>To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may either halt the progression of the cycle (cell cycle arrest) or undergo apoptosis. This choice depends on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is an S/T-Q directed kinase that is critical for the cellular response to double-stranded DNA breaks. Following DNA damage, ATM is activated and recruited to sites of DNA damage by the MRN protein complex (Mre11-Rad50-Nbs1 proteins) where ATM phosphorylates multiple substrates to trigger a cell cycle arrest. In cancer cells, this regulation may be faulty and cell division may proceed even in the presence of damaged DNA. We show here that the RSK kinase, often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that RSK disrupts the binding of the MRN complex to DSB DNA. RSK can directly phosphorylate the Mre11 protein at Ser 676 both in vitro and in intact cells and can thereby inhibit loading of Mre11 onto DSB DNA. Accordingly, mutation of Ser 676 to Ala can reverse inhibition of the DSB response by RSK. Collectively, these data point to Mre11 as an important locus of RSK-mediated checkpoint inhibition acting upstream of ATM activation.</p><p>The phosphorylation of Mre11 on Ser 676 is antagonized by phosphatases. Here, we screened for phosphatases that target this site and identified PP5 as a candidate. This finding is consistent with the fact that PP5 is required for the ATM-mediated DNA damage response, indicating that PP5 may promote DSB-induced, ATM-dependent DNA damage response by targeting Mre11 upstream of ATM.</p> / Dissertation

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.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

<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