A two-hybrid system was used to screen yeast and human expression libraries for proteins that interact with mismatch repair proteins. PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2. A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1. Mismatch repair activity was examined in human cell extracts using an assay that does not require DNA repair synthesis. Activity was inhibited by p21WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA. The data suggest a PCNA requirement in mismatch repair at a step preceding DNA resynthesis. The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between mismatch repair and replication and may be relevant to the roles of mismatch repair proteins in other DNA transactions.
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We thank John I. Risinger and Sean M. Baker for critical comments on the manuscript. We also thank Steve Ellege for the two-hybrid library, Peter Burgers for PCNA mutant plasmids and strains, and C. Eric Bonner for assistance in calculating mutation rates. This work was supported by NIH award GM45413–02 and NSF award MCB 9314116 to R. M. L. and by ACS fellowship PF-4305 to A. B. B.