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<p>DNA interstrand crosslinks provide a challenge for repair machinery given that both strands contain the lesion. Cells have evolved a sophisticated mechanism to overcome this, by recruiting proteins from several repair pathways. One protein thought to function solely in interstrand-crosslinking repair is Pso2. Pso2 deficient cells display sensitivity towards ICL agents and accumulate DNA double strand breaks upon exposure. However, Pso2 is not required for repair of DNA double strand breaks generated by other means, suggesting that these particular breaks are unique requiring Pso2 processing for successful repair. To identify what characteristics these breaks possess and what role Pso2 plays in processing theses breaks, a thorough <em>in vivo</em> and <em>in vitro </em>characterization of Pso2 was conducted.</p> <p>Pso2 was found to be a 5’-exonuclease independent of DNA structure and length but completely dependent on a 5’-phosphate. Pso2 also displayed structure-specific DNA hairpin-opening activity at the 3’ end two nucleotides from the apex. This activity was required for repair of genomic DNA capped by hairpin structures in the absence of ICL inducing agents as well those generated in response to ICL damage. The constitutively active DNA hairpin endonuclease β-CASP domain of Artemis was able to partially restore the DNA hairpin-opening deficiency and suppress the ICL defect in a <em>pso2 </em>null strain. This suggests that Pso2 acts as an endonuclease in ICL repair and that DNA hairpins may be an encountered intermediate, leading to further understanding of how this unique protein function in ICL repair as well as the repair mechanism itself.</p> / Doctor of Science (PhD)

<p><h2> </h2></p> <p>Telomeres are protein-DNA complexes found at the ends of human chromosomes. The function of telomeres is to protect chromosome ends from being recognized as damaged DNA. This protection is essential in preventing the erosion of telomeres, which has been shown to lead to genomic instability, a hallmark of cancer and aged cells. Precise regulation of telomere length and function is crucial to cell survival, and defects in this regulation are related to tumorigenesis and aging related disorders. The proteins that bind telomere DNA play an indispensable role in telomere maintenance. TRF1, <em>t</em>elomere <em>r</em>epeat binding <em>f</em>actor 1, is a protein that directly binds to mammalian telomeric DNA and participates in regulating telomere length. Post-translational modifications, such as phosphorylation, have been shown to modulate TRF1 function. The results presented here demonstrate that two phosphorylation sites on TRF1, S367 and T371, are involved in regulating the function and localization of TRF1. TRF1 S367 is phosphorylated by ATM, and this phosphorylation removes TRF1 from telomere DNA and directs TRF1 to sites of proteasome degradation. On the other hand, the phosphorylation of TRF1 at T371 prevents the association of TRF1 with telomere DNA but also protects TRF1 from degradation. We have demonstrated that the phosphorylation of T371 by CDK1 is important for the resolution of sister chromatids in mitosis. In interphase cells, in response to the induction of DNA damage, TRF1 phosphorylated at T371 is recruited to sites of damage and is involved in promoting efficient homologous recombination and in conferring checkpoint activation and cell survival. The work presented within this thesis sheds light on the regulation of TRF1 function by phosphorylation events and reveals novel functions of TRF1.</p> / Doctor of Philosophy (PhD)

<p>DNA interstrand cross-links (ICLs) occur when various chemical agents bind to chromosomal DNA and form a covalent bond between adjacent strands, preventing unwinding of the DNA double helix. The formation of an ICL is therefore extremely toxic to cells and necessitates quick removal and subsequent repair. Human SNM1A is a 5’-phosphate-dependent exonuclease that has been shown to be selectively involved in ICL repair; however the mechanism by which it processes ICL substrates remains unclear. To address this, our research is focused on the structural and functional characterization of SNM1A to determine this mechanism of substrate processing. In this thesis, we report the purification of human SNM1A_698-1040 as a His₆-NusA tagged protein from 4 L of <em>E. Coli</em> cell culture. This protein was found to possess 5’-phosphate-dependent exonuclease activity, and demonstrated a preference for ssDNA. Additionally, electrophoretic mobility shift assays performed with a D736A/H737A mutant suggest that the binding of SNM1A to DNA is independent of the presence of a 5’ phosphate. Collectively, these results provide insight into the mechanism of SNM1A substrate processing in ICL repair, and establish a platform for future studies of this protein.</p> / Master of Science (MSc)