STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF HUMAN

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 SNM1A698-1040 as a His6-NusA tagged protein from 4 L of E. Coli 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. Lastly, we have developed an in vitro fluorescence-based assay for inhibitor screening of SNM1A698-1040 with a Z ’ factor of 0.88. 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