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

Yeast DNA polymerase delta (Pol delta) 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 Pol delta, which was identical in enzymatic properties to Pol delta 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 Pol delta*:, is structurally and functionally analogous to mammalian Pol delta. The properties of Pol delta* and Pol delta were compared. A gel filtration analysis showed that Pol delta* is a heterodimer (Pol3p.Pol31p) and Pol delta a dimer of a heterotrimer, (Pol3p.Pol31p.Pol32p)(2). In the absence of proliferating cell nuclear antigen (PCNA), Pol delta* showed a processivity of 2-3 on poly(dA) oligo(dT) compared with 5-10 for Pol delta. In the presence of PCNA, both enzymes were fully processive on this template. DNA replication by Pol delta* on a natural DNA template was dependent on PCNA and on replication factor C. However, Pol delta*-mediated DNA synthesis proceeded inefficiently and was characterized by frequent pause sites. Reconstitution of Pol delta was achieved upon addition of Pol32p to Pol delta.