The interdomain connector loop of human PCNA is involved in a direct interaction with human polymerase delta

Proliferating cell nuclear antigen (PCNA) is required for processive DNA synthesis catalyzed by DNA polymerase delta (pol delta) and polymerase epsilon. We have shown that the epitope of a human PCNA inhibitory monoclonal antibody (74B1), which inhibits the PCNA stimulation of DNA synthesis catalyzed by pol delta, maps to residues 121-135, which overlap the interdomain connector loop of PCNA (residues 119-133). We have mutagenized residues 122-133 of human PCNA. The mutant proteins were expressed in Escherichia coli and purified to near-homogeneity. The interactions of the mutants with antibody 74B1 were examined; mutation of Gly-127 abolished the recognition by antibody 74B1 in a Western blot analysis, confirming the epitope assignment of 74B1. Mutations of Val-123, Leu-126, Gly-127, and Ile-128 affected the ability of PCNA to stimulate DNA synthesis by pol delta in several different assays. These mutations affected the interactions between PCNA and pol delta as determined by enzyme-linked immunosorbent assays. These mutants were also affected in their abilities to form a ternary complex with a DNA template-primer, as determined by electrophoretic mobility gel shift assays. The findings show that the interdomain connector loop region is involved in binding of pol delta. This same region is involved in the binding of p21, and our findings support the view that the mechanism of inhibition of DNA synthesis by p21 is due to a competition for PCNA binding to pol delta.