Characterization of the proliferating cell nuclear antigen (PCNA) in the context of DNA mismatch repair

Mismatch repair (MMR) corrects base-base and small insertion/deletion loop mispairs that result from errors during DNA replication. In vitro studies suggest that the proliferating cell nuclear antigen (PCNA) is involved in MMR at the DNA synthesis step as well as a prior step. In vivo studies suggest that PCNA is involved in MMR because PCNA mutants have a mutator phenotype. However, these PCNA mutants have MMR independent defects that may contribute to its imitator phenotype.; Here, we characterize the mutator phenotype of different PCNA mutants using assays that are specific for MMR defects. We show that three PCNA mutants are defective for MMR when analyzed by an in vivo MMR assay. However epistasis analysis suggests that these PCNA mutants have MMR independent defects that contribute to their mutator phenotypes.; Because these defects complicate the analysis of the mutator phenotypes of the PCNA mutants, we screened for PCNA mutants with MMR specific defects. We isolated two novel PCNA mutants, PCNA-201 and PCNA-204, that had a mutator phenotype but showed little or no MMR independent defects when compared to a wild-type strain. These mutants were defective for MMR when analyzed by an in vivo MMR assay and epistasis analysis suggests that the mutator phenotypes of these mutants are caused by MMR specific defects. These results support a role for PCNA in MMR in vivo. Consistently, in vitro, PCNA-204 was shown to be defective in binding to MSH2-MSH6 and PCNA-201 was defective in activating MSH2-MSH6 binding specificity.; The interaction between MSH2-MSH3 and MSH2-MSH6 and the stimulation of MSH2-MSH6 binding specificity by PCNA suggest a role for PCNA in mispair recognition. To investigate the interactions between PCNA, MSH2-MSH6, and DNA, we performed glycerol sedimentation and co-precipitation experiments. We found that MSH2-MSH6 and PCNA are able to form a ternary complex with homoduplex DNA but MSH2-MSH6 binding to heteroduplex DNA disrupts MSH2-MSH6 binding to PCNA. Our results support a role for PCNA in mispair recognition and are consistent with a model in which MSH2-MSH6 binds to PCNA tethered on newly replicated DNA but dissociates upon mispair binding.