Inheritance of germline pathogenic mutations in a single BRCA2 allele predisposes carriers to cancers of the breast, ovaries, pancreas, prostate and other tissues. BRCA2 encodes a complex protein of 3418 residues, with multiple distinct regions implicated in different cellular processes including homologous recombination, replication stress tolerance, maintenance of the G2 and spindle assembly checkpoints, and in completion of cytokinesis. Much evidence suggests that BRCA2 acts as a tumour suppressor by maintaining genome integrity through its functions during DNA replication, repair and chromosome segregation. Neither the mechanisms underlying these functions, nor their significance to tumour suppression, is well understood. In this thesis, I report studies that reveal two novel mechanisms underlying genome maintenance and tumour suppression by BRCA2. The best-studied cellular function of BRCA2 is its role in double strand break repair by homologous DNA recombination, wherein BRCA2 controls the DNA substrate selectivity and activity of the recombinase, RAD51. This control is exerted via evolutionarily conserved BRC repeats motifs, each comprising ~35 residues. BRCA2 homologues in simple organisms such as Caenorhabditis elegans or Ustilago maydis contain only one BRC repeat. By contrast, all known vertebrate BRCA2 orthologues contain 8 BRC repeats whose sequence as well as spacing is conserved. The functional significance of the conserved sequence and spacing of vertebrate BRC repeats in the maintenance of genome integrity is unclear. I report that the BRC1 and BRC4 repeats in chicken BRCA2 are essential for cell viability. Additionally, the BRC6 repeat has an essential, non- redundant role in tolerating replication stress by stabilizing stalled replication forks and preventing the degradation of nascent DNA by MRE11, and the mechanism underlying this function is largely RAD51-independent. Furthermore, my results imply that the BRC repeats in chicken BRCA2 are aligned in the same order as in the human BRCA2, suggesting the BRC6 repeat in chicken BRCA2 may correspond to the BRC6 repeat in human BRCA2. 2 ABSTRACT Although both alleles of BRCA2 must be inactivated to provoke defective homologous DNA recombination, accumulating evidence suggests that BRCA2 heterozygosity suffices for carcinogenesis in murine models and human patients. In a second line of research, I have explored in collaborative studies how heterozygous truncating mutations affecting BRCA2 may promote carcinogenesis. I report that in unchallenged conditions, BRCA2 heterozygous cells appear to be as functionally proficient in key cellular processes as the BRCA2 wildtype cells. However, BRCA2 heterozygosity confers selective sensitivity to replication stress induced by exposure to physiological concentrations of formaldehyde, a known genotoxic agent. Replication fork degradation is observed in BRCA2 heterozygous cells on exposure to formaldehyde, but not hydroxyurea, and this deleterious phenotype has been attributed to the ability of formaldehyde to induce BRCA2 degradation in a proteasome-dependent manner, thereby inducing haploinsufficiency. Overall, my thesis research reveals two novel mechanisms by which BRCA2 maintains genomic integrity. Identification of a novel function for the BRC6 repeat in chicken BRCA2 indicates that individual BRC repeats may have essential functions that are yet to be identified, thereby providing an explanation for the evolutionary conservation of the 8 BRC repeats observed in vertebrate BRCA2. Furthermore, results linking BRCA2 heterozygosity and formaldehyde exposure to increased genomic instability highlights the potential ability of aldehyde exposure in promoting carcinogenesis in heterozygous BRCA2 mutation carriers, which has significant preventive and therapeutic implications.

Breast cancer is one of the most common malignancies in women, affecting one in twelve. Ovarian cancer, although not as frequent, is the leading cause of death from gynaecological cancer. Inherited predisposition to breast and ovarian cancer, which accounts for approximately 5 to 10% of these cancers, has been associated with mutations in the BRCA1 and BRCA2 genes. Mutations in both of these genes increase the lifetime risk of developing breast cancer by approximately 80%. BRCA1 confers a greater predisposition of ovarian cancer than BRCA2, however, BRCA2 has been associated with male breast cancer. Polymorphisms linked to BRCA1 and BRCA2 were studied to examine whether either of these genes were linked to breast and breast/ovarian cancer families. None of the five cancer families studied generated statistically significant lod scores although the segregation of a common haplotype with the disease in each family and positive lod scores did suggest that four of these families were linked to BRCA1 and the other to BRCA2. Subsequent mutation studies identified three germline mutations, thus confirming the initial linkage results in three families. A total of four deletions and six polymorphisms were identified in BRCA1 and BRCA2 from forty-eight breast and breast/ovarian cancer families, using SSCP analysis and PTT. The functional effect of these mutations is unclear although variable expression of the cancer phenotype suggests that other genes and environmental factors play an important role in the development of breast and ovarian cancer. Evidence of an abnormal protein was detected by the presence of clonal LOH of the normal allele, using BRCA1 antibodies in familial breast and ovarian tumours. In addition, BRCA1 immunostaining was negative in a greater proportion of benign tumours compared to malignant ovarian tumours. The loss of BRCA1 does not lead to malignancy, suggesting that BRCA1 may have another role in benign ovarian epithelial tumours.

Loss of DNA damage-induced G2 checkpoint control is associated with genome instability, tumour formation and the therapeutic response of tumours to genotoxic agents. The large 3418 residue protein encoded by BRCA2 – heterozygous germline mutations in which predispose to cancer - has recently been implicated in G2 checkpoint maintenance. However, the mechanistic basis of BRCA2’s role in the G2 checkpoint remains unknown. The overall aim of my research is to understand the mechanism by which BRCA2 regulates the G2 checkpoint. Domain mapping studies, using overlapping fragments encoding the full-length BRCA2 protein, carried out in our laboratory suggest that BRCA2’s function at the G2 checkpoint is mediated through regions that span BRCA2 amino acids (aa) 1-454 and aa 2438-2824. My research has focused on understanding how these two regions contribute to G2 checkpoint function through the interrogation of two novel interactions of BRCA2 mediated at these regions. My experiments have identified that BRCA2 interacts with the serine/threonine kinase ATR (aa 2438-2824) and the deSUMOylase SENP1 (aa 1-454). ATR is known to play a key role at the G2 checkpoint and my results identify that loss of BRCA2 leads to a reduction in ATR activity at sites of damage. This leads to a downstream attenuation in the phosphorylation of Chk1 – an important effector protein of the G2 checkpoint. BRCA2 is known to function in DNA repair and is recruited to sites of DNA damage, where it displaces RPA bound to exposed single-strand DNA. RPA is required to localise and activate ATR during the G2 checkpoint and therefore I hypothesise that BRCA2’s role at the checkpoint is to substitute for RPA and mediate ATR activation. I have also shown that SENP1 interacts with BRCA2 at a region within the N-terminus (aa 290-454). SUMOylation has been increasingly recognised as an important post-translational modification (PTM) in the context of the DNA damage response, and SENP1 is involved in reversing this PTM. I have shown that BRCA2 is SUMOylated. Moreover, I have shown that loss of SENP1 prevents BRCA2 from being recruited to chromatin in a timely manner after DNA damage and that SENP1 depletion exacerbates the loss of G2 checkpoint maintenance seen in BRCA2 deficiency. Taken together, this work reveals novel insights into the mechanism by which BRCA2 maintains genomic stability through enforcement of the G2 checkpoint. This new knowledge has the potential to translate into a better understanding of how mutations in BRCA2 may lead to cancer.

BRCA2 is a highly penetrant breast cancer predisposing gene. The protein product of the BRCA2 gene mediates repair of breaks in DNA, through Homologous Recombination (HR). Understanding the mechanism(s) behind BRCA2 involvement in HR will help clarify its clinical importance and may pave the way for possible therapy. In this work we show that BRCA2 affects telomere maintenance in mammalian cells. Telomeres are physical ends of chromosomes implicated in cell senescence and carcinogenesis. In particular, the enzyme telomerase that synthesizes telomeric DNA is highly active in ~90% cancers and it is considered one of the cancer markers. The remaining 10% of cancers do not show telomerase activity and they maintain their telomeres by an alternative pathway known as Alternative Lengthening of Telomeres (ALT). We observed telomere shortening, loss of telomere function in the form of end chromosome fusions and increased incidence of Telomere Sister Chromatid Exchanges (T-SCE), one of the recognized markers of ALT, in 3 sets of Chinese hamster and human BRCA2 defective cell lines, all of which maintained telomeres by conventional mechanisms. We have also inhibited BRCA2 expression in ALT positive cells by transfecting them with si (short interfering) RNA oligonucleotides specific for BRCA2 and monitored its expression by Real Time-PCR and Western blot. Results indicate that BRCA2 knock-down in ALT positive human cells that causes reduction in T-SCE frequencies, thus suggesting that ALT cells and those that maintain telomeres by conventional mechanisms differ in this respect. One interesting scenario that emerges from these results is that BRCA2 deficiency could potentially suppress the ALT pathway. We wanted to explore this possibility further by creating a permanent BRCA2 knock-down. Our preliminary results suggest that our method for the permanent BRCA2 knock-down based on the SMARTvector 2.0 system and sh (short hairpin) iv RNA approach is still not working effectively. We identified hyper-methylation of the promoter within the vector as a possible cause. Finally, we examined repair kinetics of interstitial telomeric sites (ITSs) in BRCA2 deficient Chinese hamster cells in order to test the hypothesis that defective DNA double strand break repair may be responsible for their increased sensitivity to DNA damaging agents. Our results indicate that DNA damage within ITSs is repaired effectively thus disproving the above hypothesis. In conclusion, this work demonstrates the involvement of BRCA2 in telomere maintenance.

Thèse (M.Sc.)--Université Laval, 2005. / Titre de l'écran-titre (visionné le 14 mai 2007). Bibliogr.

Thèse (Ph. D.)--Université Laval, 2007. / Bibliogr.: f. [187]-210. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph.D.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2009/06/10). Includes bibliographical references.

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr. Publ. aussi en version électronique.

Submitted by Israel Vieira Neto (israel.vieiraneto@ufpe.br) on 2015-03-05T18:13:15Z No. of bitstreams: 2 Eduardo Ramalho.pdf: 538304 bytes, checksum: e4a30cf566174291fcd76bbfacaf4eff (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-05T18:13:15Z (GMT). No. of bitstreams: 2 Eduardo Ramalho.pdf: 538304 bytes, checksum: e4a30cf566174291fcd76bbfacaf4eff (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012 / CAPES / Sabe-se que os genes p53, BRCA1 e BRCA2 apresentam a característica em comum de serem considerados supressores tumorais. Eventos genéticos e epigenéticos são frequentes ao longo de todo o genoma humano. Mutações somáticas são passíveis de ocorrer nas regiões codificantes de genes específicos, alterando sua sequência e gerando proteínas mutantes, as quais resultam numa alteração de sua capacidade funcional ou até mesmo a perda dela. Este trabalho objetivou avaliar alterações epigenéticas nas regiões promotoras dos genes BRCA1 e BRCA2 através da técnica de PCR para Metilação Específica (MSP) e correlacionar mutações pontuais nos exons 4 e 7 do gene p53 como fator de risco para o carcinoma ductal invasivo (CDI) de mama na população feminina do Recife atendida no Hospital das Clínicas (HCUFPE). Cinquenta biópsias de mama diagnosticadas com CDI fixadas em formalina e embebidas em parafina foram obtidas do Setor de Anatomia Patológica do HC-PE e cinco amostras de tecido mamário de mulheres submetidas à mastectomia estética foram usadas como controle normal. O DNA das amostras foram extraídos e, então, amplificados por MSP. Para avaliação do perfil mutacional utilizou-se a técnica de PCR-RFLP (Restriction Fragment Length Polymorphism) com as enzimas BstUI e HaeIII para verificação dos polimorfismos nos exons 4 e 7, respectivamente. A frequência no padrão de metilação para o gene BRCA2 foi de 46,9% enquanto a frequência de mutações pontuais nos códons 72 (exon 4) e 249 (exon 7) do gene p53 foram de 91,8% e 8,1%, respectivamente. Para o BRCA1 os resultados obtidos foram inconsistentes quanto ao seu padrão de metilação. Os resultados mostraram que o polimorfismo do códon 72 apresentou-se estatisticamente significante para metástase podendo ser utilizado como um potencial biomarcador auxiliar no diagnóstico de carcinoma ductal invasivo de mama humana.

Abstract In the present study, mutations in BRCA1 and BRCA2, the two major genes predisposing individuals to hereditary breast and ovarian cancer, were screened in Finnish and Turkish cancer families. Germline BRCA1 mutations were found in 7% (6/88) and BRCA2 mutations in 6% (5/88) of the Finnish families studied in Oulu. Two distinct BRCA1 (3745delT, 4216nt-2A→G) and three BRCA2 (999delTCAAA, 6503delTT, 9346nt-2A→G) mutations were identified, all of which are recurrently found in Finland. In the 15 Turkish cancer families studied, 5382insC and 5622C→T were detected in BRCA1, and 3414delTCAG in BRCA2. The novel 3414del4 mutation was found in a family with a case of male breast cancer. In order to determine their ages and origin, 9 recurrent Finnish BRCA1 and BRCA2 mutations were studied further as regards haplotype conservation. Common origins approximately 18–80 generations (400–1600 years) ago were demonstrated for all studied mutations by partial haplotype sharing. The majority of the mutations showed geographical clustering, supporting the theory of regional founder effects. Four of the nine mutations are unique for Finland, whereas five have also been seen elsewhere. Mutations in the 5' end of BRCA1 tend to predispose individuals to ovarian cancer and those found in the 3' end to breast cancer. The age of ovarian cancer onset was significantly lower for BRCA1 (51 years) than for BRCA2 mutation carriers (61 years). Germline TP53 mutations were sought in the Finnish breast cancer families found to be negative after BRCA1 and BRCA2 screening but who exhibited some phenotypic features of the Li-Fraumeni syndrome. The Asn235Ser was found in a family displaying Li-Fraumeni syndrome phenotype and the Tyr220Cys in a family with a milder Li-Fraumeni-like phenotype. The nature of both mutations as cancer-predisposing alterations was supported by means of loss of heterozygosity (LOH) and p53 immunohistochemistry studies. Regional clustering of BRCA1 and BRCA2 founder mutations enables targeted genetic tests including especially those mutations characteristic of the birthplace of each patient. Additional genes are likely to explain a large proportion of the inherited susceptibility to breast cancer in particular. Germline TP53 mutations are expected to be found in the breast cancer families with other clinical features seen in the Li-Fraumeni syndrome.