HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 20, 20594-20606, May 14, 2004

This Article Abstract Full Text (PDF) All Versions of this Article: 279/20/20594    most recent M401841200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gong, C. Articles by Shuman, S. Search for Related Content PubMed PubMed Citation Articles by Gong, C. Articles by Shuman, S. Social Bookmarking                      What's this?

Biochemical and Genetic Analysis of the Four DNA Ligases of Mycobacteria^*

Chunling Gong, Alexandra Martins, Paola Bongiorno, Michael Glickman, and Stewart Shuman

From the Molecular Biology and Immunology Programs, Sloan-Kettering Institute, and Infectious Disease Division, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Received for publication, February 19, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 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 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 ligD cells are defective in nonhomologous DNA end joining.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
DNA ligases are grouped into two families, ATP-dependent ligases and NAD⁺-dependent ligases, according to their nucleotide substrate requirement (1, 2). The ligase reaction entails three nucleotidyl transfer steps (1). In the first step, attack by ligase on the phosphorus of ATP or NAD⁺ results in release of PP_i or NMN and formation of a covalent ligase-adenylate intermediate. In the second step, the AMP is transferred to the 5' end of the 5' phosphate-terminated DNA strand to form DNA-adenylate (AppDNA). In the third step, ligase catalyzes attack by a DNA 3'-OH on DNA-adenylate to join the two polynucleotides and liberate AMP. ATP-dependent DNA ligases are found in all three domains of life (Bacteria, Archaea, and Eukarya), whereas the NAD⁺-dependent enzymes are present only in bacteria and entomopoxviruses (1–7).

All known bacteria encode a highly conserved NAD⁺-dependent DNA ligase (LigA). A conditional mutant of Escherichia coli LigA results in growth arrest at the restrictive temperature; thus LigA is essential in E. coli (8, 9). LigA is also essential in Salmonella typhimurium, Bacillus subtilis, and Staphylococcus aureus (10–12). Some bacteria, including E. coli, S. typhimurium, Shigella flexneri, Yersinia pestis, and Pseudomonas putida, have a second NAD⁺-dependent ligase (13), the function of which is not known.

The presumption that bacteria encode only NAD⁺-dependent DNA ligases was overturned by the demonstration in 1997 of an ATP-dependent ligase in the respiratory pathogen Haemophilus influenzae (5). The 268-amino acid (aa)¹ H. influenzae ligase consists of a minimized catalytic domain. ATP-dependent ligase homologues coexist with NAD⁺-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).

Remarkably, M. tuberculosis encodes three distinct ATP-dependent ligase homologues (LigB, LigC, and LigD) plus an NAD⁺-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.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Multiple DNA ligases of M. tuberculosis. The LigA, LigB, LigC, and LigD polypeptides are depicted in cartoon form with the N termini on the left and the C termini on the right. The core ligase catalytic domains are shown as rectangles. Flanking domains of known structure or imputed function (variously drawn as ellipses or capsules) are discussed in the text. The nonessentiality of LigB, LigC, and LigB was demonstrated in the present study by targeted deletion of each lig locus as described in the text. LigA is scored as essential (Yes*) according to Ref. 36. Each of M. tuberculosis ligases was tested for complementation of the lethal yeast cdc9 mutation as described in the text.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cultures (500 ml) of E. coli BL21(DE3-RP)/pET-Lig were grown at 37 °C in Luria-Bertani medium containing 0.1 mg/ml ampicillin and 50 µg/ml chloramphenicol until the A₆₀₀ reached 0.5. The cultures were placed on ice for 10 min and then adjusted to 2% (v/v) ethanol and 0.4 mM isopropyl-1-thio--D-galactopyranoside. After further incubation for 17 h at 18 °C with constant shaking, the cells were harvested by centrifugation. The cell pellets were stored at –80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 20 ml of buffer A (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10% sucrose). Lysozyme was added to a final concentration of 50 µg/ml; the suspension was incubated on ice for 15 min, then adjusted to 0.1% Triton X-100 and sonicated to reduce the viscosity of the lysate. Insoluble material was removed by centrifugation. The soluble extracts were applied to 1-ml columns of nickel-nitrilotriacetic acid-agarose (Qiagen) that had been equilibrated with buffer A containing 0.1% Triton X-100. The columns were washed with the same buffer and then eluted stepwise with 2-ml aliquots of 50, 100, 200, and 500 mM imidazole in buffer B (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 10% glycerol, 0.1% Triton X-100). The polypeptide compositions of the column fractions were monitored by SDS-PAGE. The ligase proteins were retained on the column and recovered predominantly in the 100 and 200 mM imidazole fractions. Biochemical characterization of the mycobacterial ligases was performed using the 200 mM imidazole eluate fractions. The eluates of MtuLigA, MtuLigB, and MsmLigC1 were dialyzed overnight against buffer C (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM DTT, 10% glycerol, 0.05% Triton X-100). The enzyme preparations were stored at –80 °C. Protein concentrations were determined by using the Bio-Rad dye reagent, with BSA as the standard.

Glycerol Gradient Sedimentation—Aliquots (50 µg) of the nickel-agarose preparations of the mycobacterial ligases were mixed with catalase (50 µg), BSA (50 µg), and cytochrome c (50 µg) in 200 µl of buffer C. The mixtures were applied to 4.8-ml 15–30% glycerol gradients containing 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Triton X-100. The gradients were centrifuged for 18 h at 4 °C in a Beckman SW50 rotor at 50,000 rpm. Fractions (0.2 ml) were collected from the bottoms of the tubes. The polypeptide compositions of the gradient fractions were analyzed by SDS-PAGE. Aliquots of the fractions were assayed for adenylyltransferase and ligation activities as specified in the figure legends.

Adenylyltransferase Activity—Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 10 mM MgCl₂, 5 µM [-³²P]ATP, and enzyme as specified were incubated for 15 min at 37 °C. The reactions were quenched with SDS and the products were analyzed by SDS-PAGE. The covalent ligase-[³²P]AMP complex was visualized by autoradiography of the dried gel and quantified by scanning the gel with a Fujifilm BAS-2500 imaging apparatus.

DNA Ligation Assay—A 36-bp DNA duplex containing a centrally placed 3'-OH/5'-PO₄ nick was formed by annealing two 18-mer oligode-oxyribonucleotides to a complementary 36-mer strand as described previously (18, 19). The 18-mer constituting the 5' phosphate-terminated strand d(ATTCCGATAGTGACTACA) was 5' ³²P-labeled and gel-purified, then annealed to the complementary 36-mer DNA (the template strand) in the presence of a 3'-OH 18-mer strand d(CATATCCGTGTCGCCCTT). Ligation reaction mixtures (15 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 10 mM MgCl₂, 1 pmol of 5' ³²P-labeled nicked duplex DNA substrate, ATP or NAD⁺ as specified, and enzyme as specified were incubated at 37 °C for 15 min. The reactions were quenched by adjusting the mixtures to 60 mM EDTA and 30% formamide. The products were resolved by electrophoresis through a 15-cm 18% polyacrylamide gel containing 7 M urea in TBE (90 mM Tris borate, 2.5 mM EDTA). The extents of ligation were determined by scanning the gel with a Fujifilm BAS-2500 imaging apparatus.

Ligase Gene Disruptions in M. tuberculosis—Gene disruption was performed by specialized transduction of lig::hyg^R cassettes using temperature-sensitive mycobacteriophages as described previously (19, 20). The lig::hyg^R gene disruption cassettes were constructed by PCR amplifying genomic DNA segments flanking each ligase gene and inserting them on either side of the hygromycin resistance gene in plasmid pJSC407, a cloning vector containing the hyg^R marker, a cos site, and a unique PacI restriction site for packaging into phAE87. For ligB, the 5' flanking region was amplified from M. tuberculosis Erdman genomic DNA as a 667-bp fragment that includes the first 23 nucleotides of the predicted ligB open reading frame (ORF). The 3' flank was amplified as a 730-bp product that includes the last 13 nucleotides of the ligB ORF. For ligC, the 5' flank was a 745-bp fragment including the first 23 nucleotides of the ligC ORF. The 3' flank was a 638-bp fragment including the last 20 nucleotides of the ligC ORF. For ligD, the 5' flank was a 759-bp fragment including the first 11 nucleotides of the ligD ORF and the 3' flank was a 653-bp fragment including the last 35 nucleotides of the ORF. These lig cassettes were incorporated into phAE87 and high-titer lysates of each phage were used to infect wild-type M. tuberculosis Erdman. After 3 weeks of selection on Middlebrook 7H10 agar medium containing 50 µg/ml hygromycin, hyg^R transductants were grown in Middlebrook 7H9 liquid medium with 10% OADC supplement, 0.5% glycerol, 0.05% Tween 80, and 50 µg/ml hygromycin. Genomic DNA obtained from candidate lig disruptants was digested with diagnostic restriction endonucleases, resolved by agarose gel electrophoresis, transferred to Hybond nylon membranes (Amersham Biosciences) and genotyped at the relevant lig locus by Southern blotting with appropriate ³²P-labeled flanking DNA probes, which were prepared by random hexamer priming using Ready-to-Go DNA labeling beads (Amersham Biosciences).

Plasmid-based Assay of Nonhomologous End Joining in Vivo— M. tuberculosis lig⁺ and lig strains were transformed by electroporation with pMSG288 plasmid DNA containing the kan^R gene. Parallel transformations were performed with uncut circular plasmid (15 ng of DNA) and linear plasmids generated by digestion with EcoRV (54 ng of DNA) or Asp718I (42 ng of DNA), which cut the plasmid at a single sites well outside the kan^R gene. kan^R transformants were identified by plating on Middlebrook 7H10 agar medium containing 20 µg/ml kanamycin. The efficiency of nonhomologous end joining was quantified as the ratio of the number of kan^R colonies/ng of DNA arising in cells transformed with linear DNA to the number of kan^R colonies/ng of DNA obtained from cells transformed with uncut circular plasmid DNA.

Test of Mycobacterial Ligase Function in Vivo by Complementation of Saccharomyces cerevisiae cdc9—The M. tuberculosis lig genes were excised from the pET plasmids and inserted into the yeast vector pYX1 (CEN TRP1), in which expression of the lig gene is driven by the yeast TPI1 promoter. The ligA gene was excised from pET-MtuLigA with NdeI and EcoRI; the ends were filled in by DNA polymerase, and the blunt-ended ligA fragment was inserted into pYX1 that had been digested with EcoRI and BamHI and filled in by DNA polymerase. ligB and ligC were excised from the pET plasmids with NdeI and BamHI, then inserted between the NdeI and BamHI sites of pYX1. ligD was excised with NcoI and BamHI and inserted between the NcoI and BamHI sites of pYX1. pYX-MtuLig clones were transformed into S. cerevisiae cdc9 strain YBSL1 (MATa ura3 ade2 trp1 his3 leu2 can1 cdc9::LEU2 p360-Cdc9). YBSL1 is deleted at the chromosomal CDC9 locus and its growth is sustained by a copy of CDC9 on a CEN URA3 plasmid (p360-Cdc9) (21). Transformants were selected on agar medium lacking tryptophan. Cells from each isolate were then streaked on agar containing 0.75 mg/ml 5-fluoroorotic acid (5-FOA), a drug that selects against the URA3 gene on the CDC9 plasmid. cdc9 strains transformed with the ligA plasmid gave rise to 5-FOA-resistant colonies after 3 days of incubation at 30 °C. cdc9 cells transformed with ligB, ligC, or ligD plasmids failed to give rise to 5-FOA-resistant colonies after 10 days of incubation at 25, 30, or 37 °C. The cdc9 ligA strain grew on rich medium (YPD agar) at 25, 30, and 37 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Characterization of M. tuberculosis LigA—M. tuberculosis encodes four putative DNA ligases (Fig. 1). LigA resembles a typical bacterial NAD⁺-dependent ligase; it consists of a core ligase domain flanked by a N-terminal domain (Ia), which is implicated in NAD⁺ recognition (22), and several C-terminal modules, including a tetracysteine zinc-binding motif, a helixhairpin-helix domain, and BRCT domain (Fig. 1) (23). To evaluate the biochemical properties of MtuLigA, the protein was produced in E. coli as a His₁₀-LigA fusion and purified from a soluble extract by adsorption to nickel-agarose resin and elution with buffer containing imidazole (Fig. 2A). A synthetic duplex DNA substrate containing a single nick was then used to gauge the DNA sealing activity of LigA, which was evinced by the NAD⁺ and magnesium-dependent conversion of the 5' ³²P-labeled 18-mer strand to a 36-mer product. The extent of ligation was proportional to LigA concentration and 80% of the input substrate was sealed at saturating enzyme (Fig. 2B). The specific activity of LigA calculated from the slope of the titration curve indicated that 18 pmol of nicks were sealed per pmol of LigA during the 15-min reaction. Omission of NAD⁺ resulted in a 35-fold reduction in specific activity; the residual NAD⁺-independent ligation is attributable to the presence of preformed LigA-adenylate in the enzyme preparation. The linear dependence of nucleotide-independent strand joining on input enzyme suggested that 50% of the enzyme molecules had AMP bound at the active site. ATP had no stimulatory effect on LigA activity. Thus, MtuLigA is a bona fide NAD⁺-specific ligase.

View larger version (43K): [in this window] [in a new window]   FIG. 2. Purification and characterization of MtuLigA. A, aliquots (10 µl) of the soluble lysate of isopropyl-1-thio--D-galactopyranoside-induced bacteria (L), the nickel-agarose flow-through (F), the buffer wash (W), and the indicated imidazole eluate fractions were analyzed by SDS-PAGE. The gel was stained with Coomassie Blue dye. The positions and sizes (in kDa) of marker polypeptides are shown on the left. The LigA polypeptide is indicated by the arrowhead on the right. B, ligation reaction mixtures containing 1 pmol of 5' 32P-labeled nicked duplex DNA substrate, 0.2 mM NAD+ (), 1 mM ATP (), or no added nucleotide (), and LigA as specified were incubated at 37 °C for 15 min. The extent of ligation is plotted as a function of input LigA. C, sedimentation was performed as described under "Experimental Procedures." Aliquots (20 µl) of odd numbered gradient fractions were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown. The positions of the recombinant LigA protein and the internal standards are indicated. D, ligation reaction mixtures containing 1 pmol of 5' 32P-labeled nicked duplex DNA substrate, no added nucleotide, and 1 µl of the indicated glycerol gradient fractions were incubated at 37 °C for 15 min.

Kinetic Parameters and Inhibition of LigA by Pyridochromanone—The unique substrate specificity of LigA compared with ATP-dependent human DNA ligases recommends the NAD⁺ ligases as targets for the development of new broad-spectrum antibiotics. Brötz-Oesterhelt et al. (24) have conducted a high throughput in vitro screen that identified pyridochromanones as potent inhibitors of E. coli LigA. The most effective of the inhibitors tested was pyridochromanone compound 3, the structure of which is shown in Fig. 3B. Compound 3 was active in vitro against LigA from E. coli and Streptococcus pneumoniae, but had no effect on human DNA ligase I (24). The pyridochromanones inhibited the growth of S. aureus and B. subtilis. The identification of a mutation in S. aureus LigA that confers resistance to growth inhibition by pyridochromanone compound 3 provided strong evidence that LigA is the immediate drug target in vivo (24). Here we queried whether pyridochromanone compound 3 was active in vitro against MtuLigA.

View larger version (12K): [in this window] [in a new window]   FIG. 3. NAD+ dependence of LigA and inhibition by pyridochromanone. A, reaction mixtures (15 µl) containing 50 mM Tris-HCl (pH 7.5) 5 mM DTT, 5 mM MgCl2, 10% Me2SO, 1 pmol of 5' 32P-labeled nicked duplex DNA substrate, 0.8 ng of LigA, and NAD+ as specified were incubated for 15 min at 37 °C. B, chemical structure of pyridochromanone compound 3. C, reaction mixtures (15 µl) containing 50 mM Tris-HCl (pH 7.5) 5 mM DTT, 5 mM MgCl2, 10% Me2SO, 5 µM NAD+, 1 pmol of 5' 32P-labeled nicked duplex DNA substrate, 0.4, 0.8, or 1.6 ng of LigA, and pyridochromanone compound 3 as specified were incubated for 15 min at 37 °C. The ligase specific activities (pmol ends sealed/ng of protein) were calculated from the slopes of the titration curves and are plotted as a function of pyridochromanone concentration.

Characterization of M. tuberculosis LigB—M. tuberculosis LigB is a 507-aa polypeptide consisting of a 340-aa C-terminal catalytic domain typical of ATP-dependent DNA ligases fused to a 170-aa N-terminal domain of unknown function. The catalytic domain is defined by a set of six peptide motifs, highlighted in shaded boxes in Fig. 4, that comprise the ATP binding pocket (25–28). The closest relatives of mycobacterial LigA, which contain a homologous N-terminal domain, are a putative DNA ligase from the bacterium Streptomyces coelicolor and the ATP-dependent DNA ligases of Pyrococcus abyssii and other species of Archaea (Fig. 4).

View larger version (72K): [in this window] [in a new window]   FIG. 4. Primary structure of LigB and related ligases from bacteria and Archaea. The amino acid sequence of MtuLigB is aligned to the sequences of homologous polypeptides encoded by M. smegmatis (Msm), S. coelicolor (Sco), and P. abyssii (Pab). Gaps in the alignment are indicated by dashes. Nucleotidyl transferase motifs I, III, IIIa, IV, V, and IV are highlighted in shaded boxes. Conserved residues are indicated by . The translation start sites of serial N-terminal deletion mutants of LigB are indicated by arrows above the sequence.

View larger version (46K): [in this window] [in a new window]   FIG. 5. Purification and characterization of MtuLigB. A, aliquots (20 µl) of the soluble lysate of isopropyl-1-thio--D-galactopyranoside-induced bacteria (L), the nickel-agarose flow-through (F), the buffer wash (W), and the indicated imidazole eluate fractions were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown in the top panel. The positions and sizes (in kDa) of marker polypeptides are shown on the left. The LigB polypeptide is indicated by the arrowhead on the right. Aliquots (1 µl) of the same fractions were assayed for adenylyltransferase activity as described under "Experimental Procedures." An autoradiograph of the SDS gel is shown in the bottom panel. The positions and sizes (in kDa) of prestained marker polypeptides are shown on the left. The covalent LigB-[32P]AMP complex is indicated by the arrowhead on the right. B, sedimentation was performed as described under "Experimental Procedures." Aliquots (20 µl) of odd numbered gradient fractions were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown in the top panel. The positions of the recombinant LigB protein and the internal standards are indicated. Aliquots (1 µl) of the indicated gradient fractions were assayed for adenylyltransferase () and nick ligation in the absence of added nucleotide (). The activity profiles are plotted in the lower panel. C, ligation reaction mixtures containing 1 pmol of 5' 32P-labeled nicked duplex DNA substrate, 1 mM ATP (), 1 mM NAD+ (), or no added nucleotide (), and LigB as specified were incubated at 37 °C for 15 min. The extent of ligation is plotted as a function of input LigB. D, ATP dependence of nick ligation. Reaction mixtures (15 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 10 mM MgCl2, 1 pmol of 5' 32P-labeled nicked duplex DNA substrate, 1 ng of LigB, and ATP as specified were incubated at 37 °C for 15 min. The extent of ligation is plotted as function of ATP concentration.

The quaternary structure of LigB was gauged by glycerol gradient sedimentation with internal standards catalase, ovalbumin (45 kDa), and cytochrome c. LigB sedimented as a discrete peak (fractions 17–19) overlapping the ovalbumin peak (Fig. 5B). The adenylyltransferase and ligase activity profiles were coincident and paralleled the abundance of the LigB polypeptide and peaked at fractions 17–18 (Fig. 5B). We surmise that LigB is a monomer.

The function of the distinctive N-terminal segment of the 507-aa mycobacterial LigB protein was probed by analysis of incrementally truncated deletion mutants: LigB-(135–507), LigB-(155–507), and LigB-(172–507). The N termini of the LigB mutants are indicated by arrows in Fig. 4. The recombinant His₁₀-LigB proteins displayed the expected incremental increases in electrophoretic mobility (Fig. 6A). The purity of the LigB proteins was comparable with that of full-length LigB. All three of the LigB proteins reacted with [-³²P]ATP to form the covalent LigB-[³²P]adenylate adduct (Fig. 6B). Indeed, the loss of 134 to 171 amino acids from the N terminus had no detrimental effect on the extents of ligase adenylylation. However, the LigB proteins were severely defective in the overall nick sealing reaction, even under conditions of enzyme excess (Fig. 6C). The specific activities of the LigB mutants were determined by protein titration (data not shown) and normalized to the specific activity of full-length wild-type LigB; we thereby calculated that LigB-(135–507), LigB-(155–507), and LigB-(172–507) were 0.1, 0.2, and 0.3% as active as wild-type LigB, respectively. These results indicate that the N terminus of LigB is essential for nick joining at a step subsequent to ligase adenylylation.

View larger version (32K): [in this window] [in a new window]   FIG. 6. N-terminal deletion mutants of MtuLigB. A, aliquots (2 µg) of the nickel-agarose preparations of recombinant His10-LigB proteins (either full-length 507-aa LigB or C-terminal LigB fragments spanning residues 135–507, 155–507, or 172–507) were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. B, adenylyltransferase activity. Reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 50 µM [-32P]ATP, 5 mM MgCl2, and 0.8 µg of the indicated protein were incubated for 15 min at 37 °C. The reaction products were resolved by SDS-PAGE. An autoradiograph of the gel is shown. C, ligase activity. Reaction mixtures (15 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 1 mM ATP, 5 mM MgCl2, 1 pmol of nicked DNA substrate, and 2 pmol of the indicated protein were incubated for 15 min at 37 °C.

View larger version (85K): [in this window] [in a new window]   FIG. 7. Primary structure of mycobacterial LigD. The amino acid sequence of MtuLigD is aligned to the sequence of the homologous polypeptide of M. smegmatis (Msm). Gaps in the alignment are indicated by dashes. Nucleotidyl transferase motifs I, III, IIIa, IV, V, and IV are highlighted in shaded boxes.

View larger version (47K): [in this window] [in a new window]   FIG. 8. Purification and characterization of MtuLigD. A, aliquots (20 µl) of the soluble lysate of isopropyl-1-thio--D-galactopyranoside-induced bacteria (L), the nickel-agarose flow-through (F), the buffer wash (W), and the indicated imidazole eluate fractions were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown in the top panel. The positions and sizes (in kDa) of marker polypeptides are shown on the left. The LigD polypeptide is indicated by the arrowhead on the right. Aliquots (1 µl) of the same fractions were assayed for adenylyltransferase activity as described under "Experimental Procedures." An autoradiograph of the SDS gel is shown in the lower panel. The positions and sizes (in kDa) of prestained marker polypeptides are shown on the left. The covalent LigD-[32P]AMP complex is indicated by the arrowhead on the right. B, sedimentation was performed as described under "Experimental Procedures." Aliquots (20 µl) of odd numbered gradient fractions were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown in the top panel. The positions of the recombinant LigD protein and the internal standards are indicated. Aliquots (1.5 µl) of the indicated gradient fractions were assayed for adenylyltransferase () and nick ligation in the absence of added nucleotide (). The activity profiles are plotted in the lower panel. C, ligation reaction mixtures containing 1 pmol of 5' 32P-labeled nicked duplex DNA substrate, either 1 mM ATP (), 1 mM NAD+ (), or no added nucleotide (), and LigD as specified were incubated at 37 °C for 15 min. The extent of ligation is plotted as a function of input LigD. D, ATP-dependent trapping of DNA-adenylate. Reaction mixtures (15 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 10 mM MgCl2, 1 pmol of 5' 32P-labeled nicked duplex DNA substrate, 8 ng of LigD, and ATP as specified were incubated at 37 °C for 15 min. The extents of ligation and formation of DNA-adenylate (AppDNA) are plotted as a function of ATP concentration.

LigD sedimented as a discrete peak in a glycerol gradient (fractions 13–15) that was clearly heavier than BSA (Fig. 8B). The adenylyltransferase and ligase activity profiles were coincident (peaking at fraction 14) and paralleled the profile of the LigD polypeptide (Fig. 8B). An S value of 5.9 was determined for LigD, which is consistent with a monomeric native structure.

Further experiments showed that the failure of 1 mM ATP to stimulate nick ligation by LigD was attributable to ATP-induced accumulation of the DNA-adenylate intermediate AppDNA (Fig. 8D). In the absence of ATP, AppDNA was not detected. Overall ligation was stimulated 2-fold at low ATP concentrations (in the range of 30 to 250 µM). However, ATP also elicited a concentration-dependent trapping of the AppDNA intermediate and a concomitant suppression of the formation of a ligated phosphodiester (Fig. 8D). This ATP-dependent trapping phenomenon has been described previously for eukaryotic viral DNA ligases (17, 29) and shown to result from dissociation of the enzyme from the DNA-adenylate and immediate reaction of the free ligase with ATP to yield ligase-AMP, which cannot rebind to AppDNA and thus cannot catalyze the phosphodiester formation step (there being only one adenylate-binding pocket on the enzyme). The inference from the ATP-trapping effect during reaction of LigD with a nicked substrate (which is specific for LigD and not seen for LigB) is that LigD by itself is an inherently inefficient nick-joining enzyme, because it does not remain bound to the step 2 reaction product.

An N-terminal deletion mutant, LigD-(412–759), consisting only of the C-terminal segment containing the nucleotidyl transferase motifs, was produced in E. coli as a His₁₀ fusion and purified by affinity chromatography (Fig. 9A, N). LigD-(412–759) reacted with [-³²P]ATP to form the covalent ligase-AMP intermediate; elimination of the N-terminal 411 amino acids did not affect the extent of ligase adenylylation relative to full-length wild-type LigD (Fig. 9B). LigD-(412–759) retained nick sealing activity in the presence and absence of ATP (Fig. 9C and data not shown). Inclusion of ATP in the reaction of LigD-(412–759) with nicked DNA resulted in the accumulation of AppDNA (Fig. 9C). Thus, the ATP-dependent trapping of the DNA-adenylate intermediate is an inherent property of the C-terminal domain of MtuLigD.

View larger version (34K): [in this window] [in a new window]   FIG. 9. C-terminal ligase domain of MtuLigD. A, aliquots (1 µg) of the nickel-agarose preparations of recombinant full-length wild-type (WT) His10-LigD and the N-terminal deletion mutant LigD-(412–759) (N) were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. B, adenylyltransferase activity. Reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 50 µM [-32P]ATP, 10 mM MgCl2, and 0.2 µg of the indicated protein were incubated for 15 min at 37 °C. The reaction products were resolved by SDS-PAGE. An autoradiograph of the gel is shown. C, ligase activity. Reaction mixtures (15 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 1 mM ATP, 5 mM MgCl2, 1 pmol of nicked DNA substrate, and 40 ng of the indicated protein were incubated for 15 min at 37 °C. The extents of DNA ligation and AppDNA formation are shown.

View larger version (78K): [in this window] [in a new window]   FIG. 10. Primary structure of mycobacterial LigC. The amino acid sequence of MtuLigC is aligned to the sequence of M. smegmatis LigC1 (Msm1) and LigC2 (Msm2), and to the homologous ligase-like protein of S. coelicolor (Sco). Gaps in the alignment are indicated by dashes. Positions of side chain identity/similarity in all four proteins are indicated by . Nucleotidyl transferase motifs I, III, IIIa, IV, V, and IV are highlighted in shaded boxes.

View larger version (48K): [in this window] [in a new window]   FIG. 11. Purification and characterization of MsmLigC1. A, aliquots (10 µl) of the soluble lysate of isopropyl-1-thio--D-galactopyranoside-induced bacteria (L), the nickel-agarose flow-through (F), the buffer wash (W), and the indicated imidazole eluate fractions were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown in the top panel. The positions and sizes (in kDa) of marker polypeptides are shown on the left. The LigC1 polypeptide is indicated by the arrowhead on the right. Aliquots (1 µl) of the same fractions were assayed for adenylyltransferase activity as described under "Experimental Procedures." An autoradiograph of the SDS gel is shown in the lower panel. The positions and sizes (in kDa) of prestained marker polypeptides are shown on the left. The covalent LigC-[32P]AMP complex is indicated by the arrowhead on the right. B, sedimentation was performed as described under "Experimental Procedures." Aliquots (20 µl) of odd numbered gradient fractions were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown in the top panel. The positions of the recombinant LigC1 protein and the internal standards are indicated. Aliquots (1 µl) of the indicated gradient fractions were assay for adenylyltransferase () and nick ligation in the absence of added nucleotide (). The activity profiles are plotted in the lower panel. C, ligation reaction mixtures containing 1 pmol of 5' 32P-labeled nicked duplex DNA substrate, either 1 mM ATP (), 1 mM NAD+ (), or no added nucleotide (), and LigC1 as specified were incubated at 37 °C for 15 min. The extent of ligation is plotted as a function of input LigC1. D, ATP-dependent trapping of DNA-adenylate. Ligation reactions were performed as described in panel C. The extent of formation of DNA-adenylate (AppDNA) is plotted as function of input LigC1.

LigC sedimented as a discrete peak in a glycerol gradient at a position (fraction 17) that was clearly lighter than BSA (Fig. 11B). The adenylyltransferase and ligase activity profiles were coincident and paralleled the profile of the LigC polypeptide (Fig. 11B). An S value of 3.5 was calculated for LigC, consistent with a monomeric native structure.

Gene Knockouts of ATP-dependent DNA Ligases in M. tuberculosis—To study the function of the ATP-dependent ligases in vivo, we constructed M. tuberculosis null mutants of ligB (Rv3062), ligC (Rv3731), and ligD (Rv0938) by replacing the predicted coding sequence of each lig gene with a gene conferring resistance to hygromycin. The lig knockout cassettes containing the hyg^R marker flanked by upstream and downstream M. tuberculosis genomic DNA for each lig locus were packaged into a temperature-sensitive derivative of the mycobacteriophage TM4 and the recombinant phages were then used to transduce M. tuberculosis Erdman to hygromycin resistance (19, 20). Southern hybridization of restriction endonuclease-digested genomic DNA from hygromycin-resistant transductants revealed the predicted fragment sizes for correctly targeted allelic exchange at the ligB, ligC, and ligD loci (data not shown). The viable ligB, ligC, and ligD strains grew as well as the parental wild-type strain on agar medium and in liquid culture (data not shown). Thus, M. tuberculosis LigB, LigC, and LigD are individually dispensable for cell growth.

M. tuberculosis ligD Mutant Is Defective in Nonhomologous DNA End Joining—Nonhomologous end joining (NHEJ) was assayed by transformation of isogenic wild-type and lig strains of M. tuberculosis with a linearized plasmid containing a replication origin and a selectable kanamycin-resistance marker. Successful transformation of M. tuberculosis by the linear plasmid depends on sealing of the ends to produce a circular DNA molecule. The plasmid was linearized with either restriction endonuclease Asp718I (which leaves a 4-nucleotide 5' overhang) or EcoRV (which generates a blunt duplex end). The efficiency of NHEJ was quantified as the ratio of the number of kan^R colonies arising in cells transformed with a linearized plasmid to the number of kan^R colonies obtained from cells transformed with an equivalent amount of uncut plasmid DNA.

The first instructive finding was that efficiency of NHEJ in wild-type M. tuberculosis was apparently independent of the configuration of the ends of the linear plasmid substrate. DNA containing 5' cohesive ends formed by Asp718I was 29% as effective as uncut plasmid. The efficiency of linear transformation by blunt-ended EcoRV-cut DNA was 37%. Thus, short cohesive ends do not facilitate NHEJ in mycobacterium. This is in contrast to S. cerevisiae, where linear plasmid DNAs with 5' overhangs transform with 40-fold higher efficiency than do linear DNAs with blunt ends (30–33). The second notable finding was that deletion of LigD elicited a 79% reduction in NHEJ efficiency with blunt-ended DNA and a 70% drop in NHEJ efficiency with a 5' overhang DNA (Fig. 12). Deletion of ligB or ligC had no effect on NHEJ with either 5' overhang or bluntended linear DNA substrates (data not shown). These experiments show that, among the multiple ATP-dependent DNA ligases of M. tuberculosis, LigD is engaged in an NHEJ pathway.

View larger version (43K): [in this window] [in a new window]   FIG. 12. Effects of the ligD deletion on NHEJ in M. tuberculosis. Wild-type and ligD strains were transformed with circular plasmid DNA, plasmid DNA linearized with Asp718I (which generates a 4-nucleotide 5' overhang), or plasmid DNA linearized with EcoRV (which generates a blunt duplex end). The efficiency of NHEJ was calculated as the transformation efficiency with a linear plasmid (kanR transformants per ng of DNA) divided by the transformation efficiency with a circular plasmid. Each transformation efficiency value was the average of three independent transformations.

Here we tested by plasmid shuffle whether any of the M. tuberculosis ligases could complement growth of an S. cerevisiae cdc9 mutant. Viability of the cdc9 strain is contingent on maintenance of an extrachromosomal CDC9 gene on a CEN URA3 plasmid. Hence, cdc9 cells cannot grow on medium containing 5-FOA (a drug that selects against the URA3 CDC9 plasmid), but they can grow on 5-FOA if the cells have been transformed with a CEN TRP1 plasmid encoding CDC9 or a "biologically active" ligase from a heterologous source (21). We cloned the M. tuberculosis ligA, ligB, ligC, and ligD genes into a CEN TRP1 vector such that expression of the Mtu ligase was under the control of the constitutive yeast TPI1 promoter. The instructive finding was that cdc9 cells bearing the Mtu-ligA plasmid grew on 5-FOA. The cdc9 ligA isolates grew on YPD agar medium at 25, 30, and 37 °C (not shown; scored as "yes" in Fig. 1). Thus, the mycobacterial NAD⁺-dependent DNA ligase was functional in vivo in lieu of the ATP-dependent yeast Cdc9. In contrast, ligB, ligC, nor ligD were able to complement growth of the cdc9 mutant on 5-FOA (No in Fig. 1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
M. tuberculosis specifies four DNA ligase enzymes, which we have produced as recombinant proteins in E. coli and characterized with respect to their nick joining activities, nucleotide substrate specificities, domain architectures, and native sizes. Although all four mycobacterial ligases are capable of sealing singly nicked DNA, they differ significantly in their catalytic power. Whereas LigA and LigB are efficient at nick joining and are strongly stimulated by their respective NAD⁺ and ATP cofactors, LigC and LigD have weak intrinsic nick joining activities and accumulate DNA-adenylate. Genetic and pharmacological studies of the M. tuberculosis ligases highlight LigA as uniquely essential for cell viability and a possible drug target for treating tuberculosis.

Our characterization of recombinant MtuLigA confirms and extends a recent report that MtuLigA can seal nicked DNA in the presence of NAD⁺ (35). We show that MtuLigA is a monomeric protein with high affinity for NAD⁺, a relatively vigorous steady-state activity, and the ability to fulfill all of the essential replicative functions normally performed by Cdc9 during mitotic growth of S. cerevisiae. In these respects, MtuLigA resembles the E. coli LigA enzyme (21). We and others have suggested that inhibitors of bacterial NAD⁺-dependent LigA would, in principle, be prime candidates for broad-spectrum antibiotic therapy, given that: (i) NAD⁺-dependent ligases are present in all bacteria and have been validated as essential for growth in disease-causing species such as E. coli and S. aureus; (ii) NAD⁺-dependent ligases display unique substrate specificity and domain architecture compared with the ATP-dependent ligases of humans; and (iii) there are no homologs of LigA in any eukaryal organism. For a bacterial pathogen like M. tuberculosis, which has multiple ATP-dependent DNA ligases, this simple scenario becomes complicated if any of the ATP-dependent ligases are functionally redundant with respect to LigA.

As we show here, neither LigB, LigC, nor LigD are essential for growth of M. tuberculosis. We did not attempt to delete the Mtu ligA gene in the present study, in light of a recent genomewide transposon insertion analysis by Sassetti et al. (36) that classified ligA as a gene required for optimal growth of M. tuberculosis. Our finding here that MtuLigA is sensitive to inhibition by pyridochromanone compound 3, a newly described small molecule of inhibitor of E. coli, S. aureus, and S. pneumoniae LigA that has LigA-dependent antibacterial activity in vivo (24), augurs well for the discovery and optimization of small molecule LigA antagonists with broad-spectrum activity that embraces mycobacterial pathogens such as M. tuberculosis and M. avium.

MtuLigB has a vigorous nick sealing activity in vitro, comparable with that of LigA, yet LigB is neither functionally redundant to LigA in mycobacterium nor can it recapitulate the ability of LigA to sustain growth of S. cerevisiae. It may be the case that LigA is uniquely able to function in mycobacterial DNA replication because LigA can interact with components of the bacterial replisome in ways that LigB cannot. For example, it has been suggested that E. coli LigA interacts physically with the sliding clamp processivity factor for the bacterial replicative DNA polymerase (37). The failure of LigB to function in lieu of Cdc9 in yeast may be attributable to any of several trivial factors (e.g. inadequate steady-state levels of LigB expression in yeast, improper intracellular localization of LigB in yeast, etc.) that do not speak to intrinsic functional distinctions between LigA and LigB.

The distinctive biochemical properties of LigB are its relatively high K_m for ATP and its dependence on its N-terminal domain for nick joining activity. The C-terminal domain of LigB, which contains the six nucleotidyl transferase motifs, is competent to react with ATP to form the covalent ligase-AMP intermediate. Thus, the failure of the adenylyltransferase module to perform the composite ligation reaction cannot be attributed to global misfolding of the isolated domain. Rather, these results implicate the N-terminal domain of LigB in one or more steps of the ligation reaction subsequent to ligase adenylation. The properties of MtuLigB are reminiscent of the 552-aa vaccinia virus DNA ligase, which also has a high K_m for ATP (26, 38), and relies on its N-terminal domain flanking the C-terminal adenylyltransferase module for its efficient nick joining activity (18). MtuLigB and the poxvirus DNA ligase display primary structure similarity across the 130-aa segment of LigB from position Pro-69 to Ala-198 (32 positions of side chain identity and 22 positions of side chain similarity). The N-terminal domain of vaccinia DNA ligase is suggested to play a role in the DNA binding step of the ligation pathway (18).

MsmLigC, a monomeric protein consisting solely of the minimal ligase catalytic domain, possesses ATP-specific adenylyltransferase activity. Preformed LigC-AMP can seal nicked DNA in the absence of ATP, but ligation by LigC is suppressed when ATP (but not NAD⁺) is included in the reaction. As discussed above and in previous studies of Chlorella virus DNA ligase and T4 RNA ligase 2 (21, 39), we attribute this paradoxical trapping effect of ATP to the tendency of LigC to dissociate from the AppDNA intermediate and be trapped as ligase-adenylate, which cannot react with AppDNA. The "crippled" in vitro nick joining activity of MsmLigC contrasts with the efficient nick joining function of Chlorella virus DNA ligase, which also consists only of the catalytic domain. Although it could be that LigC is a vestigial protein with no genuine ligation function in vivo, we are reluctant to adopt this view, given that similarly minimized homologs of LigC are conserved in M. tuberculosis, M. smegmatis, and M. avium. Rather, we envision two scenarios for LigC function, as follows: (i) LigC requires a separate protein cofactor that enhances its affinity for DNA and confers the ability to efficiency seal DNA nicks, or (ii) the preferred substrate for LigC is not the singly nicked duplex DNA that we have used here to study strand sealing.

MtuLigD has a complex domain architecture composed of putative polymerase, nuclease, and ligase modules. The ligase activity of MtuLigD was demonstrated by Weller et al. (14), who inputed a role for LigD in a bacterial NHEJ pathway of DNA recombination and repair. Here we provide direct evidence for an NHEJ pathway in M. tuberculosis that depends on LigD. MtuLigD per se is a weak nick joining enzyme that accumulates high levels of AppDNA in the presence of ATP. As noted above, we interpret this behavior as indicative of a tendency to dissociate from AppDNA prior to the strand closure step. Because the isolated C-terminal ligase domain of LigD displays similar properties with respect to adenylyltransferase and nick joining as the full-length LigD protein, we surmise that LigD is not endowed with a more efficient cryptic ligase activity that is suppressed by the N-terminal domain modules. Rather, we invoke one or both of the scenarios discussed above for LigC, whereby LigD activity is either stimulated by a separate cofactor and/or LigD prefers substrates other than singly nicked DNAs.

Recent reports have highlighted the existence of putative homologs of the eukaryotic NHEJ protein Ku in certain bacteria, including M. tuberculosis (40, 41). Remarkably, the gene encoding the M. tuberculosis Ku homolog is situated adjacent to the ligD gene. Weller et al. (14) have shown that MtuLigD end joining activity in vitro is stimulated by MtuKu. In agreement with Weller et al. (14) we have seen that the sealing of singly nicked DNA is stimulated by MtuKu, albeit only modestly. Our preliminary experiments indicate that Ku did not rectify the accumulation of AppDNA by LigD in the presence of ATP. In light of the NHEJ defect of the ligD mutant, we suspect that MtuLigD activity in vivo is targeted to double-strand DNA breaks rather than single-strand nicks.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed. E-mail: glickmam{at}MSKCC.ORG. To whom correspondence may be addressed. E-mail: s-shuman{at}ski.mskcc.org.

¹ The abbreviations used are: aa, amino acid(s); DTT, dithiothreitol; BSA, bovine serum albumin; ORF, open reading frame; 5-FOA, 5-fluoroorotic acid; NHEJ, nonhomologous end joining.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Lehman, I. R. (1974) Science 186, 790–797[Abstract/Free Full Text]
2. Doherty, A. J., and Suh, S. W. (2000) Nucleic Acids Res. 28, 4051–4058[Abstract/Free Full Text]
3. Wilkinson, A., Day, J., and Bowater, R. (2001) Mol. Microbiol. 40, 1241–1248[CrossRef][Medline] [Order article via Infotrieve]
4. Kletzin, A. (1992) Nucleic Acids Res. 20, 5389–5396[Abstract/Free Full Text]
5. Cheng, C., and Shuman, S. (1997) Nucleic Acids Res. 25, 1369–1375[Abstract/Free Full Text]
6. Sriskanda, V., Kelman, Z., Hurwitz, J., and Shuman, S (2000) Nucleic Acids Res. 28, 2221–2228[Abstract/Free Full Text]
7. Sriskanda, V., Moyer, R. W., and Shuman, S. (2001) J. Biol. Chem. 276, 36100–36109[Abstract/Free Full Text]
8. Gottesman, M. M., Hicks, M. L., and Gellert, M. (1973) J. Mol. Biol. 77, 531–547[CrossRef][Medline] [Order article via Infotrieve]
9. Konrad, E. B., Modrich, P., and Lehman, I. R. (1973) J. Mol. Biol. 77, 519–529[CrossRef][Medline] [Order article via Infotrieve]
10. Park, U. E., Olivera, B. M., Hughes, K. T., Roth, J. R., and Hillyard, D. R. (1989) J. Bacteriol. 171, 2173–2180[Abstract/Free Full Text]
11. Petit, M. A., and Ehrlich, S. D. (2000) Nucleic Acids Res. 28, 4642–4648[Abstract/Free Full Text]
12. Kaczmarek, F. S., Zaniewski, R. P., Gootz, T. D., Danley, D. E., Mansour, M. N., Griffor, M., Kamath, A. V., Cronan, M., Mueller, J., Sun, D., Martin, P. K., Benton, B., McDowell, L., Biek, D., and Schmid, M. B. (2001) J. Bacteriol. 183, 3016–3024[Abstract/Free Full Text]
13. Sriskanda, V., and Shuman, S. (2001) Nucleic Acids Res. 29, 4930–4934[Abstract/Free Full Text]
14. Weller, G. R., Kysela, B., Roy, R., Tonkin, L. M., Scanlan, E., Della, M., Devine, S. K., Day, J. P., Wilkinson, A., di Fagagna, F., Devine, K. M., Bowater, R. P., Jeggo, P. A., Jackson, S. P., and Doherty, A. J. (2002) Science 297, 1686–1689[Abstract/Free Full Text]
15. Magnet, S., and Blanchard, J. S., (2004) Biochemistry 43, 710–717[CrossRef][Medline] [Order article via Infotrieve]
16. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M. A., Rajandream, M. A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J. E., Taylor, K., Whitehead, S., and Barrell, B. G. (1998) Nature 393, 537–544[CrossRef][Medline] [Order article via Infotrieve]
17. Shuman, S. (1995) Biochemistry 34, 16138–16147[CrossRef][Medline] [Order article via Infotrieve]
18. Sekiguchi, J., and Shuman, S. (1997) Nucleic Acids Res. 25, 727–734[Abstract/Free Full Text]
19. Glickman, M. S., Cahill, S. M., and Jacobs, W. R. (2001) J. Biol. Chem. 276, 2228–2233[Abstract/Free Full Text]
20. Glickman, M. S. (2003) J. Biol. Chem. 278, 7844–7849[Abstract/Free Full Text]
21. Sriskanda, V., Schwer, B., Ho, C. K., and Shuman, S. (1999) Nucleic Acids Res. 27, 3953–3963[Abstract/Free Full Text]
22. Sriskanda, V., and Shuman, S. (2002) J. Biol. Chem. 277, 9685–9700
23. Lee, J. Y., Chang, C., Song, H. K., Moon, J., Yang, J., Kim, H. K., Kwon, S. T., and Suh, S. W. (2000) EMBO J. 19, 1119–1129[CrossRef][Medline] [Order article via Infotrieve]
24. Brötz-Oesterhelt, H., Knezevic, I., Bartel, S., Lampe, T., Warnecke-Eberz, U., Ziegelbauer, K., Häbich, D., and Labischiinski, H. (2003) J. Biol. Chem. 278, 39435–39442[Abstract/Free Full Text]
25. Shuman, S., and Schwer, B. (1995) Mol. Microbiol. 17, 405–410[Medline] [Order article via Infotrieve]
26. Shuman, S., and Ru, X. (1995) Virology 211, 73–83[CrossRef][Medline] [Order article via Infotrieve]
27. Subramanya, H. S., Doherty, A. J., Ashford, S. R., and Wigley, D. B. (1996) Cell 85, 607–615[CrossRef][Medline] [Order article via Infotrieve]
28. Odell, M., Sriskanda, V., Shuman, S., and Nikolov, D. B. (2000) Mol. Cell 6, 1183–1193[CrossRef][Medline] [Order article via Infotrieve]
29. Sriskanda, V., and Shuman, S. (1988) Nucleic Acids Res. 26, 4618–4625
30. Wilson, T. E., Grawunder, U., and Lieber, M. R. (1997) Nature 388, 495–498[CrossRef][Medline] [Order article via Infotrieve]
31. Teo, S. H., and Jackson, S. P. (1997) EMBO J. 16, 4788–4795[CrossRef][Medline] [Order article via Infotrieve]
32. Schär, P., Herrmann, G., Daly, G., and Lindahl, T. (1997) Genes Dev. 11, 1912–1924[Abstract/Free Full Text]
33. Odell, M., Malinina, L., Sriskanda, V., Teplova, M., and Shuman, S. (2003) Nucleic Acids Res. 31, 5090–5100[Abstract/Free Full Text]
34. Kodama, K., Barnes, D. E., and Lindahl, T. (1991) Nucleic Acids Res. 19, 6093–6099[Abstract/Free Full Text]
35. Wilkinson, A., Sayer, H., Bullard, D., Smith, A., Day, J., Kieser, T., and Bowater, R. (2003) Proteins 51, 321–326[CrossRef][Medline] [Order article via Infotrieve]
36. Sassetti, C. M., Boyd, D. H., and Rubin, E. J. (2003) Mol. Microbiol. 48, 77–84[CrossRef][Medline] [Order article via Infotrieve]
37. Lopez de Saro, F. J., and O'Donnell, M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 8376–8380[Abstract/Free Full Text]
38. Odell, M., Kerr, S. M., and Smith, G. L. (1996) Virology 221, 120–129[CrossRef][Medline] [Order article via Infotrieve]
39. Ho, C. K., and Shuman, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 12709–12714[Abstract/Free Full Text]
40. Weller, G. R., and Doherty, A. J. (2001) FEBS Lett. 505, 340–342[CrossRef][Medline] [Order article via Infotrieve]
41. Aravind, L., and Koonin, E. V. (2001) Genome Res. 11, 1365–1374[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				K. M. Sinha, M.-C. Unciuleac, M. S. Glickman, and S. Shuman AdnAB: a new DSB-resecting motor-nuclease from mycobacteria Genes & Dev., June 15, 2009; 23(12): 1423 - 1437. [Abstract] [Full Text] [PDF]

				A. Lieber, A. Leis, A. Kushmaro, A. Minsky, and O. Medalia Chromatin Organization and Radio Resistance in the Bacterium Gemmata obscuriglobus J. Bacteriol., March 1, 2009; 191(5): 1439 - 1445. [Abstract] [Full Text] [PDF]

				A. Crut, P. A. Nair, D. A. Koster, S. Shuman, and N. H. Dekker Dynamics of phosphodiester synthesis by DNA ligase PNAS, May 13, 2008; 105(19): 6894 - 6899. [Abstract] [Full Text] [PDF]

				H. Zhu and S. Shuman Bacterial Nonhomologous End Joining Ligases Preferentially Seal Breaks with a 3'-OH Monoribonucleotide J. Biol. Chem., March 28, 2008; 283(13): 8331 - 8339. [Abstract] [Full Text] [PDF]

				J. Gu and M. R. Lieber Mechanistic flexibility as a conserved theme across 3 billion years of nonhomologous DNA end-joining Genes & Dev., February 15, 2008; 22(4): 411 - 415. [Full Text] [PDF]

				J. Aniukwu, M. S. Glickman, and S. Shuman The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends Genes & Dev., February 15, 2008; 22(4): 512 - 527. [Abstract] [Full Text] [PDF]

				N. C. Brissett, R. S. Pitcher, R. Juarez, A. J. Picher, A. J. Green, T. R. Dafforn, G. C. Fox, L. Blanco, and A. J. Doherty Structure of a NHEJ Polymerase-Mediated DNA Synaptic Complex Science, October 19, 2007; 318(5849): 456 - 459. [Abstract] [Full Text] [PDF]

				M. Korycka-Machala, E. Rychta, A. Brzostek, H. R. Sayer, A. Rumijowska-Galewicz, R. P. Bowater, and J. Dziadek Evaluation of NAD+-Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics Antimicrob. Agents Chemother., August 1, 2007; 51(8): 2888 - 2897. [Abstract] [Full Text] [PDF]

				N. C. Stephanou, F. Gao, P. Bongiorno, S. Ehrt, D. Schnappinger, S. Shuman, and M. S. Glickman Mycobacterial Nonhomologous End Joining Mediates Mutagenic Repair of Chromosomal Double-Strand DNA Breaks J. Bacteriol., July 15, 2007; 189(14): 5237 - 5246. [Abstract] [Full Text] [PDF]

				H. Zhu and S. Shuman Characterization of Agrobacterium tumefaciens DNA ligases C and D Nucleic Acids Res., June 28, 2007; 35(11): 3631 - 3645. [Abstract] [Full Text] [PDF]

				K. M. Sinha, N. C. Stephanou, F. Gao, M. S. Glickman, and S. Shuman Mycobacterial UvrD1 Is a Ku-dependent DNA Helicase That Plays a Role in Multiple DNA Repair Events, Including Double-strand Break Repair J. Biol. Chem., May 18, 2007; 282(20): 15114 - 15125. [Abstract] [Full Text] [PDF]

				L. Yakovleva and S. Shuman Nucleotide Misincorporation, 3'-Mismatch Extension, and Responses to Abasic Sites and DNA Adducts by the Polymerase Component of Bacterial DNA Ligase D J. Biol. Chem., September 1, 2006; 281(35): 25026 - 25040. [Abstract] [Full Text] [PDF]

				H. Zhu and S. Shuman Substrate Specificity and Structure-Function Analysis of the 3'-Phosphoesterase Component of the Bacterial NHEJ Protein, DNA Ligase D J. Biol. Chem., May 19, 2006; 281(20): 13873 - 13881. [Abstract] [Full Text] [PDF]

				D. Akey, A. Martins, J. Aniukwu, M. S. Glickman, S. Shuman, and J. M. Berger Crystal Structure and Nonhomologous End-joining Function of the Ligase Component of Mycobacterium DNA Ligase D J. Biol. Chem., May 12, 2006; 281(19): 13412 - 13423. [Abstract] [Full Text] [PDF]

				H. Zhu, J. Nandakumar, J. Aniukwu, L. K. Wang, M. S. Glickman, C. D. Lima, and S. Shuman Atomic structure and nonhomologous end-joining function of the polymerase component of bacterial DNA ligase D PNAS, February 7, 2006; 103(6): 1711 - 1716. [Abstract] [Full Text] [PDF]

				S. K. Srivastava, D. Dube, N. Tewari, N. Dwivedi, R. P. Tripathi, and R. Ramachandran Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I Nucleic Acids Res., December 15, 2005; 33(22): 7090 - 7101. [Abstract] [Full Text] [PDF]

				H. Zhu, L. K. Wang, and S. Shuman Essential Constituents of the 3'-Phosphoesterase Domain of Bacterial DNA Ligase D, a Nonhomologous End-joining Enzyme J. Biol. Chem., October 7, 2005; 280(40): 33707 - 33715. [Abstract] [Full Text] [PDF]

				S. K. Srivastava, R. P. Tripathi, and R. Ramachandran NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis: CRYSTAL STRUCTURE OF THE ADENYLATION DOMAIN AND IDENTIFICATION OF NOVEL INHIBITORS J. Biol. Chem., August 26, 2005; 280(34): 30273 - 30281. [Abstract] [Full Text] [PDF]

				H. Zhu and S. Shuman Novel 3'-Ribonuclease and 3'-Phosphatase Activities of the Bacterial Non-homologous End-joining Protein, DNA Ligase D J. Biol. Chem., July 15, 2005; 280(28): 25973 - 25981. [Abstract] [Full Text] [PDF]

				H. Zhu and S. Shuman Structure-guided Mutational Analysis of the Nucleotidyltransferase Domain of Escherichia coli NAD+-dependent DNA Ligase (LigA) J. Biol. Chem., April 1, 2005; 280(13): 12137 - 12144. [Abstract] [Full Text] [PDF]

				H. Zhu and S. Shuman A Primer-dependent Polymerase Function of Pseudomonas aeruginosa ATP-dependent DNA Ligase (LigD) J. Biol. Chem., January 7, 2005; 280(1): 418 - 427. [Abstract] [Full Text] [PDF]

				M. Lavesa-Curto, H. Sayer, D. Bullard, A. MacDonald, A. Wilkinson, A. Smith, L. Bowater, A. Hemmings, and R. P. Bowater Characterization of a temperature-sensitive DNA ligase from Escherichia coli Microbiology, December 1, 2004; 150(12): 4171 - 4180. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 279/20/20594    most recent M401841200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gong, C. Articles by Shuman, S. Search for Related Content PubMed PubMed Citation Articles by Gong, C. Articles by Shuman, S. Social Bookmarking                      What's this?