Ubiquitin protease Ubp1 cooperates with Ubp10 and Ubp12 to revert Lysine-164 PCNA ubiquitylation at replication forks

ABSTRACT Proliferating cell nuclear antigen (PCNA) is essential for the faithful duplication of eukaryotic genomes. PCNA orchestrates events necessary to deal with threats to genomic integrity, such as the DNA damage tolerance (DDT) response. DDT is a mechanism by which eukaryotic cells bypass replication-blocking lesions to prevent replisome instability. DDT pathways are regulated by the ubiquitylation of PCNA and the consequent recruitment of specialized polymerases and mechanisms able to guarantee the continuity of replication. We have previously described that the deubiquitylases Ubp10 and Ubp12 associate with replication forks and modulate DDT events by reverting the ubiquitylation of PCNA in Saccharomyces cerevisiae . The results of this study unveil Ubp1 as a new PCNA deubiquitylase, which cooperates with Ubp10 and Ubp12 in the regulation of DDT during DNA replication. Ubp1 is known as a cytoplasmic protein, however, we found that it also localizes to the nucleus where it binds to chromatin and associates with DNA replication forks. In addition, Ubp1 interacts with and deubiquitylates PCNA. The ablation of Ubp1, Ubp10, and Ubp12 enhances both the accumulation of ubiquitylated PCNA and the DNA replication defects observed in cells depleted for Ubp10 and Ubp12, supporting a cooperative role among the three enzymes. IMPORTANCE PCNA ubiquitylation regulates DDT mechanisms to bypass genotoxic lesions during replication and that PCNA deubiquitylation is required to limit the extent of bypass events. This study shows that Saccharomyces cerevisiae PCNA is ubiquitylated during an unperturbed S-phase progression and that three ubiquitin proteases (Ubp1, Ubp10, and Ubp12) work together facilitating DNA replication by efficiently controlling ubiquitylation of PCNA at replication forks.