Purification of Autographa californica nucleopolyhedrovirus DNA polymerase from infected insect cells

• Published in: Journal of general virology Vol. 80; no. 9; pp. 2519 - 2526
• Main Authors: Hang, X, Guarino, L.A
• Format: Journal Article
• Language: English
• Published: England Soc General Microbiol 01-09-1999
• Subjects: Animals; Autographa californica; Autographa californica nucleopolyhedrovirus; DNA Replication; DNA-Directed DNA Polymerase - isolation & purification; Exodeoxyribonuclease V; Exodeoxyribonucleases - metabolism; Molecular Weight; Noctuidae; Nuclear polyhedrosis virus; Nucleic Acid Synthesis Inhibitors; Nucleopolyhedrovirus; Nucleopolyhedroviruses - enzymology; Spodoptera
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/0022-1317-80-9-2519

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 110000 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.