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Journal of Virology, November 1998, p. 9142-9149, Vol. 72, No. 11
Department of Microbiology, Oregon State
University, Corvallis, Oregon 97331-7301
Received 21 April 1998/Accepted 14 August 1998
Sequence analysis of the Lymantria dispar multicapsid
nucleopolyhedrovirus (LdMNPV) genome identified an open
reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that
showed 35% amino acid sequence identity with vaccinia virus
ATP-dependent DNA ligase. Ligase homologs have not been reported from
other baculoviruses. The ligase ORF was cloned and expressed as an
N-terminal histidine-tagged fusion protein. Incubation of the purified
protein with [
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of a Baculovirus-Encoded
ATP-Dependent DNA Ligase
-32P]ATP resulted in formation of a
covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled
band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the
radiolabeled band occurred upon incubation of the intermediate with
pyrophosphate, poly(dA) · poly(dT)12-18, or
poly(rA) · poly(dT)12-18, characteristics of a DNA
ligase II or III. The protein was able to ligate a double-stranded
synthetic DNA substrate containing a single nick and inefficiently
ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six
plasmids containing the LdMNPV homologs of the essential
baculovirus replication genes, a plasmid containing the DNA ligase gene
was neither essential nor stimulatory. All of these results are
consistent with the activity of type III DNA ligases, which have been
implicated in DNA repair and recombination.
*
Corresponding author. Mailing address: Department of
Microbiology, Nash Hall 220, Oregon State University, Corvallis, OR
97331-7301. Phone: (541) 737-1794. Fax: (541) 737-0479. E-mail:
pearsonm{at}bcc.orst.edu.
Technical report no. 11403 from the Oregon State University
Agricultural Experiment Station.
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