Cdt1 is a conserved and essential eukaryotic protein that is required for the licensing step of DNA replication. In order to control replication licensing and ensure a single round of DNA replication occurs per cell cycle, Cdt1 is subject to strict regulation. In Metazoa and S. pombe, Cdt1 is targeted for ubiquitylation and proteolysis in S phase and after DNA damage by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 is activated in Metazoa by an unusual mechanism that requires an interaction between the substrate and chromatin-loaded proliferating cell nuclear antigen (PCNA). This study addressed the involvement of PCNA in S. pombe Cdt1 proteolysis. A mutational analysis was undertaken to establish whether the Cdt1-PCNA interaction is conserved in S. pombe and the extent to which it regulates CRL4Cdt2-dependent turnover of the protein. S. pombe Cdt1 was shown to interact with PCNA in vivo and two variant PCNA-interacting peptide (PIP) motifs were identified in the protein. The two motifs function near-redundantly to promote both the Cdt1-PCNA interaction and the CRL4Cdt2-dependent proteolysis of Cdt1 in S phase and after DNA damage. The mutational analysis also resulted in the characterisation of two in-frame AUG codons in the cdt1+ reading frame. The second in-frame AUG codon was shown to be the principal initiator codon and was required to maintain wildtype Cdt1 protein levels and cell viability. CRL4Cdt2 is emerging as an important regulator of proteins that are involved in the control of cell cycle progression and the maintenance of genome stability. However, there are a number of outstanding questions regarding the mechanism and regulation of CRL4Cdt2. In order to address these questions, a genomics approach was taken to identify novel genes involved in Cdt1 regulation. A screen of non-essential S. pombe genes identified 17 candidate genes that, when inactivated, caused up-regulation of Cdt1. Unexpectedly, deletion of genes involved in homologous recombination resulted in a Rad3-dependent up-regulation of Cdt1. Further work is required to establish the biological significance of this finding.

The final stages of replication fork termination requires the timely and orderly orchestration of catalytic and enzymatic activities. Given the complexity of this process, it is conceivable that the final stages of fork termination is susceptible to problems that could trigger recombination, which could lead to deleterious genomic rearrangements if ectopic homologous sequences are recombined. Using the site-specific RTS1 barrier in fission yeast, I demonstrated that fork termination is generally not a recombinogenic process, and that hyper-recombination-induced by fork blockage at RTS1 is largely a result of replication fork restart. To investigate the actual mechanisms and proteins, which drive and influence recombination at a replication barrier, I studied the MHF proteins, which assist Fml1 in limiting crossovers during double-strand break (DSB) repair and promoting Rad51-mediated recombination at impeded replication forks, and are also components of the constitutive centromere-associated network (CCAN). Intriguingly, structural studies revealed that the MHF can exist as an octamer in vitro. I examined the biological significance of octameric MHF by employing three mutations that disrupt the octamer configuration in vitro. In fission yeast, these mutations cause hypersensitivity to methyl methanesulfonate (MMS), suggesting that the MHF octamer may have a role in DNA repair. One of the “octamerisation” mutants, exhibits greater hypersensitivity to MMS than the other two, and biochemical experiments indicated that this is because it confers an additional defect in MHF’s interaction with Fml1. Further genetic experiments on this mutant suggest that the ability of Fml1 to unwind D-loops depends more critically on its interaction with MHF than fork reversal. Additionally, I showed a synergistic interaction between Dcr1 and MHF, and demonstrated that in the absence of Dcr1, there is a greater need for recombination to tolerate/repair DNA damage. Lastly, I uncovered a novel function for the MHF in controlling the initiation of septation.

DNA provides the blueprint for cell function and growth, as well as ensuring continuity from one cell generation to the next. In order to compact, protect, and regulate this vital information, DNA is packaged by histone proteins into nucleosomes, which are the fundamental subunits of chromatin. Reactive oxygen species, generated by both endogenous and exogenous agents, can react with DNA, altering base chemistry and generating DNA strand breaks. Left unrepaired, these oxidation products can result in mutations and/or cell death. The Base Excision Repair (BER) pathway exists to deal with damaged bases and single-stranded DNA breaks. However, the packaging of DNA into chromatin provides roadblocks to repair. Damaged DNA bases may be buried within nucleosomes, where they are inaccessible to repair enzymes and other DNA binding proteins. Previous in vitro studies by our lab have demonstrated that BER enzymes can function within this challenging environment, albeit in a reduced capacity. Exposure to ionizing radiation often results in multiple, clustered oxidative lesions. Near-simultaneous BER of two lesions located on opposing strands within a single helical turn of DNA of one another creates multiple DNA single-strand break intermediates. This, in turn, may create a potentially lethal double-strand break (DSB) that can no longer be repaired by BER. To determine if chromatin offers protection from this phenomenon, we incubated DNA glycosylases with nucleosomes containing clustered damages in an attempt to generate DSBs. We discovered that nucleosomes offer substantial protection from inadvertent DSB formation. Steric hindrance by the histone core in the nucleosome was a major factor in restricting DSB formation. As well, lesions positioned very close to one another were refractory to processing, with one lesion blocking or disrupting access to the second site. The nucleosome itself appears to remain intact during DSB formation, and in some cases, no DNA is released from the histones. Taken together, these results suggest that in vivo, DSBs generated by BER occur primarily in regions of the genome associated with elevated rates of nucleosome turnover or remodeling, and in the short linker DNA segments that lie between adjacent nucleosomes. DNA ligase IIIα (LigIIIα) catalyzes the final step in BER. In order to facilitate repair, DNA ligase must completely encircle the DNA helix. Thus, DNA ligase must at least transiently disrupt histone-DNA contacts. To determine how LigIIIα functions in nucleosomes, given this restraint, we incubated the enzyme with nick-containing nucleosomes. We found that a nick located further within the nucleosome was ligated at a lower rate than one located closer to the edge. This indicated that LigIIIα must wait for DNA to spontaneously, transiently unwrap from the histone octamer to expose the nick for recognition. Remarkably, the disruption that must occur for ligation is both limited and transient: the nucleosome remains resistant to enzymatic digest before and during ligation, and reforms completely once LigIIIα dissociates.

Genomic instability and acquisition of invasiveness through the basement membrane extracellular matrix (ECM) are two major processes for epithelial cell malignancy in breast cancer. DNA double-strand break repair (DSBR) is one of the processes that get misregulated during breast cancer progression. In addition, radiation induced breaks such as those induced during radiation therapy to treat breast cancer patients are repaired by DSBR, rendering this pathway relevant for therapy as well. DSBR can occur either by homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is accepted as the more error-free pathway. HR is regulated by the cell cycle status such that an increase is observed in G2/M, whereas NHEJ is observed throughout the cell cycle. Previous data show that ECM signaling regulates HR, as well as the kinetics of ionizing radiation (IR) induced complex formation at break sites, or foci kinetics. Both human breast epithelial cell lines and primary mouse mammary epithelial cells were used to show that the ECM receptor β1-integrin is necessary and sufficient in down regulating HR, as well as IR induced foci formation kinetics for the DSBR proteins RAD51, MRE11, and γ-H2AX in single mammary epithelial cells. RAD51 is required for most HR, whereas MRE11 and γ-H2AX function in HR as well as DNA damage signaling. Interestingly, ECM signaling up-regulates HR in cells that have “correct” in vivo-like cell-cell junctions. Based on the observation that single cells and junctioned cells respond to ECM in exact opposite manner, I hypothesized that ECM signaling may interact with cell-cell junction signaling pathways in regulating DNA repair. To test this hypothesis, I asked whether the main breast epithelial adherens junction cadherin, E-cadherin, is involved. I blocked E-cadherin function using a monoclonal antibody MB2. The function blocking was demonstrated by the loss of cell-cell junction interactions and observation of increased cell scattering using phase microscopy. I then asked whether blocking E-cadherin altered the expression and localization of proteins related to DNA repair. Indirect immuno-fluorescence showed that in the E-cadherin blocked non-tumorigenic breast epithelial cell line HMT-3522 S1 there is an up-regulation of nuclear γ-H2AX and RAD51, as well as an increase in the proliferation marker Ki67. In non-proliferative MB2 blocked cells there is an upregulation of γ-H2AX and reduced Ki67. Furthermore, in these proliferative and non-proliferative blocked cells we were able to see lower levels of β-catenin near the cell membrane and an increase in its levels inside the cell especially in the nucleus. The latter has been confirmed also by western blot technique comparing the nuclear and cytoplasmic fraction expression. In addition, western blots showed that total RAD51 level was down-regulated by E-cadherin blocking and γ-H2AX levels were found to be higher in proliferative and non-proliferative MB2 treated cells. MB2 treated cells have a higher frequency of HR in the absence of ECM and in the presence of ECM, MB2 blocking abolishes the ECM effect on HR. Furthermore, in the absence of ECM, RAD51 siRNA treated cells down-regulated HR but the absence of RAD51 did not down regulate HR in the presence of ECM. I was not able to see any difference in the phosphorylated forms of β-catenin such as Tyr-142, Ser-45 and Tyr-86 that has the ability to enter into the nucleus. Therefore, E-cadherin was found to block nuclear β-catenin, RAD51 and γ-H2AX in a proliferation-independent manner. E-cadherin also was necessary for ECM to up-regulate HR. The up-regulation of HR by ECM was only slightly dependent on RAD51 suggesting a novel E-cadherin-dependent and RAD51-independent HR component in breast epithelial cells in contact with ECM as they are in vivo in the normal breast tissue. These experiments will help us to understand the role of E-cadherin and β-catenin in DNA double-stand break repair directly, as well as in combination with ECM signaling. Both alterations in integrin mediated signaling and cell-cell junction integrity contribute to breast cancer progression by rendering breast epithelial cells more invasive. My project will shed light on whether these invasive processes also alter DNA repair and contribute to genome stability. Understanding of the interrelationships among integrin signaling, cell-cell junctions, and genome stability will contribute to understanding normal breast cell processes and open up investigations on how these may go awry in cancer progression.

Central to the progression of all organisms is the maintenance of a stable genome despite continuous insults arising from genotoxic and environmental stresses. Embryonic stem cells show promise for treatment of a variety of diseases as well as for providing normal human tissue to conduct scientific research. A major obstacle for their application is that genomic instability arises in stem cells after prolonged cell culture. The most detrimental form of DNA damage is the DNA double-strand break (DSB), which is managed by cells through complex mechanisms, designated the DNA damage response. There are two major types of DSB repair; homologous recombination repair (HRR) and non-homologous end joining (NHEJ), both of which are regulated by members of the phosphatidyl-inositol-3’-kinase-related kinase (PIKK) family, including Ataxia Telangiectasia Mutated (ATM), Ataxia Telangiectasia Mutated and Rad3-related (ATR) and the DNA dependent protein kinase (DNA-PK). The aim of this study was to define the mechanisms and important proteins involved in repair of human embryonic stem cells. Here we have also described a system to differentiate hESCs into neural progenitors and astrocytes and were able to examine their DNA damage response. In both examining DNA repair markers and using a DNA repair reporter assay, this work shows that ATR is involved in DSB repair early in development, whereas ATM is essential in DSB repair in differentiated cells. We also show that HRR, a high fidelity form of repair, is used extensively by embryonic stem cells and HRR diminishes as cells differentiate. We also further defined the extent of NHEJ and the role of high fidelity NHEJ from the embryonic to differentiated state. These findings further the basic knowledge of repair fidelity in embryonic and mature human tissue. The data gives insight into what proteins maintain stem cell genomic stability and may be important to develop safe technologies for tissue engineering. Specifically, we have defined what DNA damage signaling pathways are used as embryologic cells progress to a mature, functional state.

This thesis investigates two separate features of nonhomologous end-joining (NHEJ) DNA repair: end processing, and DNA repair kinase signaling. DNA end processing was investigated in a mouse model of hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), a congenital neurodegenerative disease. SCAN1 is caused by a homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1). To address how the H493R mutation elicits the specific pathologies of SCAN1 and to further elucidate the role of TDP1 in processing DNA end modifications, we generated a Tdp1 knockout mouse and characterized their behavior and specific repair deficiencies in extracts of embryonic fibroblasts from these animals. While Tdp1(-/-) mice appear phenotypically normal, extracts from Tdp1(-/-) fibroblasts exhibited deficiencies in processing 3'-phosphotyrosyl single-strand breaks and 3'-phosphoglycolate (PG) double-strand breaks (DSBs). Supplementing Tdp1(-/-) extracts with H493R TDP1 partially restored processing of 3'-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on PG termini on 3' overhangs of DSBs; these remained completely unprocessed. Altogether, these results suggest that for 3'-PG overhang lesions, the SCAN1 mutation confers loss of function, while for 3'-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate. Furthermore, there is evidence that TDP1 also localizes to mitochondria, and mitochondrial DNA damage should not be excluded from significantly contributing to SCAN1 pathology. The effect of ATM signaling on NHEJ was investigated via a novel vector that allows for inducing I-SceI-mediated DNA DSBs that can then be analyzed for NHEJ repair events by fluorescence- and PCR-based methods. Using highly specific DNA kinase inhibitors and the repair cassette, we showed that inhibiting ATM reduced NHEJ by 80% in a U87 glioma model. Analysis of the PCR products from the NHEJ repair vector by PsiI restriction cleavage allowed for assessment of the fidelity of the NHEJ repair: inhibiting ATM reduced high-fidelity NHEJ by 40%. Together, these results suggest that ATM is critical for NHEJ of I-SceI DSBs and for high-fidelity repair, possibly due to ATM's effects on chromatin architecture surrounding the DSB.

DNA methylation is a major form of epigenetic modification and involves the addition of a methyl group covalently to the 5-position of the cytosine pyrimidine ring, mostly within the context of CpG dinucleotides in vertebrate somatic cells. Methylation of CpG dinucleotides at promoter regions is generally associated with transcriptional repression. In this context, the methyl-CpG binding proteins (MeCPs) that are capable of recognition of methylated CpG dinucleotides are proposed to play a central role in DNA methylation associated transcriptional repression. Methyl-CpG binding domain protein 4 (MBD4) is an MeCP that possesses a glycosylase domain at its C-terminal, which can excise and repair both G:T and G:U mutations derived from DNA deamination at CpG dinucleotides, in addition to its Nterminal MBD binding domain. MBD4 has been associated with a number of pathways including DNA repair, apoptosis, transcriptional repression, and possibly DNA demethylation processes. However, the precise contribution of MBD4 to these processes remains unclear. To explore the functional repertoire of MBD4 I decided to undertake multiple protein interaction studies to identify potential partner proteins. I performed yeast 2-hybrid screens with an 11.5 day mouse embryonic cDNA library and multiple mass spectrometry of immunoprecipitates of tagged versions of MBD4 that were over-expressed in human cell lines. I detected ~380 potential interacting candidates with these assays. A significant number of candidates were detected in both assay systems. Chosen candidates were further validated by reciprocal co-IP of expressed partners and by immunofluorescence (IF) microscopy to determine their potential co-localisation in mouse and human cell lines. Subsequently, I identified the intervening domain of MBD4 as a novel protein interaction region for tested candidates. My analysis suggests that MBD4 can have a role in regulation of post-replication methyl-error repair/methylation machinery through its direct interaction with DNMT1 (previously shown), UHRF1 (novel) and USP7 (novel), as well as possible cross-talk to histone modification and chromatin remodelling pathways, through partners such as PRMT5 and ACF1. Interestingly the transcription regulatory components KAP1 and CFP1 not only interact with but also dramatically influence the stability of exogenously expressed MBD4 in human cells. In general positive validation by IP and IF demonstrates the robustness of the initial screens, and implies that MBD4 may impact upon several transcriptional and epigenetic networks along with a number of nuclear pathways that include transcriptional repression, DNA repair and RNA processing. To test for transcriptional aberration in the absence of Mbd4 function I profiled two independent mouse cell lines that lack MBD4 activity using Illumina MouseWG-6 v2.0 Expression BeadChip arrays. A number of genes were identified that are significantly up- or down- regulated in both Mbd4-/- MEFs. This included mis-expression of insulin-like growth factor-binding proteins and two paternally imprinted genes Dio3 and H19. The cohort of genes that were mis-expressed in the Mbd4-/- MEFs overlap with genes that responsed to tamoxifen exposure in an ER-positive ZR-75-1 xenograft model. In response to this observation I identified a potential interaction between MBD4 and estrogen receptor α (ERα) by co-IP and IF co-localisation. This suggests that MBD4 might potentiate transcription of estrogen regulated genes via a direct interaction with ERα, supporting a possible link between replication repair remodelling and steroid/thyroid hormone receptor transcriptional regulation. Additionally I performed a pathway analysis by which several developmental genes including Sox9, Klf2 and Klf4, were prioritised as possible MBD4 targets. On this basis I propose a role for MBD4 in acquired diseases such as cancers and autoimmune diseases via transcriptional regulation. I also performed a comparison of MBD4 DNA binding activity with MBD4 homologues from the Medaka fish (Oryzias latipes) and the amphibian, Xenopus laevis. I could show that DNA binding specificity to a series of methylated and mismatched probes is conserved regardless of the poor sequence conservation of the MBD domain of MBD4 between the species. I conclude that MBD4 is integrated in multiple pathways in the nucleus that includes DNA repair, chromatin remodelling, transcriptional regulation and genome stability.

Homologous Recombination (HR) can promote genome stability through its capacity to faithfully repair DNA gouble 2trand !;!reak2 (DSBs) and preventing the demise of stalled replication forks in part by catalysing template switching to enable DNA polymerase to bypass lesions. Despite these beneficial roles, inappropriate or untimely HR events can have deleterious consequences. HR can cause genome instability by recombining "inappropriate" homologous sequences, especially if the recombination intermediates are resolved to form crossovers. Over the past few years, study of the rare inherited chromosome instability disorder, Eanconi Anaemia (FA), has uncovered a novel DNA damage response pathway. Although the FA pathway is required primarily for interstrand DNA cross link repair, its precise role in DNA repair reactions is still unclear. FA.Qomplementation group M (FANCM) is the sole component within the FA core complex which possesses a DNA helicase/ATPase domain and an endonuclease domain (albeit non-functional), suggesting that FANCM could translocate along DNA and target the FA core complex to blocked replication forks. To further elucidate the role of FANCM in HR, I have purified Fm11, the FANCM orthologue in the fission yeast Schizosaccharomyces pombe and tested its activity on a range of synthetic replication and recombination intermediates in vitro. Fml1 binds both replication forks and Holliday Junctions (HJs) which are key intermediates of HR.

In response to DNA damage, the ATM and ATR kinases proliferate a signal that is transduced, either directly or via Chk2 and Chk1, to effector proteins, forming the DNA damage response (DDR). The effector proteins delay cell cycle progression, through checkpoints, and activate specific DNA repair mechanisms essential for preserving genome integrity and preventing cancer formation. Bloom's syndrome (BS) patients, which lack the BLM protein show genome instability and have a predisposition to cancer. BLM is phosphorylated by the DDR kinases ATM, ATR and Chk1. These phosphorylation events are essential for BLM to maintain replication fork integrity, preserve the S phase checkpoint and activate BLM to interact with other DDR proteins. In this study I have shown that BLM, isolated from mitotic cells, is phosphorylated on amino acid residue serine 26 (S26). BS cells lacking native BLM, but expressing a variant of BLM protein that cannot be phosphorylated at S26, fail to fully activate the G2/M checkpoint following UV irradiation or treatment with inhibitors of DNA topoisomerase H. Consequently, these cells are more sensitive to killing by these agents than are BS cells expressing wildtype BLM. The Chk1 and Aurora B kinases are able to phosphorylate BLM on S26 in vitro. Moreover, loss of Aurora B kinase activity leads to reduction of S26 phosphorylation in mitotic cells. Cells treated with inhibitors of Aurora B fail to fully active the G2/M checkpoint after UV DNA damage. Taken together, these data suggest, that Aurora B kinase phosphorylates BLM on S26 and that this is required to fully activate the G2/M checkpoint.

Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.