Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication.
The DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems. While less protein was obtained from the E. coli expression system, SpliNPV DNAPOL purified from E. coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system. Biochemical analyses suggested that the DNA polymerase and the 3'-5' exonuclease activities are intrinsic to the protein. Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme. Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive. Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment. Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay.
Huang
Jianhe
J
Department of Biology, University of Victoria, PO Box 3020, Victoria, British Columbia V8W 3N5, Canada1.
Levin
David B
DB
Department of Biology, University of Victoria, PO Box 3020, Victoria, British Columbia V8W 3N5, Canada1.
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005092:Exonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009154:Mutation; D017924:Nucleopolyhedroviruses / Q000187:drug effects / Q000201:enzymology*; D018411:Spodoptera / Q000821:virology*; D013997:Time Factors
D004279:DNA, Viral; D004791:Enzyme Inhibitors; D016590:Aphidicolin; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases