Purification of Autographa californica nucleopolyhedrovirus DNA polymerase from infected insect cells

J Gen Virol. 1999 Sep;80 ( Pt 9):2519-2526. doi: 10.1099/0022-1317-80-9-2519.

Abstract

Autographa californica nucleopolyhedrovirus (AcMNPV) DNA polymerase was purified from virus-infected cells using conventional chromatographic methods. The enzymatic activity of fractions eluting from single-stranded agarose gels was found to exactly coincide with a single polypeptide with an apparent molecular mass of approximately 110,000 Da on denaturing polyacrylamide gels stained with Coomassie blue. This purification scheme resulted in a 228-fold purification of AcMNPV DNA polymerase with recovery of 3.5% of the initial activity. The specific activity of the most purified fraction of DNA polymerase was 5000 units/mg, which is sufficiently high to eliminate the possibility that contaminants significantly contribute to the polymerase activity. Preparations of purified DNA polymerase had 3'-5' exonuclease activity, but no 5'-3' exonuclease activity. The proofreading activity was apparently an intrinsic property of the enzyme as the ratio of nuclease activity to polymerase activity was constant throughout purification. Using a singly-primed M13 DNA template, RF-II DNA was detected within 3 min, indicating a polymerization rate of 40 nt/s. The effects of several DNA polymerase inhibitors on the enzymatic activity of purified DNA polymerase were also determined.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • DNA Replication
  • DNA-Directed DNA Polymerase / isolation & purification*
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / metabolism
  • Molecular Weight
  • Nucleic Acid Synthesis Inhibitors
  • Nucleopolyhedroviruses / enzymology*
  • Spodoptera

Substances

  • Nucleic Acid Synthesis Inhibitors
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V