Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase δ and DNA polymerase ε

Joel C. Eissenberg, Rao Ayyagari, Xavier V. Gomes, Peter M.J. Burgers

Research output: Contribution to journalArticlepeer-review

142 Scopus citations

Abstract

The importance of the interdomain connector loop and of the carboxy- terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases δ (Polδ) and ε (Polε) was investigated by site-directed mutagenesis. Two alleles, pol30- 79 (ILI26,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro the mutant PCNAs showed defects in DNA synthesis. Interestingly the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Polδ, whereas pcna-90 was defective in replication with Polε. Protein-protein interaction studies showed that pcna- 79 and pcna-90 failed to interact with Polδ and Polε, respectively in addition, pcna-90 was defective in interaction with the FEN-1 endo- exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Polδ, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA)·oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.

Original languageEnglish
Pages (from-to)6367-6378
Number of pages12
JournalMolecular and cellular biology
Volume17
Issue number11
DOIs
StatePublished - Nov 1997

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