A ubiquitin-binding motif in the translesion DNA polymerase Rev1 mediates its essential functional interaction with ubiquitinated proliferating cell nuclear antigen in response to DNA damage.
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.
Wood
Adam
A
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Garg
Parie
P
Burgers
Peter M J
PM
D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D002474:Cell-Free System / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016296:Mutagenesis; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D025801:Ubiquitin; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases