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 K(m) for NAD(+) (1 microm) 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 K(m) 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 ligDDelta cells are defective in nonhomologous DNA end joining.
Highlights
DNA ligases are grouped into two families, ATP-dependent ligases and NADϩ-dependent ligases, according to their nucleotide substrate requirement [1, 2]
ATP-dependent ligase homologues coexist with NADϩ-dependent enzymes in several other bacterial species, including major human pathogens such as Neisseria meningitidis, Y. pestis, Vibrio cholerae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis [3, 14, 15]
LigA resembles a typical bacterial NADϩ-dependent ligase; it consists of a core ligase domain flanked by a N-terminal domain (Ia), which is implicated in NADϩ recognition [22], and several C-terminal modules, including a tetracysteine zinc-binding motif, a helixhairpin-helix domain, and BRCT domain (Fig. 1) [23]
Summary
Mycobacterium tuberculosis encodes an NAD؉-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). 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. ATP-dependent ligase homologues coexist with NADϩ-dependent enzymes in several other bacterial species, including major human pathogens such as Neisseria meningitidis, Y. pestis, Vibrio cholerae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis [3, 14, 15]. M. tuberculosis encodes three distinct ATP-dependent ligase homologues (LigB, LigC, and LigD) plus an NADϩ-dependent ligase (LigA) (Fig. 1) [16]. To begin to understand the rationale for this plethora of ligases in a single bacterium, we have produced and characterized recombinant versions of the mycobacterial DNA ligase enzymes and probed genetically the essentiality or dispensability of the ATP-dependent ligases for growth of M. tuberculosis
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