Fidelity of eucaryotic DNA polymerase δ holoenzyme from Schizosaccharomyces pombe

Abstract

The fidelity of Schizosaccharomyces pombe DNA polymerase δ was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp- loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol δ alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. 'Passive' self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol δ holoenzyme (HE) (pol δ+ PCNA + RFC) was 4.6 x 10-4 for T·G mispairs, 5.3 x 10-5 for G·G mispairs, and 4.5 x 10-6 for A·G mispairs. The T·G misincorporation frequency for pol δ without the accessory proteins was unchanged. The fidelity of pol δ HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombe polymerase-associated proofreading exonuclease. The S. pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'- terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol δ HE with E. coli pol IIIα HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair.