Barber, L.J.; (2004) The Saccharomyces cerevisiae Snm1 gene and repair of anticancer drug-induced DNA interstrand cross-links. Doctoral thesis , University of London.

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Abstract

Saccharomyces cerevisiae (budding yeast) cells defective for the PSO2/SNM1 gene are uniquely sensitive to agents that produce DNA interstrand cross-links (ICLs), but not other forms of DNA damage. In the absence of Pso2, repair is stalled after the initial incision of ICLs, resulting in an accumulation of DNA double strand breaks (DSBs). The Pso2 protein is a member of the CASP metallo-beta-lactamase family of hydrolytic enzymes, that include the mRNA processing enzyme CPSF, and Artemis, a human V(D)J recombination factor demonstrating single-strand DNA specific 5'-to-3' exonuclease and a hairpin endonuclease activity. Given the genetic epistasis of PSO2 with the nucleotide excision repair pathway (NER) confirmed here in an isogenic background, it is probable that Pso2 is involved in the processing of ICL-repair intermediates arising from NER. Consequently, the genetic interactions between PSO2 and yeast nucleases implicated in DNA repair and recombination have been characterised. PSO2 demonstrates a striking synergistic relationship with the 5'-to-3' exonuclease EXO1. Double disruptant pso2 exo1 yeast cells are significantly more sensitive to the cross-linking drug nitrogen mustard, relative to the single mutants, and accumulate a higher proportion of DSBs, which fail to be repaired. Furthermore, loss of both pso2 and exo1 results in a moderate sensitivity to ionising radiation, and a deficiency in spontaneous homologous recombination at a chromosomal inverted-repeat substrate. EXO1 is known to interact, both genetically and physically, with the mismatch repair factor MSH2. In this study, it has been established that pso2 msh2 double disruptants phenocopy pso2 exo1, consistent with an overlap of activity of Pso2 with the Exo1-Msh2 complex. This relationship involves a requirement for both MutS complexes (Msh2-Msh3 and Msh2-Msh6), but does not extend to spontaneous mismatch correction. It is proposed that Pso2 plays a role in the processing of repair intermediates for downstream recombination events, that is functionally redundant with Exo1-MutS during spontaneous recombination and double-strand break repair. As a conclusion to this study, FLAG-tagged Pso2 protein has been successfully purified from budding yeast. Western blotting shows this protein to be larger than expected, and cross-reactivity of purified Pso2 with a human polyclonal SUMO antibody suggests a role for sumoylation in the regulation of Pso2 activity.

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