Abstract
The catalytic polypeptide of human DNA polymerase delta was overexpressed in BSC-40 cells (African green monkey kidney cell line) using the vaccinia virus/pTM1 system. The recombinant human DNA polymerase delta was purified to homogeneity in two steps using an immunoaffinity column and a single-stranded DNA-cellulose column. Levels of expression were about 1% of soluble cytosolic protein. The recombinant catalytic subunit was fully active and exhibited enzymatic properties similar to that of the native two-subunit enzyme including the possession of an associated 3' to 5' exonuclease activity. Recombinant pol delta was stimulated by proliferating cell nuclear antigen (PCNA); however, the degree of stimulation was lower than that of the native human enzyme. Analysis of a double mutant of the catalytic subunit, H142R/F144S, showed that it had a greatly reduced sensitivity to PCNA, suggesting that the PCNA binding site of pol delta may be located in this region of the N terminus.
Highlights
From the Department of Medicine, University of Miami School of Medicine, Miami, Florida 33101 and the :j:Department of Microbiology and Immunology, University of Florida, Gainesville, Florida 32610
The catalytic polypeptide ofhuman DNA polymerase 0 was overexpressed in BSC-40 cells (African green monkey kidney cell line) using the vaccinia virus/pTMl system
Recombinant polo was stimulated by proliferating cell nuclear antigen (PCNA); the degree of stimulation was lower than that of the native human enzyme
Summary
7993-7998, 1995 Printed in U.S.A. Expression of the Catalytic Subunit of Human DNA Polymerase i) in Mammalian Cells Using a Vaccinia Virus Vector System*. CDNAs for the calf thymus (Zhang et al, 1991) and human (Chung et al, 1991; Yang et al, 1992) pol 8 catalytic subunit have been cloned, as have the genes for the yeast homologues of pol 8 (Boulet et al., 1989; Pignede et al, 1991). Analysis of the N-terminal regions show that there is some conservation among the members of the pol 8 group of enzymes, and that these regions are conserved to some extent in herpes simplex virus type I DNA polymerase (Yang et al, 1992). In this report we describe the successful expression of the active catalytic subunit of pol 8 using a vaccinia virus/pTMI expression system in BSC-40 cells
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