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)

Iridoviruses (IVs) are classified into five genera: Iridovirus and Chloriridovirus, whose members infect invertebrates, and Ranavirus, Lymphocystivirus, and Megalocytivirus, whose members infect vertebrates. Until now, Chloriridovirus was the only IV genus for which a representative and complete genomic sequence was not available. Here, we report the genome sequence and comparative analysis of a field isolate of Invertebrate iridescent virus type 3 (IIV-3), also known as mosquito iridescent virus, currently the sole member of the genus Chloriridovirus. Approximately 20% of the 190-kbp IIV-3 genome was repetitive DNA, with DNA repeats localized in 15 apparently noncoding regions. Of the 126 predicted IIV-3 genes, 27 had homologues in all currently sequenced IVs, suggesting a genetic core for the family Iridoviridae. Fifty-two IIV-3 genes, including those encoding DNA topoisomerase II, NAD-dependent DNA ligase, SF1 helicase, IAP, and BRO protein, are present in IIV-6 (Chilo iridescent virus, prototype species of the genus Iridovirus) but not in vertebrate IVs, likely reflecting distinct evolutionary histories for vertebrate and invertebrate IVs and potentially indicative of genes that function in aspects of virus-invertebrate host interactions. Thirty-three IIV-3 genes lack homologues in other IVs. Most of these encode proteins of unknown function but also encode IIV3-053L, a protein with similarity to DNA-dependent RNA polymerase subunit 7; IIV3-044L, a putative serine/threonine protein kinase; and IIV3-080R, a protein with similarity to poxvirus MutT-like proteins. The absence of genes present in other IVs, including IIV-6; the lack of obvious colinearity with any sequenced IV; the low levels of amino acid identity of predicted proteins to IV homologues; and phylogenetic analyses of conserved proteins indicate that IIV-3 is distantly related to other IV genera.
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PMID:Genome of invertebrate iridescent virus type 3 (mosquito iridescent virus). 1691 94

DNA ligase catalyzes the closure of single-strand nicks in double-stranded DNA that arise during replication and recombination. Inhibition of bacterial ligase is expected to cause chromosome degradation and cell death, making it an attractive target for new antibacterials. The prototypical bacterial ligase couples the hydrolysis of NAD(+) to phosphodiester bond formation between an adjacent 3'OH and 5'-terminal phosphate of nicked duplex DNA. The first step is the reversible formation of a ligase-adenylate from the reaction between apoenzyme and NAD(+). Inhibitors that compete with NAD(+) are expected to be bacterial specific because eukaryotic DNA ligases use ATP and differ in the sequence composition of their adenylation domain. We report here a high-throughput assay that measures the adenylation reaction specifically by monitoring ligase-AMP formation via scintillation proximity technologies. Escherichia coli DNA ligase was biotinylated in vivo; after reaction with radiolabeled NAD(+), ligase-[(3)H]AMP could be captured onto the streptavidin-coated surface of the solid scintillant. The method was ideal for high-throughput screening because it required minimal manipulations and generated a robust signal with minimal scatter. Certain adenosine analogs were found to inhibit the adenylation assay and had similar potency of inhibition in a DNA ligation assay.
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PMID:A high-throughput assay for the adenylation reaction of bacterial DNA ligase. 1749 75

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

DNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5' phosphate and a 3'-hydroxyl terminus in duplex DNA. This activity can repair single-stranded nicks in duplex DNA and join duplex DNA restriction fragments having either blunt ends or homologous cohesive ends. Two ligases are used for nucleic acid research and their reaction conditions and applications are described in this unit: E. coli ligase and T4 ligase. These enzymes differ in two important properties. One is the source of energy: T4 ligase uses ATP, while E. coli ligase uses NAD. Another important difference is their ability to ligate blunt ends; under normal reaction conditions, only T4 DNA ligase will ligate blunt ends.
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PMID:DNA ligases. 1826 23

Mycobacteria can repair DNA double-strand breaks (DSBs) via a nonhomologous end-joining (NHEJ) system that includes a dedicated DNA ligase (LigD) and the DNA end-binding protein Ku. Here we exploit an improved plasmid-based NHEJ assay and a collection of Mycobacterium smegmatis strains bearing deletions or mutations in Ku or the DNA ligases to interrogate the contributions of LigD's three catalytic activities (polymerase, ligase, and 3' phosphoesterase) and structural domains (POL, LIG, and PE) to the efficiency and molecular outcomes of NHEJ in vivo. By analyzing in parallel the repair of blunt, 5' overhang, and 3' overhang DSBs, we discovered a novel end-joining pathway specific to breaks with 3' overhangs that is Ku- and LigD-independent and perfectly faithful. This 3' overhang NHEJ pathway is independent of ligases B and C; we surmise that it relies on NAD(+)-dependent LigA, the essential replicative ligase. We find that efficient repair of blunt and 5' overhang DSBs depends stringently on Ku and the LigD POL domain, but not on the LigD polymerase activity, which mainly serves to promote NHEJ infidelity. The lack of an effect of PE-inactivating LigD mutations on NHEJ outcomes, especially the balance between deletions and insertions at blunt or 5' overhang breaks, argues against LigD being the catalyst of deletion formation. Ligase-inactivating LigD mutations (or deletion of the LIG domain) have a modest impact on the efficiency of blunt and 5' overhang DSB repair, because the strand sealing activity can be provided in trans by one of the other resident ATP-dependent ligases (likely LigC). Reliance on the backup ligase is accompanied by a drastic loss of fidelity during blunt end and 5' overhang DSB repair. We conclude that the mechanisms of mycobacterial NHEJ are many and the outcomes depend on the initial structures of the DSBs and the available ensemble of end-processing and end-sealing components, which are not limited to Ku and LigD.
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PMID:The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends. 1828 57

DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD(+) and assistance of a divalent metal cofactor Mg(2+). Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover (31)P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase-nucleotide complex is triggered in situ by the photo release of caged Mg(2+), and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4-5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations.
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PMID:The associative nature of adenylyl transfer catalyzed by T4 DNA ligase. 1900 96

DNA ligases are divided into two groups according to their cofactor requirement to form ligase-adenylate, ATP-dependent DNA ligases and NAD(+)-dependent DNA ligases. The conventional view that archaeal DNA ligases only utilize ATP has recently been disputed with discoveries of dual-specificity DNA ligases (ATP/ADP or ATP/NAD(+)) from the orders Desulfurococcales and Thermococcales. Here, we studied DNA ligase encoded by the hyperthermophilic crenarchaeon Sulfophobococcus zilligii. The ligase exhibited multiple cofactor specificity utilizing ADP and GTP in addition to ATP. The unusual cofactor specificity was confirmed via a DNA ligase nick-closing activity assay using a fluorescein/biotin-labelled oligonucleotide and a radiolabelled oligonucleotide. The exploitation of GTP as a catalytic energy source has not to date been reported in any known DNA ligase. This phenomenon may provide evolutionary evidence of the nucleotide cofactor utilization by DNA ligases. To bolster this hypothesis, we summarize and evaluate previous assertions. We contend that DNA ligase evolution likely started from crenarchaeotal DNA ligases and diverged to eukaryal DNA ligases and euryarchaeotal DNA ligases. Subsequently, the NAD(+)-utilizing property of some euryarchaeotal DNA ligases may have successfully differentiated to bacterial NAD(+)-dependent DNA ligases.
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PMID:Novel DNA ligase with broad nucleotide cofactor specificity from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii: influence of ancestral DNA ligase on cofactor utilization. 1864 34

Two genes encoding a NAD(+)-dependent DNA ligase (LigA) and an ATP-dependent DNA ligase (LigB) were identified in the genome of the extremely radioresistant bacterium, Deinococcus radiodurans (DR). The recombinant enzymes expressed in Escherichia coli, were purified to homogeneity and characterized. The optimal temperature and pH value of the two DNA ligases were 60 ( degrees )C and 7.0, respectively. Their optimal concentration of MgCl(2) was 5mM. Their half-lifes of heat inactivation at 100 ( degrees )C were about 3 min and 5 min, respectively. In addition, the results showed that DRLigB displayed higher activity than DRLigA at stick and blunt ended joining of DNA, indicating that DRLigB is a key DNA ligase of D. radiodurans in DNA recombination and double-strand break repair.
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PMID:Biochemical characterization of two DNA ligases from Deinococcus radiodurans. 1868 Apr 57

DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require NAD(+) for activity, but eukaryotic and viral DNA ligases require ATP. Because of their essential nature, unique structures and widespread existence in nature, bacterial DNA ligases represent a class of valuable targets for identifying novel and selective antibacterial agents. In this study, we cloned and expressed the ligA gene from Streptococcus pneumoniae, and characterized this ligA-encoded NAD(+)-dependent DNA ligase. We then screened small molecule chemical libraries using a biochemical assay and identified a new small molecule with a structure of 2,4-diamino-7-dimethylamino-pyrimido[4,5-d]pyrimidine. We show that this small molecule is a specific inhibitor of bacterial NAD(+)-dependent DNA ligases. Biochemical studies show that this molecule inhibits NAD(+)-dependent DNA ligases, but not ATP-dependent enzymes. The molecule inhibits NAD(+)-dependent DNA ligases competitively with respect to NAD(+) and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD(+)-dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.
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PMID:Identification and characterization of an inhibitor specific to bacterial NAD+-dependent DNA ligases. 1879 46

An efficient DNA strand break repair contributes to the radioresistance of Deinococcus radiodurans, which harbors the DNA repair pathways nearly identical to Escherichia coli. The molecular mechanisms of these proteins functioning in 2 diverse classes of bacteria seem to be different. The macromolecular interactions and formation of multiprotein complexes in vivo have gained significant importance in explaining the mechanism of the complex cellular processes. Here, we report the identification of a novel DNA metabolic protein complex from D. radiodurans. A similar complex has, however, not been found in E. coli. Mass spectrometric analysis showed the presence of a few known DNA repair proteins, molecular chaperones, and a large number of uncharacterized proteins from D. radiodurans R1. Biochemical and immunoblotting results indicated the presence of the protein promoting DNA repair A, DNA polymerase, Mg2+, and (or) Mn2+ -dependent 5'-->3' exonuclease activity along with protein kinase activity and phosphoproteins. DNA ligase activity was completely dependent upon the ATP requirement, as no ligase activity was seen in the presence of NAD as a cofactor. These results suggest the molecular interactions of the known DNA repair proteins with uncharacterized proteins in the macromolecular complex and the regulation of DNA degradation with the involvement of ATP and protein kinase functions.
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PMID:Identification of a DNA processing complex from Deinococcus radiodurans. 1892 46


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