High fidelity double strand break repair is paramount for the maintenance of genome integrity and faithful passage of genetic information to the following generation. Homologous recombination (HR) and non-homologous end joining (C-NHEJ) have evolved as the two major pathways for the efficient and accurate repair of double strand breaks (DSBs). In addition, a minor Ku- and Ligase IV-independent end-joining pathway has been identified and implicated in the formation of chromosomal translocations. This alternative end-joining pathway occurs by bridging the break ends through annealing between short microhomologies, hence the name microhomology-mediated end joining (MMEJ). In addition to these defined DSB repair pathways, a broken DNA end possesses immense mutagenic potential to generate chromosomal rearrangements. Diverse and complex rearrangements are a commonly observed feature amongst cancer cells. The focus of this thesis is to examine the role of Replication Protein A (RPA) in binding single-stranded DNA (ssDNA) repair intermediates to promote error free repair and to prevent mutagenic chromosomal deletions and rearrangements. RPA is a highly conserved, heterotrimeric ssDNA binding protein with a ubiquitous role in all DNA transactions involving ssDNA intermediates. RPA promotes resection at DSBs to facilitate HR and abrogation of this function has severe consequences. Defective RPA can lead to the formation of secondary structures and impair loading of homology search proteins such as Rad52 and Rad51. Using a chromosomal end-joining assay, we demonstrate that hypomorphic rfa1 mutants exhibit elevated frequencies of MMEJ by up to 350-fold. Biochemical characterization of RPAt33 and RPAt48 complexes show these mutants are compromised for their ability to prevent spontaneous annealing and the removal of secondary structures to fully extend ssDNA. These results demonstrate that annealing between MHs defines a critical control to regulate MMEJ repair. Therefore, RPA bound to ssDNA intermediates shields complementary sequences from annealing to promote error-free HR and prevents repair by mutagenic MMEJ, thereby preserving genomic integrity. RPA also impedes intrastrand annealing between short inverted repeat sequences to prevent the formation of foldback structures. Foldbacks have been proposed to drive palindromic gene amplification, a genome destabilizing rearrangement that can disrupt the protein expression equilibrium and is a prevalent phenomenon within tumor cells. Palindromic duplications are elevated ~1000-fold in rfa1-t33 sae2Δ and rfa1-t33 mre11-H125N mutants compared to sae2Δ or mre11-H125N, yet we did not detect these events in the hypomorphic rfa1-t33 mutant. This suggests that Mre11 and Sae2 play critical roles in preventing palindromic amplification through regulation of the Mre11 structure-specific endonuclease to process DNA foldbacks (also called DNA hairpins). Therefore, Mre11-Sae2 together with RPA prevent palindromic gene amplification. Together, these data focus the spotlight on RPA playing active central and supporting roles to sustain genome stability. This additionally raises that notion that secondary structures are potent instigators and mediators of many genome rearrangements and their prevention by RPA is absolutely crucial.

Genomic stability is essential for maintaining cellular function and preventing oncogenic transformation. DNA double strand breaks (DSBs) are the most severe form of DNA damage. Classical non-homologous end joining (cNHEJ) is one of two major DSB repair pathways in mammalian cells. During lymphocyte development, NHEJ is required for the repair of programmed double strand breaks (DSBs) occurring during V(D)J recombination and Class Switch Recombination (CSR). Defects in cNHEJ cause severe combined immunodeficiency (SCID) in patients and animal models. Misrepair of physiological DSBs generated during normal lymphocyte development results in clonal translocations, which is characteristic of human lymphoid malignancy: it is the most common cancer type in children and the third leading cancer type in adults. Lymphoid malignancies are characterized by clonal translocations involving the antigen receptor loci, which often arise from the misrepair of programmed double strand breaks (DSBs). Furthermore, cNHEJ also plays a critical role in aging and therapeutic responses to genotoxic cancer therapy. My thesis study focuses on the function and regulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs is a vertebrate specific NHEJ factor and one of most abundant proteins in human cells. Together with the DNA binding Ku70 and Ku80 heterodimer, DNA-PKcs forms the DNA dependent protein kinase (DNA-PK) holoenzyme. In addition to its important role in cNHEJ, DNA-PK also orchestrates the mammalian DNA damage response (DDR) together with the related ATM and ATR kinases by phosphorylating hundreds of partially overlapping substrates. My thesis goes deeper than the kinase and signaling function of DNA-PKcs during cNHEJ. We investigated the structural function of DNA-PKcs in cNHEJ (chapter 2) and A-EJ (chapter 3), using a mouse model with point mutations that lead to the expression of kinase dead (KD) DNA-PKcs. Second, we explored potential roles of DNA-PKcs outside of cNHEJ and A-EJ with a mouse model of DNA-PKcs lacking specific phosphorylation sites (chapter 4). Altogether, our results identified an unexpected structural function of DNA-PKcs in cNHEJ and the DNA damage response and expanded the purview of the function of DNA-PKcs into new areas, including hematopoiesis, alternative end-joining and potentially nucleoli stress.

DNA interstrand crosslinks (ICLs) are a potent type of DNA damage that arise as a consequence of normal cell metabolism. By covalently linking opposing strands of the double helix, ICLs block essential DNA transactions such as replication, transcription, and recombination. If unrepaired, or incorrectly repaired, ICLs can lead to gross genome instability and cell death. This cytotoxicity has been exploited in the clinic, where ICL inducing drugs are among the oldest and most widely prescribed anti-cancer therapies. However, acquired resistance is a significant limitation of these drugs, and the mechanism by which this occurs remains largely elusive. In order to develop more effective ICL-based therapies, it is imperative to first fully elucidate how healthy cells respond to and repair ICLs. Moreover, better understanding ICL repair mechanisms is necessary to fully unravel the complex DNA repair networks that govern genomic integrity, and understand the physiology of diseases such as Fanconi Anemia, which result from the inability to efficiently repair ICL lesions. Multiple mechanisms of ICL repair exist, and repair pathway choice is primarily determined by the phase of the cell cycle. In proliferating cells, the ICL repair occurs during S-phase, and in a process termed “replication coupled repair” (RCR). In contrast, slowly or non-dividing cells rely on an alternative modality of repair called “replication independent repair” (RIR). RIR is critical for homeostasis and survival in quiescent healthy cells that (for example, neurons) and in cycling cells deficient for replication coupled repair proteins (i.e. Fanconi Anemia cells). Despite its importance, little is known about RIR. This is due, in part, to the fact that ICL repair has been primarily studied in systems, such as cultured cells, that favor RCR and are therefore bias against RIR. More recently, non-replicating Xenopus cell-free extracts has emerged as a powerful system to study RIR. This system faithfully recapitulates RIR and has been instrumental in identifying DNA polymerase kappa (Pol κ) and the eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), as two critical RIR factors. However, other important RIR factors are yet to be identified. ICL repair is unique among DNA repair pathways as it harnesses proteins from diverse DNA repair pathways including, Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), and Double Strand Break Repair (DSBR). Chapter 1 provides an overview of these pathways including the types of DNA damage that each pathway responds to, key steps of the repair process, and the corresponding proteins that are involved. This chapter provides context for the rest of the thesis in which I explore the contribution of multiple DNA repair proteins on the repair of ICL lesions. In Chapter 2, I detail our studies assessing the contribution of the MMR machinery to RIR. We show that the mismatch repair sensor, MutS complex (MSH2-MSH6), is critical for ICL recognition, and the stepwise recruitment of other MMR proteins including MutL (MLH1-PMS2) and EXO1. In this chapter, I also investigate how ICL structure influences repair. I find that more distorting ICLs use an MMR-dependent ICL repair mechanism, while less distorting ICLs are repaired MMR-independently (see also Appendix A), or not repaired at all. Appendix B further explores the contribution of the MMR pathway on ICL repair in mammalian cells. Finally, in Appendix C and D we provide further evidence that RIR is fundamentally distinct from replication coupled ICL repair, as depletion of key RCR proteins from our extracts yields no phenotype. I summarize all of these findings in Chapter 3, and discuss their implications to the DNA repair field as well as the clinic, where crosslinker drugs remain a mainstay in the treatment of cancer.

DNA is continuously subjected to various types of damage during normal cellular metabolism. Among these, a DNA double-strand break (DSB) is one of the most cytotoxic lesions, and can lead to genomic instability or cell death if misrepaired or left unrepaired. The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex orchestrates the cellular response to DNA damage through its structural, enzymatic, and signaling roles. It senses DSBs and is essential for both of the two major repair mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). In addition, the complex tethers DNA ends, activates Tel1/ATM kinase, resolves hairpin capped DNA ends and maintains telomere homeostasis. Although significant progress has been made in characterizing the complex, many questions regarding the precise mechanism of how this highly conserved, multifunctional complex manages its various activities in chromosome metabolism remain to be solved. The overarching focus of this thesis is to further expand our understanding of the molecular mechanism and regulation of the MRX complex. Specifically, the contributions of Xrs2, Tel1, and Mre11 3’-5’ dsDNA exonuclease in the multiple roles of the MRX complex are examined. Xrs2/Nbs1, the eukaryotic-specific component of the complex, is required for the nuclear transport of Mre11 and Rad50 and harbors several protein-interacting domains. In order to define the role of Xrs2 as a component of the MRX complex once inside the nucleus, we fused a nuclear localization signal (NLS) to the C terminus of Mre11 and assayed for complementation of xrs2Δ defects. We found that nuclear localization of Mre11 (Mre11-NLS) is able to bypass several functions of Xrs2, including DNA end resection, meiosis, hairpin resolution, and cellular resistance to clastogens. Using purified components, we showed that the MR complex has the equivalent activity to MRX in cleavage of protein-blocked DNA ends. Although Xrs2 physically interacts with Sae2, end resection in its absence remained Sae2 dependent in vivo and in vitro. MRE11-NLS was unable to rescue the xrs2Δ defects in Tel1 kinase signaling and NHEJ, consistent with the role of Xrs2 as a chaperone and adaptor protein coordinating interactions between the MR and other repair proteins. To further characterize the role of Xrs2 in Tel1 activation, we fused the Tel1 interaction domain of Xrs2 to Mre11-NLS (Mre11-NLS-TID). Mre11-NLS-TID was sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells, indicating that Tel1 recruitment and activation are separate functions of the MRX complex. Unexpectedly, we found a role for Tel1 in stabilizing Mre11-DNA association independently of its kinase activity. This stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Moreover, while nuclear-localized MR complex is sufficient for HR without Xrs2, MR is insufficient for DNA tethering, stalled replication fork stability, and suppression of chromosomal rearrangements. Enforcing Tel1 recruitment to the MR complex fully rescued these defects, highlighting the important roles for Xrs2 and Tel1 in stabilizing the MR complex to prevent replication fork collapse and genomic instability. Lastly, in order to decipher the functional significance of the Mre11 3’-5’ dsDNA exonuclease activity in DSB repair, mre11 mutant alleles reported to be proficient endonuclease and deficient exonuclease were analyzed in vivo and in vitro. Although we did not observe a clear separation of the nuclease activities in vitro, our genetic analysis of the mutant allele is consistent with the current two-stepped, bidirectional model of end resection.

Severing the DNA double helix is a requisite step in the exchange of genetic material between homologous chromosomes in meiosis and between immunoglobulin domains during the generation of immune-receptor diversity. While these DNA transactions are essential for human fertility and the development of the immune system, misrepaired or unrepaired DNA double-strand breaks (DSBs) can lead to chromosome rearrangements or cell death. Indeed, ionizing radiation which generates DSBs in tumors is a cornerstone of cancer therapy. However, tumor cells can tolerate otherwise lethal levels of DNA damage by exploiting DNA repair pathways. Thus, discovering new strategies to selectively inhibit the repair of DSBs remains a major goal in the development of more effective cancer therapies. DSB repair may occur by multiple pathways, and the decision to use one pathway over another is influenced by cell cycle stage, the chromatin state, and the complexity of the inciting lesion. Mammalian cells primarily resolve DSBs by ligating the free ends together during a process termed “non-homologous end joining” (NHEJ). However, chemically modified or damaged DSB ends cannot be directly ligated by the NHEJ machinery. If NHEJ fails, DSBs may be nucleolytically cleaved to generate 3’ single-stranded DNA overhangs via a process called end resection. The resected DNA strands are poor substrates for NHEJ and instead search for homology in the genome to resynthesize the sequence surrounding the break site. This process is termed “homology-directed repair” (HDR). HDR is tightly coupled to cell cycle phase to ensure that resection occurs during late S and G2 when the ideal template, the sister chromatid, may be utilized. Following DNA damage, repair factors accumulate at DSB sites and form microscopically-detectable DNA repair foci. The dynamics of these foci may be observed by time-lapse microscopy making it possible to observe the behavior of breaks undergoing HDR and NHEJ. Interestingly, in yeast and mammalian cells, DNA motion is increased following DSB generation. DNA movements can lead to the clustering of DSBs into a common repair focus. DSB movements are intricately related to repair by HDR and require factors critical for resection initiation and downstream recombination. In contrast, DSBs undergoing NHEJ are relatively immobile. These observations suggest that the commitment of DSB repair to HDR regulates DSB movement and clustering; however, how DSB clustering might promote repair and whether active mechanisms drive this process remain relatively obscure. Recent studies have proposed roles for cytoskeletal proteins in genome organization and chromosomal dynamics. The Arp2/3 complex generates propulsive forces by nucleating a highly branched network of actin filaments. Genotoxic agents trigger actin polymerization in the nucleus. However, how DSB repair pathways might harness nuclear Arp2/3 machinery is unknown. Chapter 1 provides an overview of these pathways including the key steps of DSB repair, the regulation of actin nucleation, and the proteins involved in chromatin mobility. Chapter 1 provides context for the rest of the thesis in which I explore the contribution of nuclear actin polymerization to DSB repair. In Chapter 2, I detail our studies assessing the contribution of the Arp2/3 complex to DSB movement and clustering. Using Xenopus laevis cell-free extracts and mammalian cells, we show that actin nucleation machinery (WASP, Arp2/3, and actin) is recruited to damaged chromatin undergoing HDR. In this chapter, I also investigate how Arp2/3-driven DSB movements specifically promote the dynamics of HDR breaks, while Arp2/3 activity does not influence NHEJ breaks. Finally, I show that reduced DSB movement produces defects in DNA end processing and HDR efficiency, while the efficiency of end-joining is unaffected. I summarize all of these findings in Chapter 3 and discuss their implications for DNA repair, translocation formation, and clinical applications.

Introduction: The wide-ranging prognostic implications of a breast cancer diagnosis highlight the need to better enable women to make informed decisions regarding screening and treatment options. As several cancer susceptibility syndromes have been linked to germline mutations resulting in defective DNA repair, including the predisposition to breast cancer due to BRCA1 and BRCA2 mutations, more subtle defects in DNA repair capacity may contribute to the components driving differential susceptibility within the general population. Hence, understanding the role of DNA repair capacity in breast cancer onset may aid in the development of a more comprehensive risk profile, thereby furthering the effort to target relevant populations for early screening. In the studies undertaken for this dissertation, we employed various methodologies capturing endpoints across different repair pathways detectable in blood to both further elucidate the etiologic basis of breast cancer development and leverage the information into the potential development of a screening biomarker. Methods: For the phenotypic assessment of nucleotide excision repair (NER) capacity, we developed an ELISA-based method to determine benzo(a)pyrene diolepoxide (BPDE)-DNA adduct capacity in lymphoblastoid cell lines. Gene expression levels were assessed with pre-designed Taqman kits in RNA-derived cDNAs from mononuclear cells using a real-time PCR-based platform. Methylation analysis was conducted with in-house designed assays on bisulfite-converted DNA from mononuclear cells using a pyrosequencing platform. Finally, single nucleotide polymorphisms (SNP) genotyping was assessed in DNA derived from white blood cells with pre-designed Taqman SNP genotyping assays using a real-time PCR-based platform. All studies were conducted in sister-sets enrolled in the New York site within the Breast Cancer Family Registry and all statistical analysis was conducted using the R Foundation for Statistical Computing (2011). Results: We did not detect an association between the ELISA-based phenotypic assessment of NER capacity in the lymphoblastoid cells lines of the sister-sets (n=246, 114 sister-sets) and breast cancer risk (OR = 1.0, 95%CI=0.95, 1.04). Furthermore, we did not observe a correlation with previously determined NER capacity in the same population using an immunohistochemical-based method (r= -0.01, p=0.86). In our gene expression study (n=569, 218 sister-sets), women in the lowest tertile of ATM expression had a heightened risk of breast cancer compared to women in the highest tertile of expression, adjusted for age at blood draw and smoking status (OR=2.12, 95%CI=1.09, 4.12). This association was largely restricted to women with an extended family history of breast cancer (pinteraction = 0.06). Additionally, women in the lowest tertile of MSH2 expression also had a heightened risk of breast cancer compared to women in the highest tertile of expression, adjusted for age at blood draw and smoking status (OR=2.75, 95%CI=1.31, 5.79). The association observed between reductions in ATM expression level and breast cancer risk was lost upon incorporating previously determined end-joining capacity of EcoRI-generated sticky end substrates (OR=1.28, 95%CI=0.15, 11.2) and HincII-generated blunt end substrates (OR=1.55, 95%CI=0.15, 15.5) into the model, suggesting that the impact on risk due to reductions in ATM expression maybe partially driven by the reduction in double strand break repair capacity. In our study investigating breast cancer risk due to the impact of epigenetic modulation on DNA repair gene activity (n=569, 218 sister-sets), no association with risk was observed due to differential promoter methylation levels of BRCA1 (OR=1.09, 95%CI=0.98, 1.20), MLH1 (OR=1.19, 95%CI=0.91, 1.55) or MSH2 (OR=0.89, 95%CI=0.48, 1.64). Furthermore, no correlation between BRCA1 and expression (r=-0.05, p=0.39) or MSH2 methylation and expression (r=-0.04, p=0.39) was observed. Finally, our mismatch repair genotyping study (n=714, 313 sister-sets) indicated an association between the variant MutY_rs3219489 (OR=2.23, 95%CI=1.10, 4.52) and breast cancer risk, as well as a borderline association with risk due to the variant MSH2_rs2303428 (OR=1.71, 95%CI=0.99, 2.95). Furthermore, a protective effect was observed due to the variant MLH3_rs175080, restricted to women without an extended family history of breast cancer (pinteraction = 0.03). Conclusion: These studies suggest that the deregulation of targets spanning various DNA repair pathways contribute to the risk of familial breast cancer.

It is incumbent upon cellular life to ensure the faithful transmission of genetic material from mother cell to daughter cell and from parent to progeny. However, cells are under constant threat of DNA damage from sources both endogenous and exogenous, such as the products of metabolism and genotoxic chemicals. Thus, cells have evolved multiple systems of repair to ensure genome integrity. The DNA double-strand break (DSB) is among the most lethal forms of DNA damage, and a critical pathway to resolve these lesions is homologous recombination (HR). During HR, information lost at the cut site of one locus is repaired when the damaged site locates a homologous sequence in the nucleus to use as template for repair. The process by which a cut chromosome finds its homolog is known as homology search, and, while the enzymatic steps of HR have been well studied in recent years, the coordination of cell biological events like HS in the context of the crowded nucleus has remained poorly understood. Recently, our laboratory and others have studied a phenomenon known as DNA damage-induced increased chromosomal mobility, in which chromosomal loci, both damaged and undamaged, explore larger areas of the nucleus after the formation of DSBs. The increase in the mobility of cut loci is known as local mobility, and the increase in mobility of undamaged loci in response to a break elsewhere in the nucleus is known as global mobility. Here, I report that the recombination machinery and the DNA damage checkpoint cooperate in order to regulate global mobility of chromosomes following DSB formation. The RecA-like recombinase Rad51 is required for global mobility, and exerts its effect at single-stranded DNA (ssDNA), but its canonical homology search and strand exchange functions are not required. I find that Rad51 is ultimately required to displace Rad52, which is revealed to be an inhibitor of mobility when bound to ssDNA in the absence of Rad51. Thus, recombination factors can serve as DNA damage sensors, and relay information to the checkpoint apparatus in order to govern the initiation of increased mobility after DSB formation. I have also studied how the baseline confinement of loci is established, and assessed the contributions of several genes involved in repair to increased mobility. These observations offer novel insight into previously unappreciated regulatory functions performed by the recombination machinery, and demonstrate how the progression of DNA repair pathways influences nuclear organization.

Os Papilomavírus Humanos (HPV) são vírus de DNA, não envelopados que infectam as células epiteliais. A infecção persistente por alguns tipos de HPV é o principal fator de risco para o desenvolvimento do câncer cervical. A maquinaria de reparo de DNA desempenha um papel essencial em várias fases do ciclo de vida do HPV e é crucial para a sobrevivência de células tumorais. Durante a transformação maligna, as oncoproteínas E6 e E7 de HPV são capazes de induzir alterações cromossômicas e numéricas, além de modular a resposta de danos ao DNA. Estas observações sugerem que a maquinaria celular de reparo de dano ao DNA podem desempenhar um papel duplo na biologia do HPV e na sua patogênese. No presente estudo, procurou-se investigar o papel das proteínas de reparo de DNA na biologia das células derivadas de câncer cervical. A fim de alcançar este objetivo, a expressão de 189 genes foi silenciada em células HeLa (HPV 18) e em células SiHa (HPV16), bem como em queratinócitos humanos primários (QHP), utilizando vetores lentivirais que expressam shRNAs específicos. O efeito do silenciamento gênico foi determinado por ensaios de viabilidade celular, análise de proliferação celular, ensaio clonogênico e de formação de colônias em soft ágar. Observamos que o silenciamento dos genes ATM, BRCA1, CHEK2 e HMGB1 reduziu a taxa de crescimento celular, o potencial de crescimento em colônia e a capacidade de crescimento independente de ancoragem das linhagens celulares derivadas de câncer cervical transformadas por HPV, sem afetar QHP. O tratamento das linhagens celulares com fármacos capazes de inibir a atividade das proteínas ATM e CHEK2 revelou uma maior sensibilidade das células tumorais à inibição destas proteínas quando comparadas a QHP. Além disso, mostramos que QHP que expressavam E6E7 ou somente E6 de HPV16 foram mais sensíveis a estes inibidores, quando comparados ao controle QHP ou QHP expressando apenas E7. Além disso, QHP que expressavam mutantes de E6 de HPV16, defectivos para a degradação de p53, foram menos sensíveis do que QHP, que expressavam HPV16 E6 selvagem. Desta forma, estes resultados indicam que estes genes são necessários para a sobrevivência de células transformadas por HPV. Além disso, os nossos resultados sugerem que este efeito está relacionado com a expressão oncoproteína de HPV16 E6 e a sua capacidade para degradar p53. / Human Papillomaviruses (HPV) are non-enveloped DNA viruses that infect epithelial cells. Persistent infection with some HPV types is the main risk factor for the development of cervical cancer. DNA repair machinery plays an essential role in several stages of the HPV life cycle and is crucial for tumor cells survival. During malignant transformation, HPV E6 and E7 oncoproteins induce structural and numerical chromosome alterations and modulate DNA damage response. These observations suggest that cellular DNA repair machinery may play a dual role in both HPV biology and pathogenesis. In the present study, we sought to investigate the role of DNA repair proteins in cervical cancer derived cells biology. In order to achieve this goal, the expression of 189 genes was silenced in HeLa (HPV18) and SiHa (HPV16) cells as well as in primary human keratinocytes (PHK) using lentiviral vectors expressing specific shRNA. The effect of gene silencing was determined by cell viability assay, cell growth analysis, clonogenic and soft agar colony formation test. We observed that ATM, BRCA1, CHEK2 and HMGB1 down-regulation decreased growth rate, clonogenic potential and cellular anchorage-independent growth of HPV-transformed cervical cancer-derived cell lines with no effect in normal keratinocytes. Treatment of cells with drugs that inhibit ATM and CHEK2 activity showed that tumor cells are more sensitive to the inhibition of these proteins than PHK. Besides, we show that PHK expressing HPV16 E6 alone or along with HPV16 E7 were more sensitive to these inhibitors than control PHK or PHK expressing only E7. Moreover, PHK expressing E6 mutants defective for p53 degradation were less sensitive than PHK expressing E6wt. Moreover, to potentiate the effect observed by the ATM and CHEK2 inhibition, we treated cells lines with Doxorubicin and Cisplantin. We observed that tumor cells lines and PHK expressing HPV16 E6 or HPV16 E6/E7 were more sensitive to DNA damage induction. Altogether, these results indicated that these genes are required for HPV-transformed cells survival. Besides, our results suggest that this effect is related to HPV16 E6 oncoprotein expression and its capacity to degrade p53.

Lifestyle diseases represent a large burden on developed societies and account for much morbidity worldwide. Research has shown that eating a diet rich in fruit and vegetables helps to ameliorate and prevent some of these diseases. Antioxidants found in fruits and vegetables may provide a substantial benefit in reducing disease incidence. This thesis examines the antioxidant properties of resveratrol, piceatannol, and pterostilbene, and the ability of Burkitt's Lymphoma (Raji) cells to uptake these three antioxidants. It also studies the effect of the antioxidants in protecting against DNA damage and their role in DNA repair following oxygen radical exposure in Raji cells. The Oxygen Radical Absorbance Capacity (ORAC) assay was used to measure overall antioxidant contribution as well as the ability of Raji cells to uptake antioxidant following exposure to 2,2’-Azobis(2-methyl-propionamide) dihydrochloride (AAPH). The single cell gel electrophoresis (Comet) assay was used to assess DNA damage and DNA repair rates of cells. Results showed that Raji cells, following oxygen radical exposure, significantly uptake pterostilbene (p < 0.0001), but not piceatannol or resveratrol. Piceatannol provided protection against hydrogen peroxide induced DNA damage, but pterostilbene and resveratrol increased DNA damage following hydrogen peroxide treatment. None of the compounds showed any effect on DNA repair. Overall, this study indicates there is merit for further research into the bioactive roles, including antioxidant capacity, of all three compounds. Such research may provide evidence for the more widespread use of these and other food based compounds for preventing lifestyle diseases.

DNA damage represents a major challenge to the faithful replication and transmission of genetic information from one generation to the next. Cells utilize a highly integrated network of pathways to detect and accurately repair DNA damage. Mutations arise when DNA damage persists undetected, unrepaired, or repaired improperly. Mutations are a driving force of carcinogenesis and therefore many of the DNA damage surveillance and repair mechanisms guard against the transformation of normal cells into cancer cells. Central to the detection and repair of DNA damage is the relocalization of DNA damage surveillance proteins to DNA damage where they assemble into subnuclear foci and are capable to producing a signal that the cell interprets to induce cellular modifications such as cycle arrest and DNA repair which are important DNA damage tolerance. In this work, I describe my quest to understand the mechanisms underlying the assembly, maintenance, and disassembly of these DNA damage-induced foci and how they affect DNA damage signaling in Saccharomyces cerevisiae. First, I describe phenotypic characterization of a novel mutation that impairs assembly of the 9-1-1 checkpoint clamp complex into foci. Second, I describe my work to further understand the roles of the histone phosphatase Pph3 and phosphorylated histone H2A in modulating DNA damage signaling. Third, I include my work to uncover the possible mechanism by which the helicase Srs2 works to enable termination of DNA damage signaling. In summary, this thesis documents my efforts to understand the cellular and molecular nature of DNA damage signaling and how signaling is turned off in coordination with DNA damage repair.