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)

DNA from a temperate phage rho 11 and chromosomal DNA of Bacillus subtilis 168 were digested with endonuclease EcoRI and then ligated with T4 polynucleotide ligase. The ligated DNA fragments were used to transform a lysogenic strain, B. subtilis spoA12 lys21 hisA1 leuA8 p11, and Lys+, His+ or Leu+ transformants were selected. The cells of each type were then mixed, grown and treated with mitomycin C; the induced phages were tested for abilities abilities to form plaques and to tranduce the auxotrophic marker. Various types of plaque-forming or defective phages which transduce hisA or lys marker at considerably high frequencies were thus obtained.
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PMID:A method for construction of specialized transducing phage rho 11 of Bacillus subtilis. 10 55

A set of covalently closed circular duplex simian virus 40 DNA preparations of varying superhelical densities was prepared by closure of nicked duplex DNA with polynucleotide ligase in the presence of varying amounts of ethidium. The resulting molecules were tested for complex formation with the lysine-rich histone f1. The results confirmed earlier experiments in demonstrating that f1 histone reacts preferentially with superhelical DNA compared to relaxed circular DNA. Furthermore, the extent of the reaction is demonstrated to depend on the superhelical density. At the relatively low ratios of histone to DNA used in these experiments, the product of the interaction of f1 histone with superhelical DNA does not precipitate. At higher ratios of histone to DNA, an insoluble aggregate is formed.
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PMID:The effect of superhelicity on the interaction of histone f1 with closed circular duplex DNA. 126 27

Sequence analysis of the SalI g region of the genome of a virulent isolate of ASFV (Malawi Lil 20/1) has revealed an open reading frame with the potential to encode a 48 kilodalton (kD) polypeptide which has significant homology with eukaryotic and prokaryotic DNA ligases. This ASFV encoded gene also contains the putative active site region of DNA ligases including the lysine residue which is necessary for enzyme-adenylate adduct formation, but lacks the C-terminal basic region conserved in other eukaryotic DNA ligases. A novel [32P]-labelled potential DNA ligase-adenylate adduct of M(r) 45 kD was observed upon incubation of ASFV infected cell cytoplasmic extracts with alpha-[32P]-ATP and subsequent analysis of products by SDS/PAGE. These data together suggest that ASFV encodes its own DNA ligase.
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PMID:An African swine fever virus gene with homology to DNA ligases. 161 52

Nucleotide sequencing of the vaccinia virus SalI F DNA fragment identified an open reading frame of 552 amino acids encoding a protein of 63.3 kDa. The deduced amino acid sequence shares 30% identity with S. pombe and S. cerevisiae DNA ligases, with homology strongest near the carboxy terminus and around the lysine residue required for ligase-adenylate formation. Prokaryotic DNA ligases are poorly related to the vaccinia sequence. The initiation codon of the ORF forms part of a late transcriptional initiation sequence TAAATG and is preceded by two overlapping early transcriptional termination signals, TTTTTTTAT. Nonetheless, RNA mapping showed that the ligase gene is transcribed early during infection and the 5' end of the mRNA maps to the TAAATG motif. The possible roles of a DNA ligase in vaccinia virus DNA replication and recombination are discussed.
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PMID:Transcriptional mapping and nucleotide sequence of a vaccinia virus gene encoding a polypeptide with extensive homology to DNA ligases. 255 82

Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5'-monophosphate linked to the epsilon-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (epsilon-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the epsilon-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (epsilonamino)-linked adenosine monophosphoramidate.
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PMID:Structure of the DNA ligase-adenylate intermediate: lysine (epsilon-amino)-linked adenosine monophosphoramidate. 494 32

Using kinetic methods and differential spectrophotometry, the interaction between DNA and RNA ligases T4 and cibacron blue F3GA was studied. It was shown that the dye inhibits the first step of the enzymatic reaction, i. e. the formation of the AMP ligase complex. A 50% inhibition of the AMP-ligase complex by DNA ligase occurs at the dye concentration of 1 X 10(-5) M, that by RNA ligase--at 1 X 10(-4) M. Cibacron blue F3GA also inhibits the formation of end products of the reaction catalyzed by these enzymes. The dye is a noncompetitive inhibitor of RNA ligase with respect to [32P]oligoA20 and a competitive one with respect to ATP. Using differential spectrophotometry, it was found that the interaction occurs predominantly via electrostatic bonds between the SH-groups of the dye and the amino groups of lysine residues. DNA ligase possesses a higher affinity for the dye than RNA ligase.
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PMID:[Interaction of DNA and RNA ligases of phage T4 with Cibacron Blue F 3GA]. 670 48

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-L-lysine (degree of polymerization = 51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1:1 at a DNA concentration of 50 micrograms ml-1 were transgenic. The transgenic frequency for the pronuclear microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered the in vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.
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PMID:Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures. 758 16

DNA ligation entails AMP transfer from ATP to the 5' end of DNA to form a DNA-adenylate structure, A(5')pp(5')N. A similar reaction involving GMP transfer occurs during 5' capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase-AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.
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PMID:Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis. 764 38

The Apn1 DNA repair enzyme of Saccharomyces cerevisiae acts on abasic sites and oxygen radical damages. Apn1 is homologous to the repair endonuclease IV of Escherichia coli, but the yeast protein is approximately 80 residues longer at the C terminus. The Apn1 C terminus is rich in basic amino acids and includes two lysine/arginine clusters related to the nuclear transport signals of some other proteins. We show here by indirect immunofluorescence that Apn1 is localized to the yeast nucleus. Mutant Apn1 proteins were engineered with progressive deletions inward from the C terminus. Elimination of just the last 12 residues from Apn1 (to yield Apn355) did not alter the stability in yeast cells or the in vitro activity of the enzyme. Greater truncation of Apn1 produced proteins of apparently lower (Apn334) or much lower (Apn315 and Apn293) in vivo stability. Both Apn355 and Apn334 failed to concentrate in the yeast nucleus and remained in the cytoplasm. These delocalized derivatives also failed to restore wild-type resistance to oxidative or alkylating agents in a delta apn1 strain. Apn355 and Apn334 complemented repair-deficient E. coli as effectively as did wild-type Apn1. Resistance to these DNA-damaging agents in yeast was restored if Apn355 and Apn334 (but not Apn315 or Apn293) were overproduced approximately 20-fold, which suggests either weak active transport or passive diffusion of these derivatives into the nucleus. Replacement of the C-terminal 12 residues of Apn1 with the nuclear targeting sequence of SV40 T-antigen did not restore effective function or nuclear localization in yeast.
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PMID:Intracellular localization of the Apn1 DNA repair enzyme of Saccharomyces cerevisiae. Nuclear transport signals and biological role. 769 Jul 56

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


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