(An) investigation of Pso2 functions in DNA interstrand crosslink repair in Saccharomyces cerevisiae

Abstract

the N-terminal domain exerts an inhibitory effect on the nuclease activity encoded by the C-terminal catalytic domain since proteolytic removal of the N-terminal 204 amino acids of Pso2 resulted in a marked increase of exonuclease activity. In keeping with this, purified recombinant Pso2Δ204N that lacks the first 204 amino acids exhibited robust nuclease activity. Extensive analyses of preparations of full-length Pso2 and a variety of truncated versions of Pso2 revealed that full-length Pso2 was extremely low in specific activity or virtually inactive. Our findings predict the existence of a mediator that can activate Pso2 by displacing its N-terminal domain in ICL repair. Despite the robust nuclease activity observed with Pso2Δ204N, however, pso2Δ204N mutants were sensitive to psoralen/UVA, a DNA interstrand crosslinking agent. This indicates that the catalytic activity of Pso2 alone is not sufficient for its adequate function in vivo. Analysis of Pso2Δ204N enzymatic activity revealed that Pso2Δ204N required a free 5’-PO4 to hydrolyze DNA and was a DNA-structure dependent exonuclease. Furthermore, we found that Pso2 was a multiple-copy suppressor of dna2Δ405N mutant at restrictive temperature, suggesting that Pso2 interacts genetically with Dna2 in DNA repair. In fact, genetic analysis of pso2Δ and dna2Δ405N double mutants indicated that Pso2 acted synergistically with Dna2 in ICL repair. Importantly, extensive analysis of pso2Δ mutant with other mutants (dna2Δ405N and exo1Δ) critical for double strand breaks (DSBs) end resection revealed that these nucleases shared the same DNA substrates, but acting in three different repair pathways. The active involvement of each nuclease in the processing of DSBs ends is dependent on the context of DSBs formation. Pso2 is critical for the processing of ICL-induced DSBs, while Dna2 and Exo1 play more significant roles than Pso2 in non ICL-induced DSBs end resection.

Pso2, an exonuclease of the metallo-β-lactamase superfamily, plays an important role in DNA interstrand crosslink repair (ICL repair) in Saccharomyces cerevisiae. However, the enzymatic activity of Pso2 and its functions are largely unknown. Here, we report that Pso2 consists of two functionally distinct domains

an N-terminal domain (~204 amino acids) that could act as a regulatory domain, and a C-terminal domain (~457 amino acids) that possesses catalytic activity