Canadian Forest Service Publications

Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 3'–5' exonuclease. 2004. Yang, D.-H.; de Jong, J.G.; Makhmoudova, A.; Arif, B.M.; Krell, P.J. Journal of General Virology 85: 3569-3573.

Year: 2004

Available from: Great Lakes Forestry Centre

Catalog ID: 26870

Language: English

CFS Availability: PDF (request by e-mail)

Abstract

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'-32P-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 MgCl2, 2 mM dithiothreitol and 0·1 mg BSA ml–1.

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