Purification and Characterization of the Saccharomyces cerevisiae DNA Polymerase 6 Overproduced in Escherichia coZi

Abstract

In order to further define the enzymatic properties of yeast DNA polymerase 6, 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 6 (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 pg/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 a, polymerase 6 is not posttranslationally modified in yeast. Polyclonal antibody was raised against the full-length DNA polymerase 6 from bacteria and shown to cross-react with the protein purifiPd 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 characterisitics 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 6’s core catalytic subunit contains an intrinsic 3’ to 5’ exonuclease.

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@inproceedings{Brown2001PurificationAC, title={Purification and Characterization of the Saccharomyces cerevisiae DNA Polymerase 6 Overproduced in Escherichia coZi}, author={William Clay Brown and Joseph A Duncan and Judith and C S Campbell}, year={2001} }