ABSTRACT
The Saccharomyces cerevisiae REV3/7-encoded polymerase ζ and Rev1 are central to the replicative bypass of DNA lesions, a process called translesion synthesis (TLS). While yeast polymerase ζ extends from distorted DNA structures, Rev1 predominantly incorporates C residues from across a template G and a variety of DNA lesions. Intriguingly, Rev1 catalytic activity does not appear to be required for TLS. Instead, yeast Rev1 is thought to participate in TLS by facilitating protein-protein interactions via an N-terminal BRCT motif. In addition, higher eukaryotic homologs of Rev1 possess a C terminus that interacts with other TLS polymerases. Due to a lack of sequence similarity, the yeast Rev1 C-terminal region, located after the polymerase domain, had initially been thought not to play a role in TLS. Here, we report that elevated levels of the yeast Rev1 C terminus confer a strong dominant-negative effect on viability and induced mutagenesis after DNA damage, highlighting the crucial role that the C terminus plays in DNA damage tolerance. We show that this phenotype requires REV7 and, using immunoprecipitations from crude extracts, demonstrate that, in addition to the polymerase-associated domain, the extreme Rev1 C terminus and the BRCT region of Rev1 mediate interactions with Rev7.
Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions
