Abstract
Rev1 and DNA polymerase ζ (Polζ) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Polζ lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Polζ with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pull-down and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Polζ-dependent TLS pathway. Taken together, the data suggest that Polζ and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.
Highlights
The damage in either an accurate or mutagenic manner
The polymerase-associated domain (PAD) of Rev1 Interacts with proliferating cell nuclear antigen (PCNA) at the Site Marked by the pol30-113 Mutation—The first set of experiments was designed to determine whether the PAD of Rev1 binds to PCNA, and, if so, whether the interaction involves the region near the intermolecular interface of the PCNA trimer
Consistent with our hypothesis, we found that both glutathione S-transferase (GST)-Rev1-PAD and GSTRev1-catalytic core (CC) efficiently pulled down PCNA but not PCNA-113
Summary
Plasmids—Yeast expression vector pRS425-GALGST [14] was kindly provided by Peter Burgers Fragments of Saccharomyces cerevisiae REV1 encoding for amino acids 297–746 and 621– 746 were amplified by PCR and cloned in frame with glutathione S-transferase (GST) into pRS425-GALGST. The pGEX5X-1-Pol plasmid4containing the open reading frame of S. cerevisiae POL32 cloned in-frame with GST into the E. coli expression vector pGEX-5X-1 was used for the yeast Pol overproduction. The chromosomal wild-type POL30 gene of the rad, rad, rev, rev, rad, mms, ubc, and rad mutants and the corresponding wild-type strains was replaced by the pol113 allele as described previously [31]. E. coli strain BL21 (DE3) was used for overproduction and purification of GST-tagged Pol and untagged PCNA. Purification of Rev Fragments—The yeast BJ2168 strain carrying pRS425-GALGST-Rev1(297–746) or pRS425-GALGSTRev1(621–746) was grown at 30 °C in 3% glycerol/2% ethanol/ 0.1% glucose medium selective for the plasmids, and expression was induced by incubating in yeast extract/peptone/0.1% glucose/2% galactose medium for 16 –20 h.
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