<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)

<p>DNA interstrand cross-links (ICLs) constitute one of the most severe types of DNA damage. ICLs covalently tether both strands of duplex DNA, preventing unwinding and polymerase access during replication and transcription. This obstruction is exploited in cancer chemotherapy since it leads to replication fork collapse, double strand breaks (DSBs), and cell death. Mechanistic understanding of how eukaryotic cells repair these specific lesions, however, is still in its infancy. It is understood that ICL repair consists of a multitude of intersecting and connecting repair pathways that rely on interplay between critical protein factors. Interestingly, Pso2 has been identified as an integral component of the ICL repair pathway in <em>Saccharomyces cerevisiae</em>. Pso2 is a yeast nuclease from the β-CASP family of proteins that function predominantly in the repair of ICLs. It has been recognized as the only protein that does not serve a redundant function in any other DNA repair pathway. It remains unclear how the ICL repair pathway generates DNA intermediates suitable for high fidelity repair dependent on Pso2 nuclease activity. Here we show that Pso2 possesses structure-specific endonuclease activity that may be essential to its role in ICL repair. Direct <em>in vitro</em> activity assessment of the protein on a site-specific ICL proved to be inconclusive due to the heat-labile nature of the cross-linking agent employed. <em>In vitro </em>activity testing was also performed on various substrates resembling intermediates generated during ICL repair. Biochemical analysis demonstrated that Pso2 cleaves hairpins, stem loops, heterologous loops, and symmetrical bubbles. Although the precise cleavage sites vary between substrates, Pso2 demonstrates preference for the single- to double-stranded junction in the DNA backbone, with similar activity to that previously demonstrated for its human homologue, Artemis. This specific endonuclease activity is stimulated by increased concentrations of phosphate. Through two-dimensional gel electrophoresis, the presence of unique DNA intermediates generated in response to ICL damage <em>in </em><em>vivo </em>was also monitored. Results suggest the generation of hairpin-like intermediates that resemble those tested <em>in vitro</em>. These intermediates persist in the absence of Pso2 but are resolved by exogenous addition of control endonucleases. Our findings expand on previous data that established hairpin-opening activity for this protein and suggest that the structure-specific endonuclease activity demonstrated by Pso2 is important for ICL repair. We anticipate that Pso2 acts on a hairpin-containing DNA substrate in the ICL repair pathway and the resolution of this intermediate is uniquely dependent on Pso2 for the effective repair of ICL damage in yeast. Taking into consideration the current models of ICL repair, both in yeast and humans, possible roles for Pso2 have been described. Achieving a complete mechanistic perspective of this pathway is critical for the therapeutic exploitation of the human homologue, SNM1A. Implications include the potential inhibitory target for increased efficacy of chemotherapy with cross-linking agents.</p> / Master of Science (MSc)

Abasic sites (Ap site, from apurinic/apyrimidinic) are one of the most common lesions generated in DNA by spontaneous base loss or DNA repair processes. There are two equilibrating forms of an Ap site - ring-open aldehyde and cyclic hemiacetal. Ring- opened aldehydes are reactive electrophilic groups capable of formation covalent adduct with nucleophilic sites in DNA. DNA interstrand cross-link (ICL) resulting from the Ap sites is formed spontaneously as a covalent bond between ring-open aldehyde and amin group of adenin residue in the opposite strand of double stranded DNA. ICLs block DNA replication and transcription. The formation of Ap site derived ICL is relatively long process taking several hours. We assume that the ring-opening of an abasic site is the rate-limiting step in the formation of the thermodynamic ICL. However, formation, stability and DNA repair of Ap-ICL are still poorly understood processes. Here, I have set up mechanistic in vitro experiments to reveal and calculate the probability of Ap-ICl formation in vivo. In more detail, I study the rates of formation of Ap-ICLs in the sequence context of neighbouring nucleotides of freshly formed covalent bond of ICL. I focus on sequence preference, the influence of AT/ GC rich regions and the length of oligonucleotides. I have...