CHARACTERIZATION OF A BACULOVIRUS-ENCODED ATP-DEPENDENT DNA-LIGASE

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrov irus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence i dentity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cl oned and expressed as an N-terminal histidine-tagged fusion protein. I ncubation of the purified protein with [alpha-P-32]ATP resulted in for mation of a covalent enzyme-adenylate intermediate which ran as a 62-k Da labeled band on a sodium dodecyl sulfate-polyacrylamide gel, Loss o f the radiolabeled band occurred upon incubation of the intermediate w ith pyrophosphate, poly(dA).poly(dT)(12-18), or poly(rA).poly(dT)(12-1 8), 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 lig ate a 2-nt gap. It was able to ligate short, complementary overhangs b ut not blunt-ended double-stranded DNA, In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the es sential baculovirus replication genes, a plasmid containing the DNA li gase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have b een implicated in DNA repair and recombination.