Functional studies of the interstrand cross-link repair protein, SNM1A and its beta-CASP domain

Abstract

<p>Interstrand cross-linking (ICL) damage to DNA is cytotoxic as it blocks replication and transcription. This cytotoxicity is exploited in anti-cancer therapies, but increased ICL repair limits the efficacy of these chemotherapies. SNM1A (sensitive to nitrogen mustard 1A), of the beta-CASP family of nucleases, has been shown to participate in the initiation of one of the ICL repair processes. Biochemical studies of SNM1A have been limited due to insolubility and instability of SNM1A in bacteria and insect cell lines and toxicity in human cell lines. Work reported in this thesis describes a novel and efficient method of generating active protein from inclusion body expression of the beta-CASP domain of SNM1A. This refolded beta-CASP domain shows 5’ exonuclease activity on single stranded and double stranded DNA in vitro. Nevertheless, this domain alone is unable to complement <em>pso2</em> null ICL repair defects in<em> S. cerevisiae</em> after exposure to ICL agents. These functional studies of the beta-CASP domain of SNM1A will be helpful in directing future research on its role in ICL repair. Additionally, this will aid future structural and inhibitor studies of this essential interstrand cross-link repair protein, SNM1A.</p>