Stimulation of DNA Synthesis Activity of Human DNA Polymerase κ by PCNA

Abstract

Humans have three DNA polymerases, Poleta, Polkappa, and Poliota, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Polkappa (hPolkappa) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPolkappa. The processivity of hPolkappa, however, is not significantly increased in the presence of these protein factors. The efficiency (V(max)/K(m)) of correct nucleotide incorporation by hPolkappa is enhanced approximately 50- to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K(m) for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased approximately 40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolkappa would bypass an abasic site by inserting a nucleotide opposite the site.