CHARACTERIZATION OF AN ATP-DEPENDENT DNA-LIGASE ENCODED BY CHLORELLA VIRUS PBCV-1

We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dep endent DNA ligase. The PBCV-1 enzyme is the smallest member of the cov alent nucleotidyl transferase superfamily, which includes the ATP-depe ndent polynucleotide ligases and the GTP-dependent RNA capping enzymes . The specificity of PBCV-1 DNA ligase was investigated by using purif ied recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (K-m, 75 mu M). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate acro ss a 2-nucleotide gap and ligated poorly across a 2-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discrimi nated between nicked and gapped DNAs at the substrate-binding step. Th ese findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.