The Human Homologue of Fission Yeast cdc27, p66, Is a Component of Active Human DNA Polymerase δ,1.

An essential eukaryotic DNA polymerase, DNA polymerase δ(pol δ), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). Recently, a 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 was identified as a component of mouse and calf thy-mus pol δ. Our studies show that p66 interacts tightly with other subunits of pol δ during size fractionation of human cell extracts, and co-immunoprecipitates with these sub-units along with PCNA-dependent polymerase activity. Active human pol δ could be reconstituted by co-expressing pl25, p50, and p66 recombinant baculoviruses, but not by co-expressing pl25 and p50 alone. Interaction studies demonstrated that p66 tabilizes the association between pl25 and p50. Pull-down assays with PCNA-linked beads demonstrated that p66 increases the overall affinity of pol 8 for PCNA. These results indicate that p66 is a functionally important subunit of human pol 8 that stabilizes the pol 8 complex and increases the affinity of pol δ for PCNA. [ABSTRACT FROM AUTHOR]