Abstract
During normal DNA replication, the proliferating cell nuclear antigen (PCNA) enhances the processivity of DNA polymerases at the replication fork. When DNA damage is encountered, PCNA is monoubiquitinated on Lys-164 by the Rad6-Rad18 complex as the initiating step of translesion synthesis. DNA damage bypass by the translesion synthesis polymerase Rev1 is enhanced by the presence of ubiquitinated PCNA. Here we have carried out a mutational analysis of Rev1, and we have identified the functional domain in the C terminus of Rev1 that mediates interactions with PCNA. We show that a unique motif within this domain binds the ubiquitin moiety of ubiquitinated PCNA. Point mutations within this ubiquitin-binding motif of Rev1 (L821A,P822A,I825A) abolish its functional interaction with ubiquitinated PCNA in vitro and strongly attenuate damage-induced mutagenesis in vivo. Taken together, these studies suggest a specific mechanism by which the interaction between Rev1 and ubiquitinated PCNA is stabilized during the DNA damage response.
Highlights
3 The abbreviations used are: pol, DNA polymerase; TLS, translesion synthesis; RPA, replication protein A; PCNA, proliferating cell nuclear antigen; PCNAUbi, PCNA mono-ubiquitinated at Lys-164; UBM, ubiquitin-binding motif; RFC, replication factor C
After coating of the singlestranded template regions with the single-stranded binding protein RPA, PCNA or PCNAUbi was loaded by RFC prior to initiating DNA synthesis at t ϭ 0 by addition of Rev1
Two studies have recently been published [38, 39] that investigate the importance of the UBM of Y-family DNA polymerases for their function in vivo, and correlate these genetic results with in vitro interaction studies with ubiquitinated PCNA
Summary
3 The abbreviations used are: pol, DNA polymerase; TLS, translesion synthesis; RPA, replication protein A; PCNA, proliferating cell nuclear antigen; PCNAUbi, PCNA mono-ubiquitinated at Lys-164; UBM, ubiquitin-binding motif; RFC, replication factor C. TLS of most forms of DNA damage involves the participation of three DNA polymerases, pol ␦, pol , and Rev, and may require additional activation by the Cdc7/Dbf protein kinase that normally functions in cell cycle progression [12, 19]. This pathway is largely responsible for DNA damage-induced mutagenesis in eukaryotic cells. Several models for TLS of damage have been proposed in which one DNA polymerase carries out the insertion step across the lesion, and a second polymerase extends from the lesion [22, 23]
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