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 DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector. The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities. The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity. The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector. These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity. The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant. On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutagenesis of the T4 endonuclease V gene: role of lysine-130. 313 2

Mammalian cells contain three biochemically distinct DNA ligases. In this report we describe the purification of DNA ligase II to homogeneity from bovine liver nuclei. This enzyme interacts with ATP to form an enzyme-AMP complex, in which the AMP moiety is covalently linked to a lysine residue. An adenylylated peptide from DNA ligase II contains the sequence, Lys-Tyr-Asp-Gly-Glu-Arg, which is homologous to the active site motif conserved in ATP-dependent DNA ligases. The sequences adjacent to this motif in DNA ligase II are different from the comparable sequences in DNA ligase I, demonstrating that these enzymes are encoded by separate genes. The amino acid sequences of 15 DNA ligase II peptides exhibit striking homology (65% overall identity) with vaccinia DNA ligase. These peptides are also homologous (31% overall identity) with the catalytic domain of mammalian DNA ligase I, indicating that the genes encoding DNA ligases I and II probably evolved from a common ancestral gene. Since vaccinia DNA ligase is not required for DNA replication but influences the ability of the virus to survive DNA damage, the homology between this enzyme and DNA ligase II suggests that DNA ligase II may be involved in DNA repair.
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PMID:Mammalian DNA ligase II is highly homologous with vaccinia DNA ligase. Identification of the DNA ligase II active site for enzyme-adenylate formation. 798 68

Reductive methylation of the alpha NH2 moiety of the DNA repair enzyme T4 endonuclease V has been shown previously to eradicate both the N-glycosylase and apyrimidinic/apurinic lyase activities of the enzyme (Schrock, R. D., III, and Lloyd, R. S. (1991) J. Biol. Chem. 266, 17631-17639). The present study uses the technique of site-directed mutagenesis to investigate the important parameters involved in the cleavage mechanism. The prediction was that the addition of an amino acid in the immediate NH2-terminal region of the protein would alter the proximity of the alpha NH2 moiety of Thr2 to its target, thereby severely compromising the enzyme's catalytic activity. However, substitutions in this region generally should be tolerated. To test this hypothesis, three substitutions of the NH2-terminal amino acid were produced: Ser2 (T2S), Val2 (T2V), and Pro2 (T2P). An addition mutant was also produced by adding a glycine between the first and second amino acids of the protein (Thr2-Gly-Arg3) (+Gly). The T2P and +Gly mutants had negligible pyrimidine dimer-specific N-glycosylase activity as well as negligible pyrimidine dimer-specific nicking activity in vitro. Conversely, the T2S enzyme exhibited wild type levels of activity and the T2V exhibited intermediate levels of activity in vitro. Results from ultraviolet (UV) survival studies of the mutant enzymes indicated that the in vivo activities of these enzymes were directly correlated to the enzymes' ability to cleave at pyrimidine dimers in vitro. These results indicate that a critical parameter for the functionality of endonuclease V is the relative distance between the primary alpha NH2 group in the active site of the enzyme and those elements responsible for DNA binding and pyrimidine dimer recognition.
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PMID:Site-directed mutagenesis of the NH2 terminus of T4 endonuclease V. The position of the alpha NH2 moiety affects catalytic activity. 841 66

The Fpg protein of Escherichia coli is a DNA repair enzyme with DNA glycosylase, abasic site nicking, and deoxyribose excising activities. Analysis of the amino acid sequence of this protein suggests that the Fpg protein is a zinc finger protein with a Cys-X2-Cys-X16-Cys-X2-Cys motif. Competition experiments show that the Fpg protein substitutes Cu(II), Cd(II), and Hg(II), metal ions classically associated with substitutions in zinc finger proteins. The Fpg protein activities are inhibited following the reaction with a Cys-specific reagent at low protein:reagent ratios, suggesting that these residues are important for the enzymatic activities. Site-directed mutagenesis was used to produce 6 mutant Fpg proteins with Cys-->Gly mutations. Substitution of the zinc in these proteins by 65Zn(II) indicates that all the proteins bind zinc, but the Zn(II) is not retained as strongly in the zinc finger mutants. The mutations in the Fpg protein outside the zinc finger consensus sequence do not eliminate the Fapy-DNA glycosylase and abasic site nicking. One of the Fpg mutant proteins outside the zinc finger has a reduced capacity to release deoxyribose from abasic sites. Cys-->Gly mutations in the zinc finger consensus sequence reduce all three aforementioned activities substantially. The purified Fpg proteins with Cys-->Gly mutations in the zinc finger consensus sequence do not incise DNA at abasic sites with the same efficiency nor mechanism as the native Fpg protein. The wild type Fpg protein and the Fpg proteins mutated outside the zinc finger sequence bind an oligonucleotide with a unique chemically reduced abasic site in a defined sequence as assayed by retention on nitrocellulose filters, whereas the mutant Fpg proteins within the zinc finger sequence do not bind to the same oligonucleotide. Therefore, the disruption of zinc coordination in the zinc finger of the Fpg protein is associated with decreased binding capacity to DNA as well as decreased enzymatic activities.
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PMID:Fpg protein of Escherichia coli is a zinc finger protein whose cysteine residues have a structural and/or functional role. 847 47

The gene encoding Thermus filiformis (Tfi) DNA ligase was cloned and its nucleotide sequence was determined by the chain-termination method. The primary structure of Tfi DNA ligase was deduced from its nucleotide sequence. The Tfi DNA ligase comprises of 667 amino acid residues and its molecular mass was determined to be 75,936 Da. The deduced amino acid sequence of Tfi DNA ligase showed a 86.5% homology to Tth DNA ligase and 43.5% to E. coli DNA ligase. The Lys-116 of Lys-Val-Asp-Gly motif was proposed to be the active residue of Tfi DNA ligase. In comparison with the amino acid composition of DNA ligase, Tfi DNA ligase showed a significant increase in the proportion of charged residues, Arg and Glu, compared to E. coli DNA ligase. The G + C content in the first, second, and third positions of the codons used were 70.3%, 40.3%, and 90.3%, respectively. Codon usage in Tfi DNA ligase was heavily biased towards the use of G + C in the third position. Under tac promoter control, Tfi DNA ligase was overproduced to greater than 9% of E. coli BL26Blue cellular proteins.
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PMID:Cloning, nucleotide sequence, and expression of the DNA ligase-encoding gene from Thermus filiformis. 974 31

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.
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PMID:Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation. 1023 63

The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA repair enzymes.
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PMID:Effect of single mutations on the specificity of Escherichia coli FPG protein for excision of purine lesions from DNA damaged by free radicals. 1155 20

Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and 5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA-Arg(UCG), and tRNA-Gly(UCC), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into (S)-mchm(5)U in tRNA-Gly(UCC). These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.
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PMID:ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA. 2128 50