1887

Abstract

The nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 3′–5′ exonucleases. The CfMNPV 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′-P-labelled oligo(dT) 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 mM dithiothreitol and 0·1 mg BSA ml.

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2004-12-01
2020-09-28
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