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The C-terminal region of breast cancer susceptibility gene 1 (BRCA1) contains a pair of tandem BRCT repeats that are critical for the tumour suppressor function of BRCA1. BRCT repeats are present in a large of number of proteins that are implicated in the cellular response to DNA damage. A subset of tandem BRCT domains, including those of BRCA1, functions as phosphorecognition modules. Aside from BRCA1, the precise molecular mechanisms of the BRCT repeats of other proteins remain largely unknown. We determined the crystal structure of the tandem BRCT domain of human mediator of DNA checkpoint 1 (MDC1) at 1.45 Å resolution. Our structural and biochemical studies suggest that the tandem BRCT domain of MDC1 functions as the predominant histone variant, γH2AX phosphorecognition module and that the interaction is critically dependent on the free carboxylate group of the γH2AX C-terminal tail. We also determined the crystal structure of the tandem BRCT domain of human BARD1, the in vivo binding partner of BRCA1. Our structure uncovers a degenerate phosphopeptide binding pocket that lacks the key arginine critical for phosphopeptide interactions in other BRCT proteins. Our biochemical studies reveal that a flexible tether links ankyrin and BRCT domains in BARD1. Furthermore, the linker is required for the interactions between the CstF-50 WD-40 domain and BARD1, allowing the BARD1 C-terminus to convey DNA damage signals directly to RNA polymerase. Finally, using protease-based and phosphopeptide pull-down assays, we directly assessed the structural and functional effects of 117 single amino acid substitutions in the BRCA1 BRCT domain derived from breast cancer screening programs. None of the variants showing enhanced sensitivity to proteolytic digestion were found to be active in peptide binding, indicating that these missense mutations contribute to BRCA1 loss of function through protein destabilizing effects. A subset of structurally stable variants was defective in peptide binding activity, suggesting that these variants may disrupt the phosphopeptide binding pocket. Taken together, the results reveal that 32% of the variants show structural stability and peptide binding activity that were indistinguishable from those of wild type.

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Modular domains of proteins are important in cellular signaling processes. Eukaryotic cells are constantly undergoing DNA damage due to exogenous and endogenous sources of damage. The DNA damage response (DDR) involves a complex network of signaling events mediated by modular domains such as the BRCT (BRCA1 C-terminal) domains. Therefore, proteins containing BRCT domains are important for DNA damage detection and signaling. In this dissertation, we focus on two BRCT-containing proteins BRCA1 and PAXIP1. BRCA1 is a gene that is known to be associated with increased risk of hereditary breast and ovarian cancer. Germline variants of BRCA1 are assessed to determine lifetime risk of developing breast and ovarian cancer. This is performed by genetic testing of the BRCA1 sequence and the variants can be classified as pathogenic, non-pathogenic or variants of unknown significance (VUS). Using family history, segregation analysis, co-occurrence and tumor pathology, certain variants have been classified as either pathogenic or non-pathogenic. However, a large majority of the variants are classified as VUS. Functional assays are critical in providing insight in the case of VUS results. We have a developed a visualization resource to aid in functional analysis of BRCA1 missense variants that occur due to single amino acid changes. This tool is known as BRCA1 Circos (http://research.nhgri.nih.gov/bic/circos/) and it aggregates, harmonizes and allows interpretation of data from all published studies on functional analysis of BRCA1 missense variants. Therefore, this is an important tool that will aid in the meta-analysis of functional data needed to better assess VUS. Functional studies of BRCA1 also demonstrate that majority of the variants that have a functional impact on the protein lie in the BRCT region of the protein. This indicates that the BRCT region is important in cancer development. To further analyze the function of BRCT-containing proteins, a study was previously undertaken to evaluate the role of BRCT-containing proteins and their interaction partners in the DNA damage response and consequently, cancer. BRCT domains of seven BRCT-containing proteins were used as baits and their binding partners were demonstrated to be highly enriched in the DDR process. We hypothesized that members of this BRCT-centric protein-protein interaction network could constitute targets for sensitization to DNA damaging chemotherapy agents in lung cancer. Therefore, we probed this established dataset containing the protein-protein interaction network (PPIN) of seven BRCT-containing proteins to identify seventeen kinases. A systematic pharmacological screen was performed to evaluate these kinases as targets to enhance platinum-based chemotherapy in lung cancer and this revealed WEE1, a mitotic kinase, as a potential target. Of the seventeen kinases, inhibition of mitotic kinase, WEE1, was found to have the most effective response in combination with platinum-based compounds in lung cancer cell lines. In the PPIN, WEE1 was shown to interact with PAXIP1 (PTIP), a BRCT-containing protein involved in transcription and in the cellular response to DNA damage. PAXIP1 has been shown to bind DDR proteins, such as 53BP1 and γH2AX, and also shown to be an important part of immune development. In this dissertation, we observe that WEE1 binds to PAXIP1 and PAXIP1 regulates the WEE1-mediated phosphorylation of its main substrate, CDK1. We also demonstrate that ectopic expression of PAXIP1 combined with WEE1 inhibitor, AZD1775, leads to an increase in the mitotic index at the G2/M checkpoint. Overexpression of PAXIP1 combined with AZD1775 treatment in cells with prior DNA damage causes high levels of caspase-3 mediated apoptosis as compared to AZD1775 treatment alone. In summary, we identify the role of PAXIP1 in sensitizing lung cancer cells to the WEE1 inhibitor, AZD1775, in combination with platinum-based therapy and propose the use of WEE1 and PAXIP1 levels as mechanism-based biomarkers. Overall, these studies indicate that BRCT-containing proteins through their role in the DDR and the cell cycle are crucial for both cancer prevention and therapy.

<p>All living things must replicate their DNA. Despite being essential for life, this process is also inherently dangerous. Replication stress, which induces replication fork stalling, is an unavoidable risk that can trigger potentially harmful changes to the genome. Eukaryotes have a replication checkpoint pathway that stabilizes stalled replication forks to prevent damage. One of the critical protein interactions in this pathway, between Dbf4 and Rad53, pauses the cell cycle in budding yeast. This is important to give the cell time to recover from stress. The molecular details of this interaction were investigated to shed light on how this association is regulated by the cell. The structure of an N-terminal domain from Dbf4 was solved through X-ray crystallography and discovered to have a modified <em>BR</em>CA-1 <em>C</em>-<em>t</em>erminal (BRCT) fold, which included an additional N-terminal helix. This domain could interact with the <em>F</em>ork<em>H</em>ead <em>A</em>ssociated 1 (FHA1) domain from Rad53 <em>in vitro</em>, and the additional helix was necessary for complex formation. Although the FHA1 domain has a well-characterized binding site for phospho-epitopes, a combination of chemical cross-linking and NMR spectroscopy experiments demonstrated that the N-terminal domain from Dbf4 is contacting an alternative surface. However, the full-length Dbf4 protein <em>in vivo</em> may be contacting both this distal site and the phospho-epitope binding pocket. This bipartite interaction between Dbf4 and Rad53 would lend specificity to the complex and also suggests a kinase may be regulating the association. As FHA and BRCT domains are prevalent in eukaryotic nuclear proteins, these findings are instructive for how these domains mediate interactions in other signaling pathways.</p> / Doctor of Philosophy (PhD)

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