Biochemical and genetic analysis of the four DNA ligases of mycobacteria Journal Article


Authors: Gong, C.; Martins, A.; Bongiorno, P.; Glickman, M.; Shuman, S.
Article Title: Biochemical and genetic analysis of the four DNA ligases of mycobacteria
Abstract: Mycobacterium tuberculosis encodes an NAD+-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD + and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low Km for NAD+ (1 μM) and is sensitive to a recently described pyridochromanone inhibitor of NAD+-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high Km for ATP (0.34 mM) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDΔ cells are defective in nonhomologous DNA end joining.
Keywords: unclassified drug; nonhuman; chemical analysis; genetic analysis; animal cell; enzyme activity; bacteria (microorganisms); animalia; mycobacterium tuberculosis; dna; amino acid sequence; conserved sequence; molecular sequence data; sequence homology, amino acid; kinetics; genetic engineering; enzyme analysis; saccharomyces cerevisiae; enzyme inhibitors; sequence alignment; yeast; polydeoxyribonucleotide synthase; nick end labeling; enzyme specificity; biochemistry; mycobacterium; corynebacterineae; enzyme purification; enzymes; nad; archaea; enzyme mechanism; nicotinamide adenine dinucleotide; dna ligases; mycobacterium smegmatis; deoxyribonuclease; bacterial growth; bacteria; dna ligase d; mycobacteria; saccharomyces; monomers; dna ligase c; actinobacteria (class); priority journal; article; ligase enzymes; 4 chromanone derivative; dna ligase a; dna ligase b; uncultured actinomycete
Journal Title: Journal of Biological Chemistry
Volume: 279
Issue: 20
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 2004-05-14
Start Page: 20594
End Page: 20606
Language: English
DOI: 10.1074/jbc.M401841200
PROVIDER: scopus
PUBMED: 14985346
DOI/URL:
Notes: J. Biol. Chem. -- Cited By (since 1996):73 -- Export Date: 16 June 2014 -- CODEN: JBCHA -- Source: Scopus
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MSK Authors
  1. Alexandra I T Martins
    17 Martins
  2. Stewart H Shuman
    546 Shuman
  3. Michael Glickman
    110 Glickman