Repairing a chromosomal DNA double strand break is essential for survival and maintenance of genomic integrity of a eukaryotic organism. The eukaryotic cell has therefore evolved intricate mechanisms to counteract all sorts of genomic insults in the context of chromatin structure. Modulating chromatin structure has been crucial and integral in regulating a number of conserved repair processes along with other fundamental genomic processes like replication and transcription. The work in this dissertation has focused on understanding the role of chromatin remodeling enzymes in the repair of a chromosomal DNA double strand break by homologous recombination. This has been approached by recapitulating the biochemical formation of recombination intermediates on chromatin in vitro. In this study, we have demonstrated that the mere packaging of DNA into nucleosomal structure does not present a barrier for successful capture of homologous DNA sequences, a central step of the biochemical pathway of recombinational repair. It is only the assembly of heterochromatin-like more complex nucleo-protein structure that presents additional constraints to this key step. And, this additional constraint can be overcome by the activities of ATP-dependent chromatin remodeling enzymes. These findings have great implications for our perception of the mechanism of the recombinational repair process of a chromosomal DNA double strand break within the eukaryotic genome.

The function of homologous DNA recombination in human mitochondria has been a topic of ongoing debate for many years, with implications for fields ranging from DNA repair and mitochondrial disease to population genetics. While genetic and biochemical evidence supports the presence of a mitochondrial recombination activity, the purpose for this activity and the proteins involved have remained elusive. The work presented in this thesis was designed to evaluate the mitochondrial localization of the major recombinase protein in human cells, Rad51, as well as determine what function it plays in the maintenance of mitochondrial DNA (mtDNA) copy number that is critical for production of chemical energy through aerobic respiration. The combination of subcellular fractionation with immunoblotting and immunoprecipitation approaches used in this study clearly demonstrates that Rad51 is a bona fide mitochondrial protein that localizes to the matrix compartment following oxidative stress, where it physically interacts with mtDNA. Rad51 was found to be critical for mtDNA copy number maintenance under stress conditions. This requirement for Rad51 was found to be completely dependent on ongoing mtDNA replication, as treatment with the DNA polymerase gamma (Pol ϒ) inhibitor, ddC, suppresses both recruitment of Rad51 to the mitochondria following the addition of stress, as well as the mtDNA degradation observed when Rad51 has been depleted from the cell. The data presented here support a model in which oxidative stress induces a three-part response: (1) The recruitment of repair factors including Rad51 to the mitochondrial matrix, (2) the activation of mtDNA degradation systems to eliminate extensively or persistently damaged mtDNA, and (3) the increase in mtDNA replication in order to maintain copy number. The stress-induced decrease in mtDNA copy number observed when Rad51 is depleted is likely the result of failure to stabilize or repair replication forks that encounter blocking lesions resulting in further damaged to the mtDNA and its eventual degradation.

No description available.

Gliomas are the most common primary tumors of the central nervous system believed to arise from glial cells. Invasive growth and inherent propensity for malignant progression make gliomas incurable despite extensive treatment. I have developed a life-like orthotopic glioma model and used this and other in vivo models to study basic mechanisms of glioma development and treatment. Previous studies had indicated that experimental gliomas could arise from glial stem cells and astrocytes. The present thesis describes the making and characterization of a novel mouse model, Ctv-a, where gliomas are induced from oligodendrocyte progenitor cells (OPCs). Our study shows that OPCs have the capacity to give rise to gliomas and suggests in light of previous data that the differentiation state of the cell of origin affects tumor malignancy. CDKN2A encodes p16INK4a and p14ARF (p19Arf in mouse) commonly inactivated in malignant glioma. Their roles in experimental glioma have been extensively studied and both proteins have tumor suppressor functions in glial stem cells and astrocytes. Here, we demonstrate that p19Arf only could suppress gliomagenesis in OPCs while p16Ink4a had no tumor suppressive effect. Functional DNA repair is pivotal for maintaining genome integrity, eliminating unsalvageable cells and inhibiting tumorigenesis. We have studied how RAD51, a central protein of homology-directed repair, affected experimental glioma development and have found that expression of RAD51 may protect against genomic instability and tumor development. Angiogenesis, the formation of new blood vessels from pre-existing ones, is a central feature of malignant progression in glioma. Antiangiogenic treatment by inhibition of vascular endothelial growth factor receptor signaling is used in the clinic for treatment of some cancers. We have investigated the effect of an alternative antiangiogenic protein, histidine-rich glycoprotein (HRG), on glioma development and found that HRG could inhibit the formation of malignant gliomas and completely prevent the formation of glioblastoma.

My research was focused on understanding the importance of mammalian RAD51 paralogs in genome maintenance and suppression of tumorigenesis. The investigation carried out during this study has been addressed toward gaining more insights into the involvement of RAD51 paralogs in DNA damage signalling, repair of various types of lesions including double stranded breaks (DSBs), daughter strand gaps (DSGs), interstrand crosslinks (ICLs), and in the protection of stalled replication forks. My study highlights the molecular functions of RAD51 paralogs in Fanconi anemia (FA) pathway of ICL repair, in the ATM and ATR mediated DNA damage responses, in homologous recombination (HR), and in the recovery from replication associated lesions. My research also focused on the development of a novel photoinducible ICL agent for targeted cancer therapy. The thesis has been divided into following sections as follows: Chapter I: General introduction that describes about DNA damage responses and the known functions of RAD51 paralogs across species in DNA repair and checkpoint The genome of every living organism is susceptible to various types of DNA damage and mammalian cells are evolved with various DNA damage surveillance mechanisms in response to DNA damages. In response to DNA damage, activated checkpoints arrest the cell cycle progression transiently and allow the repair of damaged DNA. Upon completion of DNA repair, checkpoints are deactivated to resume the normal cell cycle progression. Defective DNA damage responses may lead to chromosome instability and tumorigenesis. Indeed, genome instability is associated with several genetic disorders, premature ageing and various types of cancer in humans. The major cause of chromosome instability is the formation of DSBs and DSGs. Both DSBs and DSGs are the most dangerous type of DNA lesions that arise endogenously as well as through exogenous sources such as radiations and chemicals. Spontaneous DNA damage is due to generation of reactive oxygen species (ROS) through normal cellular metabolism. Replication across ROS induced modified bases and single strand breaks (SSBs) leads to DSGs and DSBs, respectively. Such DNA lesions need to be accurately repaired to maintain the integrity of the genome. To understand the various cellular responses that are triggered after different types of DNA damage and the possible roles of RAD51 paralogs in these processes, chapter I of the thesis has been distributed in to multiple sections as follows: Briefly, the initial portion of the chapter provides a glimpse of various types of DNA damage responses and repair pathways to deal with the lesions arising from both endogenous as well as exogenous sources. Owing to the vast range of cellular responses and pathways, the following section provides the detailed description and mechanisms of various pathways involved in taking care of wide range of DNA lesions from SSBs to DSBs. Subsequent section of chapter I provides a comprehensive description of maintenance of genome stability at the replication fork and telomeres. Germline mutations in the genes that regulate genome integrity cause various genetic disorders and cancer. Mutations in ATM, ATR, MRE11, NBS1, BLM and FANC (1-16), BRCA1 and BRCA2 that are known to regulate DNA damage signaling, DNA repair and genome integrity lead to chromosome instability disorders such as ataxia-telangiectasia, ATR-Seckel syndrome, AT-like disorder, Nijmegen breakage syndrome, Bloom syndrome, FA, and breast and ovarian cancers respectively. Interestingly, RAD51 paralog mutations are reported in patients with FA-like disorder and various types of cancers including breast and ovarian cancers. Mono-allelic germline mutations in all RAD51 paralogs are reported to cause cancer in addition to the reported cases of FA-like disorder with bi-allelic germline mutations in RAD51C and XRCC2. In accordance, the last section of the chapter has been dedicated to describe the genetics of breast and ovarian cancers and the known functions of tumor suppressors such as BRCA1, BRCA2 and RAD51 paralogs in the protection of genome. Despite the identification of five RAD51 paralogs nearly two decades ago, the molecular mechanism(s) by which RAD51 paralogs regulate HR and genome maintenance remain obscure. To gain insights into the molecular mechanisms of RAD51 paralogs in DNA damage responses and their link with genetic diseases and cancer, the following objectives were laid for my PhD thesis: 1) To understand the functional role of RAD51 paralog RAD51C in FA pathway of ICL repair and DNA damage signalling. 2) To dissect the ATM/ATR mediated targeting of RAD51 paralog XRCC3 in the repair of DSBs and intra S-phase checkpoint. 3) To uncover the replication restart pathway after transient replication pause and the involvement of distinct complexes of RAD51 paralogs in the protection of replication forks. 4) To design photoinducible ICL agent that can be activated by visible light for targeted cancer therapy. Chapter II: Distinct roles of FANCO/RAD51C protein in DNA damage signaling and repair: Implications for Fanconi anemia and breast cancer susceptibility RAD51C, a RAD51 paralog has been implicated in HR. However, the underlying mechanism by which RAD51C regulates HR mediated DNA repair is elusive. In 2010, a study identified biallelic mutation in RAD51C leading to FA-like disorder, whereas a second study reported monoallelic mutations in RAD51C associated with increased risk of breast and ovarian cancers. However, the role of RAD51C in the FA pathway of DNA cross-link repair and as a tumor suppressor remained obscure. To understand the role of RAD51C in FA pathway of ICL repair and DNA damage response, we employed genetic, biochemical and cell biological approaches to dissect out the functions of RAD51C in genome maintenance. In our study, we observed that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G2/M accumulation, which are hallmarks of the FA phenotype. We found that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrated that RAD51C plays a vital role in the HR-mediated repair of DSBs associated with replication. Finally, we showed that RAD51C participates in ICL and DSB induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C displayed that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor. Chapter III: ATM-and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair The RAD51 paralogs XRCC3 and RAD51C have been implicated in HR and DNA damage responses, but the molecular mechanism of their participation in these pathways remained obscured. In our study, we showed that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We found that RAD51C in CX3 complex but not in BCDX2 complex is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation was found to be required for chromatin loading and stabilization of RAD51 and HR-mediated repair of DSBs. Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we found that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation was required for the HR-mediated recovery of collapsed replication forks but is dispensable for the recovery of stalled replication forks. Together, our findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair. Chapter IV: RAD51 paralogs protect stalled forks and mediate replication restart in an FA-BRCA independent manner Mammalian RAD51 paralogs RAD51 B, C, D, XRCC2 and XRCC3 are critical for genome maintenance. To understand the crucial roles of RAD51 paralogs during spontaneously arising DNA damage, we have studied the RAD51 paralogs assembly during replication and examined the replication fork stability and its restart. We found that RAD51 paralogs are enriched onto the S-phase chromatin spontaneously. Interestingly, the number of 53BP1 nuclear bodies in G1-phase and micro-nucleation which serve as markers for under replicated lesions increases after genetic ablation of RAD51C, XRCC2 and XRCC3. Furthermore, we showed that RAD51 paralogs are specifically enriched at two major fragile sites FRA3B and FRA16D after replication fork stalling. We found that all five RAD51 paralogs bind to nascent DNA strands after replication fork stalling and protect the fork. Nascent replication tracts created before fork stalling with hydroxyurea degrade in the absence of RAD51 paralogs but remain stable in wild-type cells. This function was dependent on ATP binding at the walker A motif of RAD51 paralogs. Our results also suggested that RAD51 paralogs assemble into BCDX2 complex to prevent generation of DSBs at stalled replication forks, thereby safeguarding the pre-assembled replisome from the action of nucleases. Strikingly, we showed that RAD51C and XRCC3 in complex with FANCM promote the restart of stalled replication forks in an ATP hydrolysis dependent manner. Moreover, RAD51C R258H mutation that was identified in FA-like disorder abrogates the interaction of RAD51C with FANCM and XRCC3, and prevents fork restart. Thus, assembly of RAD51 paralogs in different complexes prevents nucleolytic degradation of stalled replication forks and promotes restart to maintain genomic integrity. Chapter V: Trans-dichlorooxovandium(IV) complex as a potent photoinducible DNA interstrand crosslinker for targeted cancer therapy Although DNA ICL agents such as MMC, cisplatin and psoralen are known to serve as anticancer drugs, these agents affect normal cells as well. Moreover, tumor resistance to these agents has been reported. We have designed and synthesized a novel photoinducible DNA crosslinking agent (ICL-2) which is a derivative of oxovanadiumterpyridine complex with two chlorides in trans position. We found that ICL-2 can be activated by UV-A and visible light to enable DNA ICLs. ICL-2 efficiently activated FA pathway of ICL repair. Strikingly, photoinduction of ICL-2 induces prolonged activation of cell cycle checkpoint and high degree of cell death in FA pathway defective cells. Moreover, we showed that ICL-2 specifically targets cells that express pathological RAD51C mutants. Our findings suggest that ICL-2 can be potentially used for targeted cancer therapy in patients with gene mutations in FA and HR pathway.

Le cancer de la prostate est le cancer le plus diagnostiqué et représente la troisième cause de mort par cancer chez les hommes au Canada. Environ un quart des patients auront une récidive biochimique suite aux traitements de première ligne (chirurgie ou radiation). Le traitement systématique subséquent est la thérapie de déprivation à l’androgène qui permettra, dans un premier temps, un ralentissement de la croissance de la tumeur dite hormonosensible. Puis, dans un second temps, cette thérapie mènera à une progression vers un stade résistant à la castration avec ou sans métastases. De plus, environ 10% des patients recevront un diagnostic de cancer de la prostate métastatique. Cette forme du cancer de la prostate est une des formes les plus agressives et, à ce jour, il n’existe aucun traitement curatif. C’est pourquoi il est important de mieux déterminer les facteurs impliqués dans la progression du cancer de la prostate. De nombreuses études menées par le laboratoire ont permis d’identifier la kinase IKKe comme un facteur impliqué dans la progression du cancer de la prostate. Ainsi, il a été montré que les lignées résistantes à la castration expriment constitutivement IKKe sécrètent IL-6 et IL-8, cytokines impliquées dans la transactivation du récepteur à l’androgène, un des mécanismes de progression de ce cancer. Pour permettre cette sécrétion, IKKe phosphoryle C/EBP-b, facteur de transcription, ce qui conduit à l’activation de la transcription des gènes IL-6 et IL-8. Par ailleurs, C/EBP-b joue un rôle dans le contrôle de la sénescence induite par la thérapie de déprivation à l’androgène. Nous émettons donc l’hypothèse que IKKe interfère avec les mécanismes de sénescence induit par la thérapie de déprivation à l’androgène et que cibler IKKe permettrait de contrôler la croissance du cancer de la prostate résistant à la castration. Le premier objectif fut d’évaluer l’impact de l’inhibition de IKKe sur le destin cellulaire lors de la progression du cancer de la prostate. La déplétion de IKKe induit un phénotype de sénescence dans la lignée PC-3. L’utilisation d’inhibiteurs de IKKe, le BX795 et l’Amlexanox, induit un phénotype de sénescence dans les cellules résistantes à la castration, où IKKe a une expression constitutive, accompagnée d’une forte induction de dommages à l’ADN et d’une instabilité génomique, de façon dose-dépendent. Dans les iii cellules hormonosensibles, les inhibiteurs n’ont que très peu d’effet puisque l’expression de IKKe n’est pas constitutive. De plus, les inhibiteurs de IKKe ralentit la croissance tumorale des xénogreffes PC-3 et DU145, alors qu’ils n’ont aucun effet sur la croissance tumorale de la xénogreffe hormonosensible 22Rv1. Le deuxième objectif avait pour but de caractériser le rôle de IKKe dans l’échappement de la sénescence induite par la thérapie de déprivation à l’androgène. Nos travaux de recherche montrent que la déplétion ou l’utilisation de l’Amlexanox induit une diminution du recrutement de C/EBP-b au niveau du promoteur du gène de Rad51, protéine indispensable pour l’efficacité des mécanismes de réparation des dommages à l’ADN. De plus, bloquer la voie de réparation médiée par Rad51 par l’intermédiaire de l’Amlexanox améliore la sensibilité à l’Olaparib des cellules résistantes à la castration in vitro. De même, dans un modèle de xénogreffes résistantes à la castration, la combinaison Amlexanox – Olaparib montre un meilleur effet sur le ralentissement de la croissance tumorale. En conclusion, ce projet de doctorat aura permis de préciser les mécanismes impliquant IKKe dans la progression du cancer de la prostate. Les résultats apportent un nouvel éclairage sur le rôle de IKKe dans la régulation des dommages à l’ADN, particulièrement sur la transcription du gène Rad51 via C/EBP-b. De plus, les expériences in vivo montrent le potentiel thérapeutique de l’Amlexanox, notamment en le combinant avec l’Olaparib, afin de contrôler la croissance des tumeurs résistantes à la castration. / Prostate cancer is the most frequently diagnosed cancer and is the third leading cause of cancer death in men in Canada. About a quarter of patients will have a biochemical recurrence following first-line treatments (surgery or radiation). The subsequent systematic treatment is androgen deprivation therapy which will initially slow the growth of hormone- sensitive tumors. Almost inevitably this therapy will lead to progression towards castrate resistance with or without metastases. In addition, approximately 10% of patients will be initially diagnosed with metastatic prostate cancer. This form of prostate cancer is one of the most aggressive forms and, to date, there is no curative treatment. This underscores the important of better understanding the factors involved in the progression of prostate cancer. Numerous studies conducted by our laboratory have identified IKKe kinase as a factor involved in the progression of prostate cancer. It has been shown that castrate resistant cell lines that constitutively express IKKe secrete IL-6 and IL-8, cytokines involved in the transactivation of the androgen receptor, one of the mechanisms leading to cancer progression. IKKe contributes to this through the phosphorylation of C/EBP-b, a transcription factor, which leads to the activation of the transcription of the IL-6 and IL-8 genes. Furthermore, C/EBP-b plays a role in the control of senescence induced by androgen deprivation therapy. We therefore hypothesized that IKKe interferes with the mechanisms of senescence induced by androgen deprivation therapy and that targeting IKKe would control the growth of castration-resistant prostate cancer. The first objective was to assess the impact of IKKe inhibition on cell fate during prostate cancer progression. Depletion of IKKe induces a senescence phenotype in the PC- 3 cell line. The use of the IKKe inhibitors, BX795 and Amlexanox, induces a senescence phenotype in castrate resistant cell lines, where IKKe is constitutively expressed, accompanied by a strong induction of DNA damage and genomic instability in a dose- dependent manner. Since IKKe expression is not constitutive in hormone-sensitive cell lines, IKKe inhibitors have very little effect in these. In addition, IKKe inhibitors slow the growth of the PC-3 and DU145 xenografts, while they have no effect on the growth of hormone-sensitive 22Rv1 xenografts. v The second objective was to study the role of IKKe in the senescence escape induced by androgen deprivation therapy. Our research shows that the IKKe depletion or the use of Amlexanox induces a decrease in the C/EBP-b recruitment on the promoter of the Rad51 gene, a protein essential for the efficiency of the mechanisms of DNA damage repair. In addition, blocking the Rad51-mediated repair pathway through Amlexanox enhances susceptibility to Olaparib in castrate resistant cell lines in vitro. Likewise, in a castrate resistant xenograft model, the combination Amlexanox - Olaparib has a stronger effect on tumor growth as compared to a control or each treatment individually. In conclusion, this doctoral research has made it possible to identify a new mechanism implicating IKKe in the progression of prostate cancer. The results show the role of IKKe in the regulation of DNA damage, particularly on the transcription of the Rad51 gene via C/EBP-b. In addition, in vivo experiments show the therapeutic potential of Amlexanox, particularly in combination with Olaparib, to control the growth of castrate resistant tumors.

No / Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611-20. (c)2012 AACR.