Functional analysis of Pso2 reveals a novel DNA hairpin endonuclease activity: Implications for interstrand crosslink repair

Abstract

<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>