Purification and characterization of the Saccharomyces cerevisiae DNA polymerase delta overproduced in Escherichia coli.

W.C Brown,J.A Duncan,J.L Campbell

doi:10.1016/s0021-9258(18)54030-8

Abstract

In order to further define the enzymatic properties of yeast DNA polymerase delta, the Saccharomyces cerevisiae POL3 gene, whose expression is highly toxic to bacteria in most cloning vectors, was cloned into a new T7 expression vector (W. C. Brown and J. L. Campbell, submitted for publication) which allowed efficient overexpression in bacteria. Fifteen mg of polymerase were obtained from 3 g of cells. Since the protein is produced in insoluble form, to obtain active polymerase, inclusion bodies were solubilized with urea. DNA polymerase delta (124 kDa) was purified in the presence of urea and then renatured by dialysis against buffers containing decreasing concentrations of urea. Optimal protein concentration for refolding was 5 micrograms/ml. By several criteria the enzyme obtained is comparable with that from yeast: specific activity, electrophoretic mobility, template preference, sensitivity to inhibitors, and processivity. The electrophoretic mobility suggests that, unlike DNA polymerase alpha, polymerase delta is not posttranslationally modified in yeast. Polyclonal antibody was raised against the full-length DNA polymerase delta from bacteria and shown to cross-react with the protein purified from yeast on protein blots. The renatured protein also exhibits an exonucleolytic activity. Further examination of this nuclease determined it to be a 3' to 5' exonuclease with the characteristics of a proofreading activity. The presence of this nuclease in the highly purified bacterial polymerase provides biochemical confirmation of earlier genetic evidence (Simon, M., Giot, L., and Faye, G. (1991) EMBO J. 10, 2165-2170) that suggested that DNA polymerase delta's core catalytic subunit contains an intrinsic 3' to 5' exonuclease.

Highlights

Biochemical analysis of S. cereuisiae cdc2 mutants (Sitney et al, 1989) and DNA sequence analysis (Boulet et al, 1989) botharrived at the conclusion that DNApolymerase 6 is encoded bythe CDC2 gene
This nuclease in the highly purified bacterial polymer- An accessory factor was discovered during thepurification ase provides biochemical confirmation of earlier genetic evidence (SimonM, ., Giot, L., and FayeG, . (1991) EMBO J. 10, 2165-2170) that suggested that DNA polymerase 6’s core catalytic subunit contains an intrinsic 3’to 5’ exonuclease
In order to fully repress transcription priortoinduction, a new T7-based expressionvector was developed. This was used to clone the full-lengthPOL3 gene and deletions of either the amino terminus or the putative exonuclease domains or both.' Only the full-lengthcopy and the gene truncated at the NdeI site (Fig. l),which removes the 220 amino acids of the amino terminus but retains alolf the polymerase conserved regions including the exonuclease domains in region IV, were found to be overexpressed

Summary

Introduction

Biochemical analysis of S. cereuisiae cdc2 mutants (Sitney et al, 1989) and DNA sequence analysis (Boulet et al, 1989) botharrived at the conclusion that DNApolymerase 6 is encoded bythe CDC2 gene. The enzymatic activities thought to be associated with the 124-kDa subunit were characterized and compared with a DNA polymerase 6 preparation purified from yeast cells. Aliquots were withdrawn and placed into reaction buffer on ice. Reactions were started by the addition of substrate and incubated at 37 "C for 30 min (DNA synthesis) or 20 min (exonuclease).Reactions were processed as described above.

Results

Conclusion