Structure and processivity of two forms of saccharomyces cerevisiae DNA polymerase δ

Abstract

Yeast DNA polymerase δ (Polδ) consists of three subunits encoded by the POL3, POL31, and POL32 genes. Each of these genes was cloned under control of the galactose-inducible GAL1-10 promoter and overexpressed in various combinations. Overexpression of all three genes resulted in a 30- fold overproduction of Polδ, which was identical in enzymatic properties to Polδ isolated from a wild-type yeast strain. Whereas overproduction of POL3 together with POL32 did not lead to an identifiable Pol3p·Pol32p complex, a chromatographically distinct and novel complex was identified upon overproduction of POL3 and POL31. This two-subunit complex, designated Polδ*, is structurally and functionally analogous to mammalian Polδ. The properties of Polδ* and Polδ were compared. A gel filtration analysis showed that Polδ* is a heterodimer (Pol3p·Pol31p) and Polδ a dimer of a heterotrimer, (Pol3p·Pol31p·Pol32p)2. In the absence of proliferating cell nuclear antigen (PCNA), Polδ* showed a processivity of 2-3 on poly(dA)·oligo(dT) compared with 5-10 for Polδ. In the presence of PCNA, both enzymes were fully processive on this template. DNA replication by Polδ* on a natural DNA template was dependent on PCNA and on replication factor C. However, Polδ*-mediated DNA synthesis proceeded inefficiently and was characterized by frequent pause sites. Reconstitution of Polδ was achieved upon addition of Pol32p to Polδ*.