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-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step. These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.
Ho
C K
CK
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Van Etten
J L
JL
Shuman
S
S
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent; D002708:Chlorella / Q000821:virology*; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D017862:Phycodnaviridae / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D002413:Cations, Divalent; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases