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.

[1]  J. V. Van Etten,et al.  Analysis of 74 kb of DNA located at the right end of the 330-kb chlorella virus PBCV-1 genome. , 1997, Virology.

[2]  D. Wigley,et al.  Crystal Structure of an ATP-Dependent DNA Ligase from Bacteriophage T7 , 1996, Cell.

[3]  J. V. Van Etten,et al.  Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182. , 1996, Virology.

[4]  S. Shuman,et al.  Vaccinia virus DNA ligase: specificity, fidelity, and inhibition. , 1995, Biochemistry.

[5]  S. Shuman,et al.  Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA , 1995, Molecular and cellular biology.

[6]  J. V. Van Etten,et al.  Analysis of 43 kb of the Chlorella virus PBCV-1 330-kb genome: map positions 45 to 88. , 1995, Virology.

[7]  S. Shuman,et al.  Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis. , 1995, Virology.

[8]  S. Shuman,et al.  RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases , 1995, Molecular microbiology.

[9]  D. Barnes,et al.  Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination , 1995, Molecular and cellular biology.

[10]  J. V. Van Etten,et al.  Analysis of 45 kb of DNA located at the left end of the chlorella virus PBCV-1 genome. , 1995, Virology.

[11]  A. Tomkinson,et al.  Mammalian DNA ligase II is highly homologous with vaccinia DNA ligase. Identification of the DNA ligase II active site for enzyme-adenylate formation. , 1994, The Journal of biological chemistry.

[12]  S. Shuman,et al.  Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[13]  S. Buratowski,et al.  Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Shuman,et al.  Mutational analysis of yeast mRNA capping enzyme. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  E. G. Niles,et al.  Identification of the vaccinia virus mRNA guanyltransferase active site lysine. , 1993, The Journal of biological chemistry.

[16]  S. Shuman,et al.  Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases. , 1993, The Journal of biological chemistry.

[17]  A. Tomkinson,et al.  DNA ligase I from Saccharomyces cerevisiae: physical and biochemical characterization of the CDC9 gene product. , 1992, Biochemistry.

[18]  A. Kletzin.  Molecular characterisation of a DNA ligase gene of the extremely thermophilic archaeon Desulfurolobus ambivalens shows close phylogenetic relationship to eukaryotic ligases. , 1992, Nucleic acids research.

[19]  L. Dixon,et al.  An African swine fever virus gene with homology to DNA ligases. , 1992, Nucleic acids research.

[20]  J. V. Van Etten,et al.  Viruses and viruslike particles of eukaryotic algae , 1991, Microbiological reviews.

[21]  T. Lindahl,et al.  In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I. , 1991, Nucleic acids research.

[22]  A. Tomkinson,et al.  Location of the active site for enzyme-adenylate formation in DNA ligases. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[23]  A. Montecucco,et al.  Use of ATP, dATP and their alpha-thio derivatives to study DNA ligase adenylation. , 1990, The Biochemical journal.

[24]  D. Barnes,et al.  Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Geoffrey L. Smith,et al.  Transcriptional mapping and nucleotide sequence of a vaccinia virus gene encoding a polypeptide with extensive homology to DNA ligases. , 1989, Nucleic acids research.

[26]  S. Heaphy,et al.  Effect of single amino acid changes in the region of the adenylylation site of T4 RNA ligase. , 1987, Biochemistry.

[27]  K. Kleppe,et al.  Effect of ATP analogues on T4 polynucleotide ligase. , 1978, Biochemical and biophysical research communications.

[28]  I. R. Lehnman,et al.  DNA Ligase: Structure, Mechanism, and Function , 1974, Science.

[29]  A. Riggs,et al.  Lac repressor-operator interaction. I. Equilibrium studies. , 1970, Journal of molecular biology.

[30]  J. V. Van Etten,et al.  Analysis of 76 kb of the chlorella virus PBCV-1 330-kb genome: map positions 182 to 258. , 1996, Virology.