Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The single-stranded DNA-binding proteins (SSBs) are vital to virtually all DNA functions. Here, we report on the biochemical properties of SSB from a fast-growing mycobacteria, Mycobacterium smegmatis, and the interaction of the homotetrameric SSBs with uracil DNA glycosylases (UDGs) from M. smegmatis (Msm), Mycobacterium tuberculosis (Mtu) and Escherichia coli (Eco). UDG is a crucial DNA repair enzyme, which removes the promutagenic uracil residues. MsmSSB stimulates activity of the homologous Msm UDG and of the heterologous Mtu-, and Eco-UDGs. On the contrary, while the MtuSSB stimulates the Mtu UDG, it inhibits the other two UDGs. Although the MsmSSB shares 84% identity with MtuSSB, the two are strikingly different, in that MsmSSB contains a glycine-rich segment (11 out of 13 residues) in the spacer connecting the N-terminal DNA-binding domain with the C-terminal acidic tail. While the DNA-binding properties of MsmSSB, such as its affinity to oligomeric DNA, requirement of minimum size DNA and the modes of interaction are indistinguishable from those of Eco-, and Mtu-SSBs, it is unclear if the glycine-rich segment confers structural advantage to MsmSSB, responsible for its stimulatory effect on all UDGs tested. More importantly, by using a small polypeptide inhibitor of UDGs, and the deletion mutants of SSBs, we suggest that the C-terminal acidic tail of the SSBs interacts within the DNA-binding groove of the UDGs, and propose a role for SSBs in the recruitment of UDGs to the damaged DNA.
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PMID:Biochemical properties of single-stranded DNA-binding protein from Mycobacterium smegmatis, a fast-growing mycobacterium and its physical and functional interaction with uracil DNA glycosylases. 1208 15

Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD(+)-dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD(+) as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperature-sensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD(+)-dependent DNA ligases.
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PMID:NAD+-dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor. 1269 44

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.
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PMID:Biochemical and genetic analysis of the four DNA ligases of mycobacteria. 1498 46

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates.
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PMID:Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism. 1504 60

In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3' to 5' exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair.
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PMID:Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine. 1549 16

A prokaryotic non-homologous end-joining (NHEJ) system for the repair of DNA double-strand breaks (DSBs), composed of a Ku homodimer (Mt-Ku) and a multidomain multifunctional ATP-dependent DNA ligase (Mt-Lig), has been described recently in Mycobacterium tuberculosis. Mt-Lig exhibits polymerase and nuclease activity in addition to DNA ligation activity. These functions were ascribed to putative polymerase, nuclease and ligase domains that together constitute a monomeric protein. Here, the separate polymerase, nuclease and ligase domains of Mt-Lig were cloned individually, over-expressed and the soluble proteins purified to homogeneity. The polymerase domain demonstrated DNA-dependent RNA primase activity, catalysing the synthesis of unprimed oligoribonucleotides on single-stranded DNA templates. The polymerase domain can also extend DNA in a template-dependent manner. This activity was eliminated when the catalytic aspartate residues were replaced with alanine. The ligase domain catalysed the sealing of nicked double-stranded DNA designed to mimic a DSB, consistent with the role of Mt-Lig in NHEJ. Deletion of the active-site lysine residue prevented the formation of an adenylated ligase complex and consequently thwarted ligation. The nuclease domain did not function independently as a 3'-5' exonuclease. DNA-binding assays revealed that both the polymerase and ligase domains bind DNA in vitro, the latter with considerably higher affinity. Mt-Ku directly stimulated the polymerase and nuclease activities of Mt-Lig. The polymerase domain bound Mt-Ku in vitro, suggesting it may recruit Mt-Lig to Ku-bound DNA in vivo. Consistent with these data, Mt-Ku stimulated the primer extension activity of the polymerase domain, suggestive of a functional interaction relevant to NHEJ-mediated DSB repair processes.
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PMID:Domain structure of a NHEJ DNA repair ligase from Mycobacterium tuberculosis. 1602 71

Non homologous end-joining (NHEJ)-mediated repair of DNA double-strand breaks in prokaryotes requires Ku and a specific multidomain DNA ligase (LigD). We present crystal structures of the primase/polymerisation domain (PolDom) of Mycobacterium tuberculosis LigD, alone and complexed with nucleotides. The PolDom structure combines the general fold of the archaeo-eukaryotic primase (AEP) superfamily with additional loops and domains that together form a deep cleft on the surface, likely used for DNA binding. Enzymatic analysis indicates that the PolDom of LigD, even in the absence of accessory domains and Ku proteins, has the potential to recognise DNA end-joining intermediates. Strikingly, one of the main signals for the specific and efficient binding of PolDom to DNA is the presence of a 5'-phosphate group, located at the single/double-stranded junction at both gapped and 3'-protruding DNA molecules. Although structurally unrelated, Pol lambda and Pol mu, the two eukaryotic DNA polymerases involved in NHEJ, are endowed with a similar capacity to bind a 5'-phosphate group. Other properties that are beneficial for NHEJ, such as the ability to generate template distortions and realignments of the primer, displayed by Pol lambda and Pol mu, are shared by the PolDom of bacterial LigD. In addition, PolDom can perform non-mutagenic translesion synthesis on termini containing modified bases. Significantly, ribonucleotide insertion appears to be a recurrent theme associated with NHEJ, maximised in this case by the deployment of a dedicated primase, although its in vivo relevance is unknown.
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PMID:Structure and function of a mycobacterial NHEJ DNA repair polymerase. 1717 32

Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD(+)-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD(+)-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD(+)-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD(+)-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.
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PMID:Evaluation of NAD(+) -dependent DNA ligase of mycobacteria as a potential target for antibiotics. 1754 1

Mycobacterium tuberculosis codes for an essential NAD+-dependent DNA ligase (MtuLigA) which is a novel, validated, and attractive drug target. We created mutants of the enzyme by systematically deleting domains from the C-terminal end of the enzyme to probe for their functional roles in the DNA nick joining reaction. Deletion of just the BRCT domain from MtuLigA resulted in total loss of activity in in vitro assays. However, the mutant could form an AMP-ligase intermediate that suggests that the defects caused by deletion of the BRCT domain occur primarily at steps after enzyme adenylation. Furthermore, genetic complementation experiments using a LigA deficient E. coli strain demonstrates that the BRCT domain of MtuLigA is necessary for bacterial survival in contrast to E. coli and T. filiformis LigA, respectively. We also report the identification, through virtual screening, of a novel N-substituted tetracyclic indole that competes with NAD+ and inhibits the enzyme with IC50 in the low muM range. It exhibits approximately 15-fold better affinity for MtuLigA compared to human DNA ligase I. In vivo assays using LigA deficient S. typhimurium and E. coli strains suggest that the observed antibacterial activity of the inhibitor arises from specific inhibition of LigA over ATP ligases in the bacteria. In silico ligand-docking studies suggest that the exquisite specificity of the inhibitor arises on account of its mimicking the interactions of NAD+ with MtuLigA. An analysis of conserved water in the binding site of the enzyme suggests strategies for synthesis of improved inhibitors with better specificity and potency.
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PMID:NAD+-dependent DNA ligase (Rv3014c) from Mycobacterium tuberculosis: novel structure-function relationship and identification of a specific inhibitor. 1755 28

Unlike Escherichia coli, Mycobacterium tuberculosis (Mt) expresses a Ku-like protein and an ATP-dependent DNA ligase that can perform non-homologous end-joining (NHEJ). We have expressed the Mt-Ku and Mt-Ligase D in E. coli using an arabinose-inducible promoter and expression vectors that integrate into specific sites in the E. coli chromosome. E. coli strains have been generated that express the Mt-Ku and Mt-Ligase D on a genetic background that is wild-type for repair, or deficient in either the RecA or RecB protein. Transformation of these strains with linearized plasmid DNA containing a 2bp overhang has demonstrated that expression of both the Mt-Ku and Mt-Ligase D is required for DNA end-joining and that loss of RecA does not prevent this double-strand break repair. Analysis of the re-joined plasmid has shown that repair is predominantly inaccurate and results in the deletion of sequences. Loss of RecB did not prevent the formation of large deletions, but did increase the amount of end-joining. Sequencing the junctions has revealed that the majority of the ligations occurred at regions of microhomology (1-4bps), eliminating one copy of the homologous sequence at the junction. The Mt-Ku and Mt-Ligase D can therefore function in E. coli to re-circularize linear plasmid.
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PMID:Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in RecA and RecB-independent DNA end-joining at regions of microhomology. 1756 Jan 74


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