Purification and characterization of the Saccharomyces cerevisiae DNA polymerase delta overproduced in Escherichia coli.
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.
Brown
W C
WC
Braun Laboratories, California Institute of Technology, Pasadena 91125.
Duncan
J A
JA
Campbell
J L
JL
D016590:Aphidicolin / Q000494:pharmacology; D003001:Cloning, Molecular; D004258:DNA Polymerase III; D004271:DNA, Fungal / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003854:Deoxyribonucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005092:Exonucleases / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013942:Thymine Nucleotides / Q000378:metabolism; D014316:Tritium
D004271:DNA, Fungal; D003854:Deoxyribonucleotides; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D014316:Tritium; D016590:Aphidicolin; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; C024157:thymidine 5'-triphosphate