DNA polymerase δ (pol δ) has a major and essential role in eukaryotic nuclear DNA replication (1
). Pol δ also performs DNA synthesis during homologous recombination and fills DNA gaps during mismatch repair, long patch base excision repair of damaged bases and nucleotide excision repair of bulky DNA lesions [reviewed in (2
)]. Because all these transactions influence eukaryotic genome stability, it is of interest to understand the fidelity of DNA synthesis conducted by pol δ. Previous studies (3
) have shown that pol δ is a highly accurate enzyme whose fidelity derives from high nucleotide selectivity at the polymerase active site and from proofreading by its intrinsic 3′ exonuclease activity.
In performing its roles in replication and repair, pol δ is assisted by accessory proteins. The three-subunit replication protein A (RPA) complex (6
) binds single-stranded DNA and coordinates the exchange of pol δ and other proteins at template–primer termini (7
). In addition, the processivity of pol δ is enhanced by proliferating cell nuclear antigen (PCNA) (8
), the sliding clamp that is loaded onto template–primers by the five-subunit RFC complex (9
). There are several reasons to consider whether RPA or PCNA modulate the fidelity of DNA synthesis by pol δ. Genetic studies have identified mutations in the genes encoding the large subunit of RPA (10
), RFC subunits (12
) and PCNA (14
) that elevate mutation rates. Among several possible explanations for these mutator effects, one is that they may result from reduced DNA synthesis fidelity by pol δ during replication (15
), repair or recombination. Single-stranded DNA-binding proteins have been shown previously to affect the fidelity of other DNA polymerases [e.g. see (16
) and references therein]. Proteins that enhance polymerase processivity promote the extension of mismatches (18
), which could reduce base substitution fidelity by preventing partitioning of mismatches to the active sites of proofreading exonucleases (21
). On the other hand, several studies [reviewed in (22
)] have shown that the processivity of DNA polymerases correlates with their insertion/deletion (indel) fidelity in mononucleotide repeat sequences, such that proteins that increase processivity may improve indel fidelity.
Here we test these ideas by examining the fidelity of DNA synthesis by three-subunit yeast DNA polymerase δ alone and its fidelity in the presence of RPA alone, PCNA (plus its loader RFC) and all three accessory protein complexes. To evaluate the effects of these accessory proteins on both nucleotide selectivity and proofreading, we compare error rates of proofreading-proficient (wild-type) pol δ to those observed with two different proofreading-deficient derivatives. To obtain a comprehensive view of the effects of accessory proteins on pol δ fidelity, we use an assay (23
) that scores a variety of base substitution and indel errors. The results indicate that fidelity for errors involving single base pairs is largely determined by pol δ itself. However, the accessory proteins strongly modulate the ability of pol δ to delete large numbers of nucleotides between directly repeated sequences. The results are discussed in relation to earlier studies (cited below) on the effects of accessory proteins, and in light of models for how accessory proteins may modulate fidelity.