title: The non-canonical protein binding site at the monomer-monomer interface of yeast proliferating cell nuclear antigen (PCNA) regulates the Rev1-PCNA interaction and Polζ/Rev1-dependent translesion DNA synthesis.
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.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
authors.forename: Neeru M
authors.initials: NM
authors.lastname: Sharma
authors.forename: Olga V
authors.initials: OV
authors.lastname: Kochenova
authors.forename: Polina V
authors.initials: PV
authors.lastname: Shcherbakova
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects; D004271:DNA, Fungal / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D050296:Microbial Viability / Q000528:radiation effects; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
chemical_list: D034961:Antigens, Nuclear; D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase; D050505:Mutant Proteins; D009713:Nucleotidyltransferases; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C546594:POL30 protein, S cerevisiae; C522758:Pol32 protein, S cerevisiae; C425929:DNA polymerase zeta; C100951:REV1 protein, S cerevisiae; D000099270:Y-Family DNA Polymerases