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.

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Maintaining genomic integrity is crucial for cell survival. In Escherichia coli, Rec-Amediated homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSB) and the SOS response through a series of highly dynamic interactions with the chromosome. A greater understanding of the mechanism of homologous recombination requires quantitative analysis of genomic studies in live cells. The aim of this thesis was to investigate the dynamics of the RecA-DNA interactions in vivo following the induction of a site-specific DSB in the chromosome of E. coli. This DSB is caused by the cleavage of a DNA hairpin by the hairpin-specific endonuclease, SbcCD. The DNA hairpin is formed only on the lagging strand template of replication by a 246 bp-interrupted palindrome. As a result cleavage only occurs on one sister chromosome, leaving one unbroken chromosome to serve as a template for repair by HR. Here, this system has been used as a basis to develop a method that combines chromatin immunoprecipitation with quantitative PCR (ChIP-qPCR) and next-generation sequencing (ChIP-Seq) to quantify RecA protein binding during the active repair of a single chromosomal DSB. This study reports that DSB-dependent RecA binding is stimulated in response to the eight base DNA sequence Chi (5’-GCTGGTGG-3’). Increasing the number of Chi sites close to the DSB stimulates more RecA loading to DNA, with ChIP-Seq analysis also revealing a role for subsequent Chi sites in RecA binding during DSBR. If the Chi sites close to the DSB are removed then Chi-dependent RecA binding to DNA can be observed at distances greater than 100 kb from the DSB, suggesting that these subsequent Chi sites can be engaged in DSBR. Through collaboration, these in vivo data were combined with stochastic modeling to determine that, in vivo, Chi is recognised by the RecBCD complex with an efficiency of 20- 35%. The genomic analysis also revealed two unexpected aspects of RecA protein binding. First, ChIP-Seq analyses identified that following a DSB at lacZ there is RecA enrichment detected in the terminus region of the E. coli chromosome. This RecA binding is Chi-dependent, indicating a role for HR. Second, DSB-independent binding was observed at the RNA encoding genes dispersed throughout the chromosome. A temporal analysis of RecA dynamics was also performed. These analyses revealed that RecA binding to DNA near the DSB is extremely dynamic, cycling between periods of high RecA enrichment and periods of low RecA enrichment. This is the first in vivo study of DSB-dependent RecA-DNA distribution and dynamics in recombination proficient E. coli cells.

DNA double strand break (DSB) repair is critical to maintain genomic integrity and cell viability. DSBs can occur during the course of cell cycle during replication or transcription, or by exogenous agents such as chemicals or ionizing radiation. For my thesis, I studied homologous recombination (HR), which has two sub-pathways: Homology Directed Repair (HDR) and Single Strand Annealing (SSA). HDR involves strand invasion of a homologous template to prime DNA synthesis; SSA involves annealing of homologous segments flanking a DSB. Background data showed that depletion of XAB2 protein by RNA interference reduced both HDR and SSA events. XAB2 protein contains 15 tetratricopeptide repeat (TPR) motifs, which likely enable protein-protein interactions. While XAB2 is speculated to have a role in transcription coupled repair and pre-mRNA splicing, its role in HR pathway is uncertain. The overall hypothesis for my thesis is that XAB2 mediates a specific step of HR (5’-3’ end resection), and the TPR motifs present in XAB2 enable the protein to function in a complex during HR. By using an end resection assay and cell biology analysis, I found that XAB2 is essential for 5’ – 3’ end resection, an intermediate step common to both HDR and SSA pathways. With a functional complementation assay I developed, I have shown that specific TPR regions are critical for XAB2 functions in HR. Overall, my research demonstrates that XAB2 protein has a key role in the 5’-3’ end resection step of HR, and its function in HR requires specific sets of its TPR regions.

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Radiation therapy targets cancer cell death by overwhelming cells with harmful DNA damage. Understanding how cells repair radiation damage and in particular how they become resistant to radiation therapy is important for effective cancer treatment. Our lab made the novel discovery that Bcl10, a cytoplasmic protein important for NF-B activation, localizes to endogenous H2AX foci in the nucleus of breast cancer cells. We determined that following radiation treatment Bcl10 is recruited to ionizing radiation-induced foci in a dose-dependent matter and that it is important for the repair of radiation-induced DNA damage. We also observed that breast cancer cells are extremely sensitive to Bcl10 knockdown, causing cellular senescence, while normal breast epithelial cells are insensitive. Our findings identify Bcl10 as potent anti-cancer target. / Experimental Oncology

Abstract As the defects in DNA repair and cell cycle control are known to promote tumorigenesis, a proportion of inherited breast cancers might be attributable to mutations in the genes involved in these functions. In the present study, three such genes, TP53, CHK2 and ATM, which are also associated with known cancer syndromes, were screened for germline mutations in Finnish breast cancer patients. In combination with our previous results, three TP53 germline mutations, Tyr220Cys, Asn235Ser and Arg248Gln, were detected in 2.6% (3/108) of the breast cancer families. The only observed CHK2 alteration with a putative effect on cancer susceptibility, Ile157Thr, segregated ambiguously with the disease, and was also present in cancer-free controls. The available functional data, however, suggests that the altered CHK2 in some way promote tumorigenesis. Furthermore, compared to the other studied populations, Ile157Thr seems to be markedly enriched in Finland. Thus, the clinical significance of Ile157Thr requires further investigation among Finnish cancer patients. ATM germline mutations appear to contribute to a small proportion of the hereditary breast cancer risk, as two distinct ATM mutations, Ala2524Pro and 6903insA, were found among three families (1.9%, 3/162) displaying breast cancer. They all originated from the same geographical region as the AT families with the corresponding mutations, possibly referring to a founder effect concerning the distribution of these mutations in the Finnish population. The genes important for tumorigenesis in sporadic disease might also contribute to familial breast cancer. Therefore, four putative LOH targets genes in chromosome 11q21-q24 were screened for intragenic mutations, and five were analyzed for epigenetic inactivation in sporadic breast tumors. The lack of somatic intragenic mutations in MRE11A, PPP2R1B, CHK1 and TSLC1 led us next to investigate promoter region hypermethylation as a mechanism capable of silencing these genes, as well as the ATM gene. Only TSLC1 demonstrated involvement of CpG island methylation, which was especially prominent in three tumors. This suggests that together with LOH, methylation could result in biallelic inactivation of the TSLC1 gene in breast cancer.

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No description available.

Nucleotide synthesis is a conserved and highly regulated response to DNA damage, required for the efficient repair of DNA double strand breaks (DSB) by homologous recombination (HR). This is essential to prevent loss of heterozygosity (LOH) and maintain genome stability. The aim of this study was to identify new genes important for HR through roles in damage-induced nucleotide synthesis. A screen was performed to identify S. pombe gene deletion strains whose DSB sensitivity was suppressed by deleting the ribonucleotide reductase (RNR) inhibitor spd1⁺ to promote nucleotide synthesis. The screen identified a number of genes including ddb1⁺, cdt2⁺, rad3⁺ and csn1⁺ which have known roles in nucleotide synthesis. Distinct roles were identified for the DNA damage checkpoint in suppressing LOH. rad3⁺, rad26⁺, rad17⁺ and the rad9⁺, rad1⁺ and hus1⁺ genes encoding the 9-1-1 complex were required for DNA damage-induced nucleotide synthesis through Cdt2 induction to promote Spd1 degradation. The HR repair defect of rad3⁺ and rad26⁺ deletion strains was partially suppressed by spd1⁺ deletion. However, the HR repair defect of rad17⁺, rad9⁺, rad1⁺ and hus1⁺ deletion strains was not suppressed. An additional role was confirmed for Rad17 and the 9-1-1 complex in preventing LOH by promoting DSB resection. A role was identified for the Gcn5 histone acetyl transferase (HAT) protein module, consisting of Gcn5, Ngg1, Ada2 and Sgf29, in suppressing DSB sensitivity by promoting nucleotide synthesis. This was independent of Cdt2 or RNR protein levels. The Gcn5 HAT module was also found to regulate DSB repair pathway choice consistent with previous observations. Deletion of gcn5⁺, ngg1⁺ or ada2⁺ decreased HR and increased non-homologous end joining. Surprisingly, deletion of spd1⁺ in a gcn5∆, ngg1∆ or ada2∆ background also promoted HR. This predicts a role for nucleotide pools in regulating DSB repair pathway choice. Eleven other candidates showed repeatable suppression of DSB sensitivity following spd1⁺ deletion. However many of these candidates did not show reduced nucleotide levels. This suggests deleting spd1⁺ may also suppress DSB sensitivity by a different mechanism.