PCNA binding domains in all three subunits of yeast DNA polymerase δ modulate its function in DNA replication

DNA polymerase delta (Pol delta) plays an essential role in replication from yeast to humans. Pold in Saccharomyces cerevisiae is comprised of three subunits, the catalytic subunit Pol3 and the accessory subunits Pol31 and Pol32. Yeast Pold exhibits a very high processivity in synthesizing DNA with the proliferating cell nuclear antigen (PCNA) sliding clamp; however, it has remained unclear how Pold binds PCNA to achieve its high processivity. Here we show that PCNA interacting protein (PIP) motifs in all three subunits contribute to PCNA-stimulated DNA synthesis by Pol delta, and mutational inactivation of all three PIP motifs abrogates its ability to synthesize DNA with PCNA. Genetic analyses of mutations in these PIPs have revealed that in the absence of functional Pol32 PIP domain, PCNA binding by both the Pol3 and Pol31 subunits becomes essential for cell viability. Based on our biochemical and genetic studies we infer that yeast Pol delta can simultaneously utilize all three PIP motifs during PCNA-dependent DNA synthesis, and suggest that Pold binds the PCNA homotrimer via its three subunits. We consider the implications of these observations for Pol delta's role in DNA replication.