Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 3'-5' exonuclease.
The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 3'-5' exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 3'-5' exonuclease that cleaves oligonucleotides from the 3' end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 5'-digoxigenin- or 5'-(32)P-labelled oligo(dT)(30) substrate was observed at increasing incubation times or increased amounts of V-TREX. The 3'-excision activity of V-TREX was maximal at alkaline pH (9.5) in the presence of 5 mM MgCl(2), 2 mM dithiothreitol and 0.1 mg BSA ml(-1).
Yang
Dan-Hui
DH
Department of Microbiology, University of Guelph, Guelph, ON, Canada, N1G 2W1.
de Jong
Jondavid G
JG
Department of Microbiology, University of Guelph, Guelph, ON, Canada, N1G 2W1.
Makhmoudova
Amina
A
Department of Microbiology, University of Guelph, Guelph, ON, Canada, N1G 2W1.
Arif
Basil M
BM
Great Lakes Forestry Centre, Sault Ste Marie, ON, Canada, P6A 2E5.
Krell
Peter J
PJ
Department of Microbiology, University of Guelph, Guelph, ON, Canada, N1G 2W1.
D000818:Animals; D004261:DNA Replication; D005092:Exonucleases / Q000235:genetics / Q000502:physiology*; D009036:Moths / Q000821:virology*; D017924:Nucleopolyhedroviruses / Q000201:enzymology* / Q000235:genetics
D005092:Exonucleases