The tumor suppressor Breast Cancer Susceptibility Protein 1 (BRCA1) protects our cells from genomic instability in part by facilitating the efficient repair of DNA double-strand breaks. Other functions of BRCA1 include transcriptional regulation, apoptosis, DNA damage signaling, chromatin remodeling and protein ubiquitination. The major contribution of BRCA1 to maintaining genomic stability is thought to be through its role in DNA repair. BRCA1 promotes the error-free repair of double-strand breaks by homologous recombination, and is also implicated in the regulation of non-homologous end joining repair. Here we investigated the role of BRCA1 in maintaining the fidelity of non-homologous end joining repair following a double-strand break. We also examined the frequency of microhomology-mediated end joining (MMEJ) and the fidelity of double-strand break repair relative to BRCA1 protein levels in both control and tumorigenic breast epithelial cells. In addition to altered BRCA1 protein levels, we tested the effects of cellular exposure to mirin, an inhibitor of Meiotic recombination enzyme 11 (Mre11) 3' to 5' exonuclease activity. Knockdown or loss of BRCA1 protein resulted in an increased frequency of overall plasmid DNA repair mutagenesis and MMEJ following a double-strand break. Inhibition of Mre11 exonuclease activity with mirin significantly decreased the occurrence of MMEJ, but did not considerably affect the overall mutagenic frequency of plasmid double-strand break repair, although some of our data indicate that the size of sequence deletions may be reduced by mirin inhibition. The results suggest that BRCA1 protects DNA from mutagenesis during non-homologous double strand break repair in plasmid-based assays. The increased frequency of double-strand break mutagenesis and MMEJ repair in the absence of BRCA1 suggests a potential mechanism for carcinogenesis.

Ovarian cancer is serious and one of the most common gynecologic cancers. Carboplatin is the therapeutic agent of the first choice in the ovarian cancer therapy. However, after the primary therapeutic response to carboplatin, the relapse of the disease may occur with developed resistance to carboplatin. Chemoresistance and insufficient therapy response are considered to be the reason of the high mortality rate of ovarian cancer. The DNA damage response pathways play an important role in the therapeutic response and chemoresistance development. Restoration of homologous recombination function in cancers is the key mechanism of resistance development to platinum agents. Based on this knowledge, we formed our hypothesis, that the inhibition of homologous recombination could increase the sensibility to carboplatin. The main goal of this thesis was to define the role of double-strand breaks repair in response to chemotherapy of ovarian cancer. Protein MRE11 is part of the MRN complex, that participates in double-strand breaks repair. Using mirin as a pharmaceutic inhibitor of MRE11 we were aiming to determine the impact of homologous recombination on the effect of carboplatin and its role in resistant development to carboplatin. In the practical part of the thesis, we described the association between...

Due to the inevitable reality that most patients diagnosed with cancer will eventually relapse, modern oncology research has been forced to tackle this outcome primitively using combination therapies. Adoptive T-cell transfer with Oncolytic Virus Vaccination represents a new class of combination therapies that can facilitate the crosstalk of multiple aspects of the immune system such that they work in concert to prevent this outcome for many types of cancer. Despite this, immunosuppressive systems like those characterized in the B16F10-gp33 melanoma model pose a new problem for this approach. Typically, this model has total regression but is subsequently followed by relapse. Previous work from the Wan lab has suggested that this may be an outcome of total target gene deletion. Here we present two approaches to tackle this through the targeting of DNA repair pathways of the host cell. Our data can show that both VSV and Vaccinia infection/ propagation does lead to the generation of DNA damage but in the case of VSV this leads to incomplete cell lysis, and ultimately target gene loss via double-stranded DNA repair mechanisms. We were able to tackle the phenomenon following VSV administration by adding DNA repair inhibitors to the mix and showed that the proportion of cells that escaped after the loss of the target antigen was decreased by half when compared to the standard procedures. Additionally, this work also gave a preliminary understanding of how Vaccinia may achieve a similar outcome to this via its unique cytoplasmic replication mechanisms. / Thesis / Master of Science (MSc)