Genetic Dissection of Budding Yeast PCNA Mutations Responsible for the Regulated Recruitment of Srs2 Helicase

DNA-damage tolerance (DDT) is a mechanism by which eukaryotes bypass replication-blocking lesions to resume DNA synthesis and maintain cell viability. In Saccharomyces cerevisiae, DDT is mediated by sequential ubiquitination and sumoylation of proliferating cell nuclear antigen (PCNA, encoded by POL30) at the K164 residue. Deletion of RAD5 or RAD18, encoding two ubiquitin ligases required for PCNA ubiquitination, results in severe DNA-damage sensitivity, which can be rescued by inactivation of SRS2 encoding a DNA helicase that inhibits undesired homologous recombination. In this study, we isolated DNA-damage resistant mutants from rad5Δ cells and found that one of them contained a pol30-A171D mutation, which could rescue both rad5Δ and rad18Δ DNA-damage sensitivity in a srs2-dependent and PCNA sumoylation-independent manner. Pol30-A171D abolished physical interaction with Srs2 but not another PCNA-interacting protein Rad30; however, Pol30-A171 is not located in the PCNA-Srs2 interface. The PCNA-Srs2 structure was analyzed to design and create mutations in the complex interface, one of which, pol30-I128A, resulted in phenotypes reminiscent of pol30-A171D. This study allows us to conclude that, unlike other PCNA-binding proteins, Srs2 interacts with PCNA through a partially conserved motif, and the interaction can be strengthened by PCNA sumoylation, which turns Srs2 recruitment into a regulated process.