Les cellules utilisent deux stratégies principales pour réparer les cassures double-brin (CDB) de l’ADN : la recombinaison homologue (RH) et la ligature d’extrémités non homologues (NHEJ). D’autres voies de réparation plus minoritaires existent qui mènent nécessairement à des altérations génétiques : Single Strand Annealing (SSA) et Alternative End Joining (A-EJ). Nous avons proposé que le choix entre les mécanismes de réparation des CDB nécessite deux étapes : 1) la compétition entre C-NHEJ et la résection, 2) sur les extrémités d’ADN résectées, la compétition entre RH, A-EJ et SSA. Ici, nous avons étudié la régulation de la deuxième étape de ce choix. En outre, la létalité synthétique a été décrite entre RAD52 et BRCA2/PALB2. Étant donné que BRCA2 et PALB2 sont nécessaires pour le chargement de RAD51 sur l’ADN simple brin, cela suggère que la formation d’un nucléofilament RAD51/ADNsb ordonné et RAD52 sont des acteurs essentiels dans le choix de la réparation à la deuxième étape. Nous avons trouvé que l’extinction de RAD51 ou BRCA2 stimule à la fois le SSA et EJ, d’une manière épistatique et que RAD52 contrôle la stimulation de SSA et A-EJ, en absence de RAD51. De plus, par séquençage haut débit, nous montrons que l’inhibition de RAD51 induit une instabilité génomique médiée par la microhomologie au niveau du génome. Cependant l’inhibition de RH n’est pas la réparation directe suffisante vers SSA et A-EJ. En effet, en utilisant des mutants dominants négatifs de RAD51, nous avons trouvé que les mutants du site de fixation/hydrolyse de l’ATP inhibent la RH et stimulent le SSA et que la chimère SMRAD51, qui inhibe la RH, inhibe également le SSA et EJ. Par TEM, nous avons observé que SMRAD51 perturbe spécifiquement la structure de l’ADNsb/SMRAD51. De l’autre côté, deux mutants d’hydrolyse de l’ATP de RAD51 ont montré que la liaison à l’ATP et l’hydrolyse d’ATP sont nécessaires pour une charge efficace de RAD51 sur l’ADN endommagé, dans les cellules vivantes. Ces deux mutants d’ATP ne se fixent pas à l’ADN en opposition à SMRAD51. Enfin, nous montrons que RAD51 n’empêche pas la résection étendue, mais que, in vitro, la protéine RAD51 empêche l’annealing de l’ADNsb complémentaire. Au total, les données montrent que RAD51 joue effectivement un rôle crucial dans la deuxième étape du choix de la voie de réparation des CDB à travers deux mécanismes distincts : 1- il déclenche la RH par son activité catalytique, 2- mail il empêche également les mécanismes non conservateurs dépendants de RAD52, SSA et A-EJ, en altérant l’étape de l’annealing. Par conséquent, le choix en deuxième étape entre la RH et les mécanismes mutagènes, SSA et A-EJ, est orchestré par un antagonisme entre RAD51 et RAD52. / Cells use two primary strategies to repair DNA double-strand break (DSB): Homologous Recombination (HR) and Non-homologous end joining (NHEJ). Beside other mechanisms exist that necessarily lead to genetic alterations: Single Strand Annealing (SSA) and Alternative End Joining (A-EJ). We have proposed that the choice between DSB repair mechanisms requires two steps: 1) competition between C-NHEJ and resection; 2) on resected DNA ends, competition between HR, A-EJ and SSA. Herein we investigated the regulation of the second step of this choice. Furthermore, synthetic lethality has been described between RAD52 and BRCA2/PALB2. Since BRCA2/PALB2 are required for the loading of RAD51 onto the ssDNA, suggesting that both the formation of an ordered RAD51/ssDNA nucleofilament and RAD52 are central players in the choice of repair at the 2nd step.We found that silencing RAD51 or BRCA2 stimulate both SSA and EJ, in an epistatic manner and that silencing RAD51 induced microhomology mediated genomic instability at a genome wide level. Moreover, we show that RAD52 controls the stimulation of SSA and A-EJ, upon RAD51 silencing. However inhibition of HR is not sufficient redirect repair toward SSA and A-EJ. Indeed, using dominant negative mutants of RAD51 we found that the chimera SMRAD51, which inhibits HR, also inhibits SSA and EJ. By TEM we observed that SMRAD51 specifically disrupts the structure of the ssDNA/SMRAD51. On the other side, two ATP hydrolysis mutants of RAD51 showed that ATP binding and hydrolysis is required for efficient loading of RAD51 on damaged DNA, in living cells. These two ATP mutants that do not bind DNA in opposition to SMRAD51, do not inhibit A-EJ and stimulate SSA. Finally we show RAD51 do not prevents extended resection, but that, in vitro, RAD51 protein prevents the annealing of complementary ssDNA.Altogether the data show that RAD51 indeed plays a pivotal role in the second step of DSB repair pathway choice through two separable mechanisms: 1- it triggers HR through its catalytic HR activity 2- but it also prevents RAD52-dependent non-conservative mechanisms SSA and A-EJ, by impairing the annealing step. Therefore, the choice between HR and alternative mutagenic mechanisms A-EJ and SSA (2nd step) is orchestrated by an antagonism between RAD51 and RAD52

Thesis advisor: Anthony T. Annunziato / The histone acetyltransferase Hat1 is an enzyme that specifically acetylates newly synthesized histone H4 at positions K5 and K12 (or their homologous positions) in all eukaryotes. In Schizosaccharomyces pombe, the deletion of hat1 presents a mutant phenotype. The telomeres in a hat1-del strain become permissive for transcription, as analyzed by a telomeric ura4 marker gene. In this study, we evaluate the efficacy of high copy suppression of this hat1 deletion. Due to high-frequency recombination events in the telomere, it became necessary to create a hat1-rad52 double deletion strain that also contains a telomeric ura4 reporter. High copy suppressor screens for recovery of telomeric silencing yielded several promising transformants. Multiple rounds of testing were performed to assess the recovery of transcriptional repression at the telomere. It was found that despite the anti-recombination effect of deleting rad52, the ura4 reporter was still lost from the telomere through recombination. Additional observation of the hat1-del rad52-del ura4-tel strain revealed a significant synthetic slow-growth phenotype. The double mutant displays a greatly decreased growth rate compared to hat1-del, as well as increased cellular length. Further study showed unique phenotypes on various media, and gene expression studies showed unique patterns of regulation in this double mutant when compared to both a wild- type and its single mutant counterparts (hat1-del, rad52-del). In summary, the telomeric ura4 marker in a hat1-del strain of S. pombe is not stable and is lost by recombination at a high frequency. This has led to the discovery of a double mutant (hat1-del rad52-del) that displays a severe synthetically sick phenotype. / Thesis (MS) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Defects in BRCA1 and BRCA2 tumor suppressors predispose one to breast and ovarian cancer. The current treatment for BRCA-deficient cancers is mastectomy. Because both copies of the tumor suppressor need to be defective for cancer to occur, identifying cellular mechanisms that specifically target BRCA-deficient cells is of paramount importance. Luckily, recent experiments have shown that depletion of a protein named RAD52 in BRCA1 or BRCA2 cancer cells causes them to die. Therefore, we can use small molecules to stop the RAD52 protein from functioning. We need, however, to know which of the RAD52 activities to inhibit and how. One function of RAD52 that likely underlies all cellular activities is its ability to bind single-stranded DNA (ssDNA). To identify if small molecules could inhibit the RAD52-ssDNA complex, I screened a small library of compounds and found 13 potential inhibitors. We validated that these small molecules bind to RAD52 and inhibit RAD52 DNA binding and annealing activities. The identification of these small molecules is important because we can use them to dissect the function of RAD52 in normal and malignant cells, which to date remains elusive. In an attempt to further advance our understanding of RAD52 function and regulation we are also investigating how a novel binding partner, DSS1, interacts with RAD52 and modulates its activities. My data show that this protein enhances the way RAD52 finds separate complementary DNA templates and anneals them to make a double-stranded product. At least in part, these studies have identified some residues likely involved in the binding site of DSS1 on RAD52. In aggregate, the outcome of the two projects deepens our understanding of the complex and interconnected cellular pathways that support the integrity of genomes.

Le génome est sans cesse menacé dans sa structure par des stress génotoxiques d’origine endogène (stress oxydant, blocage de la réplication…) ou exogène (irradiations, produits chimiques, métaux lourds…). La voie de réponse aux dommages de l’ADN coordonne un réseau d’événements en cascades qui incluent les points de surveillance du cycle cellulaire, la réplication/réparation/recombinaison de l’ADN et la mort cellulaire programmée. Ces voies collaborent pour assurer la transmission fidèle du génome et empêcher la prolifération des cellules qui aurait accumulé des altérations génétiques. En général, des défauts dans une de ces voies entraînent un changement de sensibilité aux agents génotoxiques, une instabilité génétique et une prédisposition au cancer. La recombinaison homologue (RH) est une voie essentielle pour la réparation de l’ADN ; elle joue un rôle fondamental dans le maintien de la stabilité du génome. Le stress oxydant, résultant d’une augmentation de la concentration intracellulaire de ERO, est une des causes majeures de dommages aux lipides, aux protéines et à l’ADN et pour cela, il représente un défi pour la survie cellulaire et pour la stabilité du génome. Les ERO apparaissent physiologiquement lors de la respiration cellulaire ou résultent d’un stress environnemental, comme l’exposition aux radiations UV ou agents chimiques oxydants. Elles contribuent à certains processus comme la croissance cellulaire, l’activité et au repliement des protéines, la sénescence et la mort cellulaire programmée. Il est important de souligner qu’un état redox altéré est souvent associé à un fonctionnement anormal des cellules, comme il est observé pour les cellules cancéreuses et les cellules sénescentes. L’objectif de ce projet a été d’analyser l’interface entre les voies DDR et SAPK (Stress activated protein kinase) évolutivement conservées et d’en étudier les conséquences sur la stabilité du génome en utilisant comme organisme modèle la levure à fission. Nos résultats montrent que la voie SAPK joue un rôle sur la RH en promouvant la phosphorylation de Rad52, une protéine impliquée dans différentes sous-voies de la RH. Nous avons aussi montré que Rad52 est phosphorylée sur différents acides aminés, parmi lesquels certains sont les cibles de kinases inconnues qui n’ont aucun lien avec la voie SAPK. Nous avons observé que Rad52 est phosphorylée soit après un stress oxydant, soit dans des cellules génétiquement sujettes à des perturbations de la RH. Nous avons identifié deux sites de phosphorylation de la protéine Rad52, dont un seul est dépendant de la voie SAPK. En étudiant la phosphorylation de Rad52 dans les cellules invalidées pour la voie SAPK ou mutées pour un des sites de phosphorylation de Rad52, nous avons pu montrer que la RH est modulée par la voie SAPK même en absence de insulte externe. Notre travail ouvre le chemin vers une nouvelle compréhension des mécanismes fondamentaux du maintien de l’intégrité du génome. / Genomes are routinely submitted to injuries from either endogenous stress (oxidative stress, DNA replication block…) or from exogenous sources (radiations, chemicals, heavy metals…). The DNA damage response (DDR) coordinates a network of pathways including cell cycle checkpoints, DNA replication/repair/recombination, and programmed cell death, ensuring faithful genome transmission and preventing from the proliferation of cells bearing genetic alterations. Defect in one of these pathways generally results in altered sensitivity to genotoxins, genetic instability and cancer predisposition. Homologous recombination (HR) is an essential DNA repair pathway playing pivotal role to maintain genome stability.Oxidative stress, resulting from increased intracellular concentration of ROS, is one of the major causes of lipid, protein and DNA damage, and therefore a challenge for cell survival and genome stability. ROS are generated physiologically as by-products of cellular respiration, or as result of environmental stresses, such as exposure to solar UV radiations or to oxidant chemicals, and they actively participate in processes such as cellular growth, protein activity and folding, senescence and programmed cell death. It is noteworthy that an altered redox homeostasis is often associated to abnormally functioning cells, such as cancer and senescent cells. The aim of this project was to study the interface between two major evolutionarily conserved pathways, DDR and SAPK (Stress activated protein kinase) and the consequences on genome stability using fission yeast as a model organism. We report data showing that SAPK pathway impinges on HR by promoting phosphorylation of Rad52, a key protein involved in all sub-pathways of HR. We also revealed that Rad52 is phosphorylated at multiple different sites some of which are substrate for unidentified kinases unrelated to the SAPK pathway. Rad52 phosphorylation occurs either after oxidative stress or in cells genetically prone to HR perturbation. We identified two sites of phosphorylation, one of which is dependent on functional SAPK pathway. By studying Rad52 phosphorylation in cells mutated in the SAPK pathway or mutated at the Rad52 site of phosphorylation, we showed that HR is modulated by SAPK even in the absence of external insults. Our work pave the way to a novel understanding of fundamental mechanisms required for genome integrity maintenance.

Submitted by Chaylane Marques (chaylane.marques@ufpe.br) on 2015-03-12T19:35:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Jaqueline de Azevedo Silva.pdf: 1480397 bytes, checksum: b38b99c8bc543a2c2b3933d0bb630f45 (MD5) / Made available in DSpace on 2015-03-12T19:35:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Jaqueline de Azevedo Silva.pdf: 1480397 bytes, checksum: b38b99c8bc543a2c2b3933d0bb630f45 (MD5) Previous issue date: 2012 / CNPQ; CAPES; FACEPE / O Lúpus Eritematoso Sistêmico (LES) é uma das mais relevantes desordens autoimunes no mundo com a prevalência variando entre 20 a 150 casos a cada 100.000 indivíduos. A formação de autoanticorpos e a deposição de imunocomplexos na circulação sanguínea é um dos principais mecanismos patogênicos da doença. Além disso, o LES é caracterizado por um diversificado quadro de manifestações clínicas que varia de paciente para paciente. A genética do LES é complexa e caracteriza-se pela participação de diversos genes atuando em conjunto na etiopatogênese da doença. Neste trabalho foi estudada a susceptibilidade dos polimorfismos nos genes LIG4, RAD52, VDR e IFIH1 ao LES e suas manifestações clínicas. Os genes LIG4 e RAD52 são codificadores de enzimas de reparo do DNA e os danos ao DNA são potenciais ativadores da resposta imune. O VDR (receptor de vitamina D) atua como modulador da resposta imune através da ação da vitamina D. Uma vez que pacientes com LES apresentam com frequência alterações dos níveis séricos da vitamina D e o VDR está presente em importantes células do sistema imune, o VDR aparece como um candidato promissor à susceptibilidade ao LES. O gene IFIH1 é capaz de induzir a ativação do IFN e, como esta citocina apresenta papel chave na patogênese do LES, a ação deste gene tem papel importante na resposta imune. Nos gene LIG4 e RAD52 foram analisados quatro (rs10131, rs1805386, rs1805388 e rs3093740) e três (rs1051669, rs1106467 e rs3748522) SNPs respectivamente, em 158 pacientes e 212 controles da população do Sudeste Brasileiro. Os polimorfismos nos genes LIG4 e RAD52 não apresentaram associação ao LES nem às suas manifestações clínicas na população analisada. Os polimorfismos analisados no gene VDR (rs11168268, rs2248098, rs1540339, rs4760648 e rs3890733) não apresentaram associação com o LES, no entanto apresentaram associação com as seguintes manifestações clínicas: alterações cutâneas com genótipo G/G (rs11168269, OR=3,01e p=0,035), anticorpo anti ds-DNA com o genótipo C/T (rs4760648, OR=0,369 e p=0,03), alterações imunológicas com o genótipo G/G (rs2248098, OR=2,82 e p=0,04) e artrite com o genótipo T/T (rs3890733, OR=17,05 e p= 0,001). No gene IFIH1 foram analisados dois polimorfismos (rs6432714 e rs10930046) e o genótipo C/C (rs10930046) foi associado com ao LES (p=0.032), no entanto, não foi encontrada associação com as manifestações clínicas. Os resultados obtidos neste estudo forneceram dados para o primeiro estudo de associação com LES e os genes de reparo LIG4 e RAD52. Além disso, os genes VDR e IFIH1 foram testados pela primeira vez na população brasileira, contribuindo como marcadores não somente na doença, mas nas manifestações clínicas do LES.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-10T17:17:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-JaquelineSilva.pdf: 1487628 bytes, checksum: 1f5fec25235b035e5719c320cf633d92 (MD5) / Made available in DSpace on 2017-04-10T17:17:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-JaquelineSilva.pdf: 1487628 bytes, checksum: 1f5fec25235b035e5719c320cf633d92 (MD5) Previous issue date: 2012 / O Lúpus Eritematoso Sistêmico (LES) é uma das mais relevantes desordens autoimunes no mundo com a prevalência variando entre 20 a 150 casos a cada 100.000 indivíduos. A formação de auto anticorpos e a deposição de imunocomplexos na circulação sanguínea é um dos principais mecanismos patogênicos da doença. Além disso, o LES é caracterizado por um diversificado quadro de manifestações clínicas que varia de paciente para paciente. A genética do LES é complexa e caracteriza-se pela participação de diversos genes atuando em conjunto na etiopatogênese da doença. Neste trabalho foi estudada a susceptibilidade dos polimorfismos nos genes LIG4, RAD52, VDRe IFIH1ao LES e suas manifestações clínicas. Os genes LIG4e RAD52 são codificadores de enzimas de reparo do DNA e os danos ao DNA são potenciais ativadores da resposta imune. O VDR (receptor de vitamina D)atua como modulador da resposta imune através da ação da vitamina D. Uma vez que pacientes com LES apresentam com frequência alterações dos níveis séricos da vitamina D e oVDR está presente em importantes células do sistema imune, o VDR aparece como um candidato promissor à susceptibilidade ao LES. O gene IFIH1 é capaz de induzir a ativação do IFN e,como esta citocina apresenta papel chave na patogênese do LES,a ação deste gene tem papel importante na resposta imune. Nos gene LIG4e RAD52 foram analisados quatro (rs10131, rs1805386, rs1805388 e rs3093740) e três (rs1051669, rs1106467 e rs3748522)SNPs respectivamente, em 158 pacientes e 212 controles da população do Sudeste Brasileiro. Os polimorfismos nos genes LIG4e RAD52 não apresentaram associação ao LES nem às suas manifestações clínicas na população analisada. Os polimorfismos analisados no gene VDR (rs11168268, rs2248098, rs1540339, rs4760648 e rs3890733) não apresentaram associação com o LES, no entanto apresentaram associação com as seguintes manifestações clínicas: alterações cutâneas com genótipo G/G (rs11168269, OR=3,01e p=0,035), anticorpo anti ds-DNA com o genótipo C/T(rs4760648, OR=0,369e p=0,03), alterações imunológicas com o genótipo G/G(rs2248098, OR=2,82e p=0,04)e artrite com o genótipo T/T(rs3890733, OR=17,05e p=0,001). No gene IFIH1foram analisados dois polimorfismos (rs6432714 e rs10930046) e o genótipo C/C (rs10930046)foi associado com ao LES (p=0.032), no entanto, não foi encontrada associação com as manifestações clínicas. Os resultados obtidos neste estudo forneceram dados para o primeiro estudo de associação com LES e os genes de reparo LIG4 e RAD52. Além disso, os genes VDR e IFIH1 foram testados pela primeira vez na população brasileira, contribuindo como marcadores não somente na doença, mas nas manifestações clínicas do LES. / Systemic lupus erythematosus (SLE) is one of the most importantautoimmune disordersworldwideand the prevalence ranges from 20 to 50 cases per 100.000 individuals. The formation of autoantibodies and deposition of immune complexesthroughout the body are the major pathogenic mechanisms in thedisease. In addition, SLEis characterized by a heterogeneousclinical manifestations varying from patient to patient. The genetic component in SLE is characterized by the participation of several genes acting together in the etiopathogenesis of the disease. This work studied thegenes LIG4, RAD52, VDRand IFIH1and the susceptibility to systemic lupus erythematosus and its clinical manifestations. The LIG4and RAD52genesproducts areDNA repair enzymes and DNA damage are potential activators of the immune response. The VDRacts as a modulator of the immune response through the action of vitamin D. Since SLE patients often have decreasedserum levels of vitamin D and VDR is present in important immune system cells, VDRappears as a promising candidate to SLE susceptibility. The IFIH1gene is able to induce IFN activation and, since IFN is akey cytokine in the pathogenesis of SLE, the action of this gene plays an important role in immune response. In this thesis weanalyzed SNPs in LIG4, RAD52, VDRand IFIH1genes and the susceptibility to SLE and its clinical manifestations.We analyzed four SNPs in LIG4gene(rs10131, rs1805386, rs1805388 and rs3093740) and three in RAD52(rs1051669, rs1106467 and rs3748522)in 158 SLE patients and 212 healthy controls in a Southeast Brazilian population. Polymorphisms in the LIG4and RAD52genes were not associated toSLE or to their clinical manifestations in the studied population. The analyzed polymorphisms in the VDRgene (rs11168268, rs2248098, rs1540339, rs4760648 and rs3890733) were not associated with SLE, but were associated to the following clinical manifestations: cutaneous alterationsto genotype G/G(rs11168269, OR = 3.01 and p= 0.035), antibody anti dsDNAto genotype C/T(rs4760648, OR = 0.369andp= 0.03), immunological alterationsto genotypeG/G (rs2248098, OR = 2.82, p= 0.04) and arthritisto genotypeT/T(rs3890733, OR = 17.05 andp= 0.001). In IFIH1gene weanalyzed two polymorphisms (rs6432714 and rs10930046) and the genotype C/C atrs10930046 was associated toSLE (p = 0.032), however, no association was found with the clinical manifestations. The results obtained in this thesis provide data for the first association study with SLE andDNA repairgenes LIG4and RAD52. In addition, VDRand IFIH1geneswere tested for the firsttime in the Brazilian population, contributing not only as disease markers butas disease activity predictors

Meiosis is necessary for sexual reproduction in eukaryotes. Genetic recombination between non-sister homologous chromosomes is needed in most organisms for successful completion of the first meiotic division. Proteins that function during meiotic recombination have been studied extensively in model organisms. However, less is known about the evolution of these proteins, especially among protists. We searched the genomes of diverse eukaryotes, representing all currently recognized supergroups, for 26 genes encoding proteins important for different stages of interhomolog recombination. We also performed phylogenetic analyses to determine the evolutionary relationships of gene homologs. At least 23 of the genes tested (nine that are known to function only during meiosis in model organisms) are likely to have been present in the Last Eukaryotic Common Ancestor (LECA). These genes encode products that function during: i) synaptonemal complex formation; ii) interhomolog DNA strand exchange; iii) Holliday junction resolution; and iv) sister-chromatid cohesion. These data strongly suggest that the LECA was capable of these distinct and important functions during meiosis. We also determined that several genes whose products function during both mitosis and meiosis are paralogs of genes whose products are known to function only during meiosis. Therefore, these meiotic genes likely arose by duplication events that occurred prior to the LECA. The Rad51 protein catalyzes DNA strand exchange during both mitosis and meiosis, while Dmc1 catalyzes interhomolog DNA strand exchange only during meiosis. To study the evolution of these important proteins, we performed degenerate PCR and extensive nucleotide and protein sequence database searches to obtain data from representatives of all available eukaryotic supergroups. We also performed phylogenetic analyses on the Rad51 and Dmc1 protein sequence data obtained to evaluate their utility as phylogenetic markers. We determined that evolutionary relationships of five of the six currently recognized eukaryotic supergroups are supported with Bayesian phylogenetic analyses. Using this dataset, we also identified ten amino acid residues that are highly conserved among Rad51 and Dmc1 protein sequences and, therefore, are likely to confer protein-specific functions. Due to the distributions of these residues, they are likely to have been present in the Rad51 and Dmc1 proteins of the LECA. To address an important issue with the gene inventory method of scientific inquiry, we developed a heuristic metric for determining whether apparent gene absences are due to limitations of the sequence search regimen or represent true losses of genes from genomes. We collected RNA polymerase I (Pol I), Replication Protein A (RPA), and DNA strand exchange (SE) sequence data from 47 diverse eukaryotes. We then compared the numbers of apparent absences to a single measure of protein sequence length and sequence conservation (Smith-Waterman pairwise alignment (S-W) scores) obtained by comparing yeast and human protein sequence data. Using Poisson correlation regression to analyze the Pol I and RPA subunit datasets, we confirmed that S-W scores and apparent gene absences are correlated. We also determined that genes encoding products that are critical for interhomolog SE in model organisms (Rad52, Rad51, Dmc1, Rad54, and Rdh54) have been lost frequently during eukaryotic evolution. Saccharomyces cerevisiae null rad52, dmc1, rad54, and rdh54 mutant phenotypes are suppressed by rad51 overexpression or mutation. If rad51 overexpression or mutation affects other eukaryotes in a similar fashion, this phenomenon may account for frequent losses of genes whose products are critical for the completion of meiosis in model organisms. Finally, we place this work into greater context with a review of hypotheses for the selective forces and mechanisms that resulted in the origin of meiosis. The review and the data presented in this thesis provide the basis for a model of the origin of meiotic genes in which meiosis arose from mitosis by large-scale gene duplication, following a preadaptation that served to reduce increased numbers of chromosomes (from diploid to haploid) caused by erroneous eukaryotic cell-cell fusions.

PARP inhibitors (PARPi) have been used to induce synthetic lethality in BRCA-deficient tumors in clinical trials with limited success due to the development of resistance to PARPi. BRCA-deficient cells are unable to repair DNA double strand breaks by the accurate homologous recombination repair (HR), and therefore rely on alternative DNA repair pathways for survival. We hypothesized that RAD52-mediated DNA repair mechanisms remain active and are thus protecting some PARPi-treated BRCA-deficient tumor cells from apoptosis, and that targeting RAD52 should enhance the synthetic lethal effect of PARPi. We show here that RAD52 inhibitors (RAD52i) attenuated single-strand annealing (SSA) and residual HR activity in BRCA-deficient cells. Simultaneous targeting of PARP1 and RAD52 with small molecule inhibitors or via expression of dominant-negative mutants induced an accumulation of DSBs and selective eradication of BRCA-deficient solid tumor and leukemia cells, while BRCA-proficient cells were unaffected. Parp1-/-Rad52-/- transgenic mice are healthy and indistinguishable from wild-type mice due to the presence of the BRCA-pathway, and Parp1-/-Rad52-/- mice with inducible BRCA1-deficient leukemia displayed significantly prolonged survival when compared to Parp1-/- and Rad52-/- counterparts. Finally, PARPi + RAD52i selectively targeted BRCA1-deficient solid tumors in immunodeficient mice with minimal toxicity to normal cells and tissues which are protected by the BRCA-pathway, indicating minimal side effects. In conclusion, our data indicate that combination treatment of RAD52i and PARPi will significantly improve therapeutic outcome of BRCA-deficient malignancies compared to treatment with PARPi monotherapy, while leaving healthy cells and tissues unharmed. / Biomedical Sciences

DNA double strand breaks (DSB) are the most toxic of all types of DNA lesions. In Saccharomyces cerevisiae, DNA DSBs are predominantly repaired by the homologous recombination repair (HRR) pathway. The initial step of HRR requires extensive processing of DNA ends from the 5' to 3' direction by specific endo-exonuclease(s) (EE) at the DSB sites, but no endo-exonuclease(s) has yet been conclusively determined for such processing of DSBs. S. cerevisiae TRM2/RNC1 is a candidate endo-exonuclease that was previously implicated for its role in the HRR pathway and was also shown to have methyl transferase activity primarily located at its c-terminus. / In this dissertation, we provided compelling biochemical and genetic evidence that linked TRM2/RNC1 to the DNA end processing role in HRR. Trm2/Rnc1p purified with a small calmodulin binding peptide (CBP) tag displayed single strand (ss) specific endonuclease and double strand (ds) specific 5' to 3' exonuclease activity characteristic of endo-exonucleases involved in HRR. Intriguingly, purified Trm2/Rnc1p deleted for its C-terminal methyl transferase domain retained its nuclease activity but not the methyl transferase activity indicating that the C-terminal part responsible for its methyl transferase function is not required for its nuclease activity. / Our genetic and functional studies with S. cerevisiae trm2/rnc1 single mutants alone or in combination with other DNA DSB repair mutants after treatment with the DNA damaging drug methyl methane sulfonate (MMS) or IR that is believed to produce DSBs, or with specific induction of DNA DSBs at the MAT locus by HO-endonuclease demonstrated an epistatic relationship of TRM2/RNC1 with the major recombination factor RAD52. These studies suggested that TRM2/RNC1 probably acts at an earlier step than RAD52 in the HRR pathway. The genetic evidence also indicated a possible functional redundancy with the bona fide endo-exonuclease EXO1 in the processing of DNA ends at the DSB sites. / In a recent report, the immuno-purified mouse homologue of TRM2/RNC1 exhibited similar enzymatic properties as the endo-exonucleases involved in HRR. A small molecular inhibitor pentamidine specifically inhibited the nuclease activity of the mouse EE and sensitized various cancer cells to DNA damaging agents commonly used in cancer chemotherapy. We specifically suppressed expression of the mouse EE using small interfering RNA (siRNA) that conferred sensitivity of B16F10 melanoma cells to a variety of DNA damaging drugs often used in cancer treatment. This further validated our earlier claim of the endo-exonuclease as a potential therapeutic target in treating cancer.

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