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 reaction catalyzed by the DNA repair enzyme uracil DNA glycosylase (UDG) proceeds through an unprecedented stepwise mechanism involving a positively charged oxacarbenium ion sugar and uracil anion leaving group. Here we use a novel approach to evaluate the catalytic contribution of electrostatic interactions between four essential phosphodiester groups of the DNA substrate and the cationic transition state. Our strategy was to substitute each of these phosphate groups with an uncharged (R)- or (S)-methylphosphonate linkage (MeP). We then compared the damaging effects of these methylphosphonate substitutions on catalysis with their damaging effects on binding of a cationic 1-azadeoxyribose (1-aza-dR(+)) oxacarbenium ion analogue to the UDG-uracil anion binary complex. A plot of log k(cat)/K(m) for the series of MeP-substituted substrates against log K(D) for binding of the 1-aza-dR(+) inhibitors gives a linear correlation of unit slope, confirming that the electronic features of the transition state resemble that of the 1-aza-dR(+), and that the anionic backbone of DNA is used in transition state stabilization. We estimate that all of the combined phosphodiester interactions with the substrate contribute 6-8 kcal/mol toward lowering the activation barrier, a stabilization that is significant compared to the 16 kcal/mol catalytic power of UDG. However, unlike groups of the enzyme that selectively stabilize the charged transition state by an estimated 7 kcal/mol, these phosphodiester groups also interact strongly in the ground state. To our knowledge, these results provide the first experimental evidence for electrostatic stabilization of a charged enzymatic transition state and intermediate using the anionic backbone of DNA.
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PMID:Powering DNA repair through substrate electrostatic interactions. 1259 May 78

Tyrosyl-DNA phosphodiesterase (Tdp1) is a member of the phospholipase D superfamily and acts as a DNA repair enzyme that removes stalled topoisomerase I- DNA complexes by hydrolyzing the bond between a tyrosine side chain and a DNA 3' phosphate. Despite the complexity of the substrate of this phosphodiesterase, vanadate succeeded in linking human Tdp1, a tyrosine-containing peptide, and a single-stranded DNA oligonucleotide into a quaternary complex that mimics the transition state for the first step of the catalytic reaction. The conformation of the bound substrate mimic gives compelling evidence that the topoisomerase I-DNA complex must undergo extensive modification prior to cleavage by Tdp1. The structure also illustrates that the use of vanadate as the central moiety in high-order complexes has the potential to be a general method for capturing protein-substrate interactions for phosphoryl transfer enzymes, even when the substrates are large, complicated, and unusual.
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PMID:Crystal structure of a transition state mimic for Tdp1 assembled from vanadate, DNA, and a topoisomerase I-derived peptide. 1261 86

DNA of all living organisms is constantly modified by exogenous and endogenous reagents. The mutagenic threat of modifications such as methylation, oxidation, and hydrolytic deamination of DNA bases is counteracted by base excision repair (BER). This process is initiated by the action of one of several DNA glycosylases, which removes the aberrant base and thus initiates a cascade of events that involves scission of the DNA backbone, removal of the baseless sugar-phosphate residue, filling in of the resulting single nucleotide gap, and ligation of the remaining nick. We were interested to find out how the BER process functions in hyperthermophiles, organisms growing at temperatures around 100 degrees C, where the rates of these spontaneous reactions are greatly accelerated. In our previous studies, we could show that the crenarchaeon Pyrobaculum aerophilum has at least three uracil-DNA glycosylases, Pa-UDGa, Pa-UDGb, and Pa-MIG, that can initiate the BER process by catalyzing the removal of uracil residues arising through the spontaneous deamination of cytosines. We now report that the genome of P. aerophilum encodes also the remaining functions necessary for BER and show that a system consisting of four P. aerophilum encoded enzymes, Pa-UDGb, AP endonuclease IV, DNA polymerase B2, and DNA ligase, can efficiently repair a G.U mispair in an oligonucleotide substrate to a G.C pair. Interestingly, the efficiency of the in vitro repair reaction was stimulated by Pa-PCNA1, the processivity clamp of DNA polymerases.
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PMID:Enzymology of base excision repair in the hyperthermophilic archaeon Pyrobaculum aerophilum. 1273 Feb 26

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.
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PMID:Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli. 1275 14

Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent DNA ligase known; it suffices for yeast cell growth in lieu of the essential yeast DNA ligase Cdc9. The Chlorella virus ligase-adenylate intermediate has an intrinsic nick sensing function and its DNA footprint extends 8-9 nt on the 3'-hydroxyl (3'-OH) side of the nick and 11-12 nt on the 5'-phosphate (5'-PO4) side. Here we establish the minimal length requirements for ligatable 3'-OH and 5'-PO4 strands at the nick (6 nt) and describe a new crystal structure of the ligase-adenylate in a state construed to reflect the configuration of the active site prior to nick recognition. Comparison with a previous structure of the ligase-adenylate bound to sulfate (a mimetic of the nick 5'-PO4) suggests how the positions and contacts of the active site components and the bound adenylate are remodeled by DNA binding. We find that the minimal Chlorella virus ligase is capable of catalyzing non-homologous end-joining reactions in vivo in yeast, a process normally executed by the structurally more complex cellular Lig4 enzyme. Our results suggest a model of ligase evolution in which: (i) a small 'pluripotent' ligase is the progenitor of the much larger ligases found presently in eukaryotic cells and (ii) gene duplications, variations within the core ligase structure and the fusion of new domains to the core structure (affording new protein-protein interactions) led to the compartmentalization of eukaryotic ligase function, i.e. by enhancing some components of the functional repertoire of the ancestral ligase while disabling others.
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PMID:Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and a new crystal structure of the ligase-adenylate intermediate. 1293 Sep 60

T4 DNA ligase is an Mg2+-dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a 'processive' ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a 'nonprocessive' ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (<10 microM) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (approximately 0.2 min(-1)) and a subsequent slow ligation (approximately 2x10(-3) min(-1)). The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+. The activation energies of self-adenylation (16.2 kcal.mol(-1)), transadenylation of the nick (0.9 kcal.mol(-1)), and nick-sealing (16.3-18.8 kcal.mol(-1)) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase-AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis.
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PMID:Kinetics and thermodynamics of nick sealing by T4 DNA ligase. 1462 96

The gene from Neisseria meningitidis serogroup A, encoding a putative, secreted ATP-dependent DNA ligase was cloned and overexpressed, and the soluble protein was purified. Mass spectrometry indicated that the homogeneous protein was adenylated as isolated, and sedimentation velocity experiments suggested that the enzyme exists as a monomer in solution. The 31.5 kDa protein can catalyze the ATP-dependent ligation of a singly nicked DNA duplex but not blunt-end joining. The first step of the overall reaction, the ATP-dependent formation of an adenylated ligase, was studied by measuring the formation of the covalent intermediate and isotope exchange between [alpha-32P] ATP and PPi. Mg2+ was absolutely required for this reaction and was the best divalent cation to promote catalysis. Electrophoretic gel mobility shift assays revealed that the enzyme bound both unnicked and singly nicked double stranded DNA with equivalent affinity (Kd approximately 50 nM) but cannot bind single stranded DNA. Preadenylated DNA was synthesized by transferring the AMP group from the enzyme to the 5'-phosphate of a 3'-dideoxy nicked DNA. The rate of phosphodiester bond formation at the preadenylated nick was also Mg(2+)-dependent. Kinetic data showed that the overall rate of ligation, which occurred at 0.008 s(-1), is the result of three chemical steps with similar rate constants (approximately 0.025 s(-1)). The Km values for ATP and DNA substrates, in the overall ligation reaction, were 0.4 microM and 30 nM, respectively.
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PMID:Mechanistic and kinetic study of the ATP-dependent DNA ligase of Neisseria meningitidis. 1473 Sep 75

Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3' phosphate and functions as a DNA repair enzyme that cleaves stalled topoisomerase I-DNA complexes. We previously determined a procedure to crystallize a quaternary complex containing Tdp1, vanadate, a DNA oligonucleotide, and a tyrosine-containing peptide that mimics the transition state for hydrolysis of the Tdp1 substrate. Here, the ability of vanadate to accept a variety of different ligands is exploited to produce several different quaternary complexes with a variety of oligonucleotides, and peptides or a tyrosine analogue, in efforts to explore the binding properties of the Tdp1 DNA and peptide binding clefts. Eight crystal structures of Tdp1 with vanadate, oligonucleotides, and peptides or peptide analogues were determined. These structures demonstrated that Tdp1 is able to bind substituents with limited sequence variation in the polypeptide moiety and also bind oligonucleotides with sequence variation at the 3' end. Additionally, the tyrosine analogue octopamine can replace topoisomerase I derived peptides as the apical ligand to vanadate. The versatility of this system suggests that the formation of quaternary complexes around vanadate could be adapted to become a useful method for structure-based inhibitor design and has the potential to be generally applicable to other enzymes that perform chemistry on phosphate esters.
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PMID:Explorations of peptide and oligonucleotide binding sites of tyrosyl-DNA phosphodiesterase using vanadate complexes. 1476 Nov 85

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

The Ape1 protein initiates the repair of apurinic/apyrimidinic sites during mammalian base excision repair (BER) of DNA. Ape1 catalyzes hydrolysis of the 5'-phosphodiester bond of abasic DNA to create nicks flanked by 3'-hydroxyl and 5'-deoxyribose 5-phosphate (dRP) termini. DNA polymerase (pol) beta catalyzes both DNA synthesis at the 3'-hydroxyl terminus and excision of the 5'-dRP moiety prior to completion of BER by DNA ligase. During BER, Ape1 recruits pol beta to the incised apurinic/apyrimidinic site and stimulates 5'-dRP excision by pol beta. The activities of these two enzymes are thus coordinated during BER. To examine further the coordination of BER, we investigated the ability of Ape1 to modulate the deoxynucleotidyltransferase and 5'-dRP lyase activities of pol beta. We report here that Ape1 stimulates 5'-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. We also demonstrate a second mechanism, independent of Ape1, in which conditions that support DNA synthesis by pol beta also enhance 5'-dRP excision. Ape1 modulates the gap-filling activity of pol beta by specifically inhibiting synthesis on an incised abasic substrate but not on single-nucleotide gapped DNA. In contrast to the wild-type Ape1 protein, a catalytically impaired mutant form of Ape1 did not affect DNA synthesis by pol beta. However, this mutant protein retained the ability to stimulate 5'-dRP excision by pol beta. Simultaneous monitoring of 5'-dRP excision and DNA synthesis by pol beta demonstrated that the 5'-dRP lyase activity lags behind the polymerase activity despite the coordination of these two steps by Ape1 during BER.
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PMID:Modulation of the 5'-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase beta by apurinic/apyrimidinic endonuclease 1. 1507 79


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