The human homologue of fission yeast cdc27, p66, is a component of active human DNA polymerase delta

An essential eukaryotic DNA polymerase, DNA polymerase delta (pol delta), s ynthesizes DNA processively in the presence of proliferating cell nuclear a ntigen (PCNA), Recently, a 66 kDa polypeptide (p66) that displays significa nt homology within its PCNA binding domain to that of fission yeast cdc27 w as identified as a component of mouse and calf thymus pol delta. Our studie s show that p66 interacts tightly with other subunits of pol delta during s ize fractionation of human cell extracts, and co-immunoprecipitates with th ese subunits along with PCNA-dependent polymerase activity. Active human po l delta could be reconstituted by co-expressing p125, p50, and p66 recombin ant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrated that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrated that p66 increase s the overall affinity of pol delta for PCNA. These results indicate that p 66 is a functionally important subunit of human pol delta that stabilizes t he pol delta complex and increases the affinity of pol delta for PCNA.