Proliferating cell nuclear antigen promotes misincorporation catalyzed by calf thymus DNA polymerase δ

Abstract

A proliferating cell nuclear antigen (PCNA)-dependent complex, detectable after nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus DNA polymerase δ (pol δ) and synthetic oligonucleotide template-primers containing a mispaired nucleotide at the 3'-terminal position of the primer. This complex is indistinguishable in composition from that formed with a fully base paired template-primer. Extension of a mispaired primer terminus is a component of DNA polymerase fidelity. The fidelity of pol δ on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol δ misincorporates less than one nucleotide for every 100,000 nucleotides incorporated correctly. Addition of PCNA to reactions reduces fidelity by at least 27-fold. PCNA also confers upon pol δ, the ability to incorporate (and/or not excise) the dTTP analog, 2'-deoxythymidine-5'-O-(α- phosphonomethyl)-β,γ-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol δ·template-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol δ. This model suggests an explicit mechanistic requirement for the intrinsic 3'-5'- exonuclease of pol δ.