Structure and processivity of two forms of Saccharomyces cerevisiae DNA polymerase delta.
Yeast DNA polymerase delta (Poldelta) 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 Poldelta, which was identical in enzymatic properties to Poldelta 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 Poldelta*, is structurally and functionally analogous to mammalian Poldelta. The properties of Poldelta* and Poldelta were compared. A gel filtration analysis showed that Poldelta* is a heterodimer (Pol3p.Pol31p) and Poldelta a dimer of a heterotrimer, (Pol3p.Pol31p.Pol32p)2. In the absence of proliferating cell nuclear antigen (PCNA), Poldelta* showed a processivity of 2-3 on poly(dA). oligo(dT) compared with 5-10 for Poldelta. In the presence of PCNA, both enzymes were fully processive on this template. DNA replication by Poldelta* on a natural DNA template was dependent on PCNA and on replication factor C. However, Poldelta*-mediated DNA synthesis proceeded inefficiently and was characterized by frequent pause sites. Reconstitution of Poldelta was achieved upon addition of Pol32p to Poldelta*.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA. burgers@biochem.wustl.edu
P M
PM
Burgers
K J
KJ
Gerik
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004258:DNA Polymerase III / Q000737:chemistry*; D004261:DNA Replication / Q000235:genetics; D019281:Dimerization; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011067:Poly dA-dT / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; C016069:poly (dA).oligo((d)T); D011067:Poly dA-dT; D004258:DNA Polymerase III