Budding Yeast Ubiquitin Ligases Rad5 and Rad18 Bind a Novel PCNA Surface, which Is Required for their Functions in DNA-Damage Tolerance.

DNA-damage tolerance (DDT) is a highly regulated pathway to resume DNA synthesis in the presence of replication-blocking lesions. In budding yeast Saccharomyces cerevisiae, DDT is mediated by sequential ubiquitination and sumoylation of proliferating cell nuclear antigen (PCNA). Rad18 and Rad5 are two E3 ligases responsible for PCNA mono- and poly-ubiquitination, respectively, at its K164 residue. Although Rad18 and Rad5 have been reported to interact with PCNA, they do not contain known PCNA-binding domains like a PCNA interaction peptide (PIP) box. In this study, we performed extensive mapping by a yeast two-hybrid assay and delineated PCNA-binding regions within 40 residues in both Rad18 and Rad5; amino acid sequence alignment revealed that they share a previously uncharacterized sequence defined as a LxLF motif in this study. Interestingly, Rad5 and Rad18 interact with PCNA on same surface distinct from the PIP box-binding sites. Site-specific mutagenesis confirmed that this LxLF motif is required for the DDT functions of Rad18 and Rad5 possibly through affecting PCNA mono- and poly-ubiquitination, respectively. In addition, an adjacent HIRAN domain in Rad5, known to bind 3' DNA ends to drive replication fork reversal, is also required for the PCNA interaction and DDT functions, consistent with previous reports.