Activation of Ubiquitin-Dependent DNA Damage Bypass Is Mediated by Replication Protein A

• DOI: 10.1016/j.molcel.2007.12.016
• Publication Date: 2008-04-11T19:38:36Z
• Authors: Adelina A. Davies; Diana Huttner; Yasukazu Daigaku; Shuhua Chen; Helle D. Ulrich
• Keywords: DNA Replication; 0301 basic medicine; Saccharomyces cerevisiae Proteins; Ubiquitin; Cell Cycle; DNA, Single-Stranded; Cell Biology; Saccharomyces cerevisiae; Article; Cell Line; DNA-Binding Proteins; 03 medical and health sciences; Proliferating Cell Nuclear Antigen; Replication Protein A; Two-Hybrid System Techniques; Animals; Humans; Molecular Biology; DNA Damage

Replicative DNA damage bypass, mediated by the ubiquitylation of the sliding clamp protein PCNA, facilitates the survival of a cell in the presence of genotoxic agents, but it can also promote genomic instability by damage-induced mutagenesis. We show here that PCNA ubiquitylation in budding yeast is activated independently of the replication-dependent S phase checkpoint but by similar conditions involving the accumulation of single-stranded DNA at stalled replication intermediates. The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation. We found that RPA directly interacts with the ubiquitin ligase responsible for the modification of PCNA, Rad18, both in yeast and in mammalian cells. Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro. Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.