Direct interaction of proliferating cell nuclear antigen with the p125 catalytic subunit of mammalian DNA polymerase delta

The formation of a complex between DNA polymerase delta (pol delta) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible f or the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the ability of the p125 catalytic subunit of DNA polymerase delta to engage in protein-protein interactions with PCN A was established by biochemical and genetic methods. p125 and PCNA were sh own to co-immunoprecipitate from either calf thymus or HeLa extracts, or wh en they were ectopically co-expressed in Cos 7 cells. Because pol delta is a multimeric protein, this interaction could be indirect. Thus, rigorous ev idence was sought for a direct interaction of the p125 catalytic subunit an d PCNA. To do this, the ability of recombinant p125 to interact with PCNA w as established by biochemical means. p125 co-expressed with PCNA in Sf9 cel ls was shown to form a physical complex that can be detected on gel filtrat ion and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)), An interactio n between p125 and PCNA could also be demonstrated in the yeast two hybrid system. Overlay experiments using biotinylated PCNA showed that the free p1 25 subunit interacts with PCNA The PCNA overlay blotting method was also us ed to demonstrate the binding of synthetic peptides: corresponding to the N 2 region of pol delta and provides evidence for a site on pol delta that is involved in the protein-protein interactions between PCNA and pol delta, T his region contains a sequence that is a potential member of the PCNA bindi ng motif found in other PCNA-binding proteins. These studies provide an une quivocal demonstration that the p125 subunit of pol delta interacts with PC NA.