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)

A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.
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PMID:Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans. 1525 Dec 7

The crystal structure of NAD+-dependent DNA ligase from Thermus filiformis (Tfi) revealed that the protein comprised four structural domains. In order to investigate the biochemical activities of these domains, seven deletion mutants were constructed from the Tfi DNA ligase. The mutants Tfi-M1 (residues 1-581), Tfi-M2 (residues 1-448), Tfi-M3 (residues 1-403) and Tfi-M4 (residues 1-314) showed the same adenylation activity as that of wild-type. This result indicates that only the adenylation domain (domain 1) is essential for the formation of enzyme-AMP complex. It was found that the zinc finger and helix-hairpin-helix (HhH) motif domain (domain 3) and the oligomer binding (OB)-fold domain (domain 2) are important for the formation of enzyme-DNA complex. The mutant Tfi-M1 alone showed the activities for in vitro nick-closing and in vivo complementation in Escherichia coli as those of wild-type. These results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the enzyme activity. The enzymatic properties of Tfi-M1 mutant (deleted the BRCT domain) were slightly different from those of wild-type and the nick-closing activity of Tfi-M1 mutant was approximately 50% compared with that of wild-type.
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PMID:Mutational analyses of the thermostable NAD+-dependent DNA ligase from Thermus filiformis. 1526 45

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.
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PMID:Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay. 1528 77

DNA ligase is an enzyme important for DNA repair and replication. Eukaryotic genomes encode ligases requiring ATP as the cofactor; bacterial genomes encode NAD(+)-dependent ligase. This difference in substrate specificities and the essentiality of NAD(+)-dependent ligase for bacterial survival make NAD(+)-dependent ligase a good target for designing highly specific anti-infectives. Any such structure-guided effort would require the knowledge of the precise mechanism of NAD+ recognition by the enzyme. We report the principles of NAD+ recognition by presenting the synthesis of NAD+ from nicotinamide mononucleotide (NMN) and AMP, catalyzed by Enterococcus faecalis ligase within the crystal lattice. Unprecedented conformational change, required to reorient the two subdomains of the protein for the condensation to occur and to recognize NAD+, is captured in two structures obtained using the same protein crystal. Structural data and sequence analysis presented here confirms and extends prior functional studies of the ligase adenylation reaction.
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PMID:Structural rearrangement accompanying NAD+ synthesis within a bacterial DNA ligase crystal. 1529 24

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.
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PMID:Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA). 1567 Oct 15

Archaea encode a DNA ligase composed of a C-terminal catalytic domain typical of ATP-dependent ligases plus an N-terminal domain similar to that found in eukaryotic cellular and poxvirus DNA ligases. All archaeal DNA ligases characterized to date have ATP-dependent adenylyltransferase and nick-joining activities. However, recent reports of dual-specificity ATP/NAD+ ligases in two Thermococcus species and Pyrococcus abyssi and an ATP/ADP ligase in Aeropyrum pernix raise the prospect that certain archaeal enzymes might exemplify an undifferentiated ancestral stage in the evolution of ligase substrate specificity. Here we analyze the biochemical properties of Pyrococcus horikoshii DNA ligase. P. horikoshii ligase catalyzes auto-adenylylation and nick sealing in the presence of a divalent cation and ATP; it is unable to utilize NAD+ or ADP to promote ligation in lieu of ATP. P. horikoshii ligase is thermophilic in vitro, with optimal adenylyltransferase activity at 90 degrees C and nick-joining activity at 70 to 90 degrees C. P. horikoshii ligase resembles the ligases of Methanobacterium thermautotrophicum and Sulfolobus shibatae in its strict specificity for ATP.
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PMID:Characterization of a thermophilic ATP-dependent DNA ligase from the euryarchaeon Pyrococcus horikoshii. 1619 59

Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant phiKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with phiKZ proteins. Comparative EL and phiKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and phiKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences.
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PMID:Genome comparison of Pseudomonas aeruginosa large phages. 1625 35

DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile Haloferax volcanii as a model system, we describe the first genetic analysis of archaeal DNA ligase function. We show that the Hfx. volcanii ATP-dependent DNA ligase family member, LigA, is non-essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that Hfx. volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that LigN is also non-essential for cell viability. Simultaneous inactivation of both proteins is lethal, however, indicating that they now share an essential function. Thus the LigN protein acquired by LGT appears to have been co-opted as a back-up for LigA function, perhaps to provide additional ligase activity under conditions of high genotoxic stress.
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PMID:ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii. 1642 Mar 48

Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication, modification, and repair. Here we produced, purified, and characterized mimivirus DNA ligase (MimiLIG), an NAD+-dependent nick joining enzyme homologous to bacterial LigA and entomopoxvirus DNA ligase. MimiLIG is a 636-aa polypeptide composed of an N-terminal NAD+ specificity module (domain Ia), linked to nucleotidyltransferase, OB-fold, helix-hairpin-helix, and BRCT domains, but it lacks the tetracysteine Zn-binding module found in all bacterial LigA enzymes. MimiLIG requires conserved domain Ia residues Tyr36, Asp46, Tyr49, and Asp50 for its initial reaction with NAD+ to form the ligase-AMP intermediate, but not for the third step of phosphodiester formation at a preadenylylated nick. MimiLIG differs from bacterial LigA enzymes in that its activity is strongly dependent on the C-terminal BRCT domain, deletion of which reduced its specific activity in nick joining by 75-fold without affecting the ligase adenylylation step. The DeltaBRCT mutant of MimiLIG was impaired in sealing at a preadenylylated nick. We propose that eukaryal DNA viruses acquired the NAD+-dependent ligases by horizontal transfer from a bacterium and that MimiLIG predates entomopoxvirus ligase, which lacks both the tetracysteine and BRCT domains. We speculate that the dissemination of NAD+-dependent ligase from bacterium to eukaryotic virus might have occurred within an amoebal host.
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PMID:Characterization of mimivirus NAD+-dependent DNA ligase. 1684 79

Concurrent activation of poly (ADP-ribose) polymerase (PARP) and DNA ligase was observed in cultured human epidermal keratinocytes (HEK) exposed to the DNA alkylating compound sulfur mustard (SM), suggesting that DNA ligase activation could be due to its modification by PARP. Using HEK, intracellular 3H-labeled NAD+ (3H-adenine) was metabolically generated and then these cells were exposed to SM (1 mM). DNA ligase I isolated from these cells was not 3H-labeled, indicating that DNA ligase I is not a substrate for (ADP-ribosyl)ation by PARP. In HEK, when PARP was inhibited by 3-amino benzamide (3-AB, 2 mM), SM-activated DNA ligase had a half-life that was four-fold higher than that observed in the absence of 3-AB. These results suggest that DNA repair requires PARP, and that DNA ligase remains activated until DNA damage repair is complete. The results show that in SM-exposed HEK, DNA ligase I is activated by phosphorylation catalysed by DNA-dependent protein kinase (DNA-PK). Therefore, the role of PARP in DNA repair is other than that of DNA ligase I activation. By using the DNA ligase I phosphorylation assay and decreasing PARP chemically as well as by PARP anti-sense mRNA expression in the cells, it was confirmed that PARP does not modify DNA ligase I. In conclusion, it is proposed that PARP is essential for efficient DNA repair; however, PARP participates in DNA repair by altering the chromosomal structure to make the DNA damage site(s) accessible to the repair enzymes.
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PMID:Poly (ADP-ribose) polymerase (PARP) is essential for sulfur mustard-induced DNA damage repair, but has no role in DNA ligase activation. 1690 6


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