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

, 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.