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)

Monovalent cations such as Na+ and K+ inhibit the activity of T4 DNA ligase. However, the extent of inhibition varies with the terminal sequence of the duplex DNA used as substrate; in many cases, ligation of DNA is completely inhibited at 200 mM. The activity of the ligase is stimulated by raising the concentration of polyethylene glycol 6000 from 0 to 15% (w/v) when NaC1 and KC1 were both absent. Ligation was reduced as the concentration of NaC1 or KC1 was raised in a mixture containing 5 or 15% PEG 6000. With 10% PEG 6000, both cohesive- and blunt-end ligation of this ligase increased at high concentrations of salt (150-200 mM NaC1, or 200-250 mM KC1). Further, with 10% PEG 6000, inter- and intramolecular ligation occurred at low salt concentrations (0-100 mM NaC1, or 0-150 mM KC1); only linear oligomers were formed by intermolecular ligation at the high concentrations.
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PMID:Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol. 298 79

Embryos of Drosophila melanogaster contain two distinct DNA ligases (DNA ligase I and II). DNA ligase I was eluted at 0.2 M KCl and DNA ligase II at 0.6 M KCl on phosphocellulose column chromatography. The former was rich in early developing embryos and its activity decreased during embryonic development. The latter was found constantly throughout the developing stages of embryos. DNA ligase I existed in a cytoplasmic fraction and DNA ligase II is concentrated in nuclei. Both enzymes ligate 5'-phosphoryl and 3'-hydroxyl groups in oligo(dT) in the presence of poly(dA). DNA ligase II is also able to join oligo(dT)(poly(rA). Both enzymes require ATP and Mg2+ for activity. The Km for ATP is 2.7 X 10(-6) M for DNA ligase I, and 3.0 X 10(-5) M for DNA ligase II. DNA ligase I requires dithiothreitol and polyvinyl alcohol, but DNA ligase II does not. Both enzymes are inhibited in the presence of N-ethylmaleimide. DNA ligase I is active at a low salt concentration (0-30 mM KCl), but DNA ligase II is active at high salt concentrations (50-100 mM). DNA ligase I is more labile than DNA ligase II. The molecular masses of DNA ligase-AMP adducts were determined as 86 and 75 kDa for DNA ligase I, and as 70 (major protein) and 90 kDa (minor protein) for DNA ligase II under denaturing conditions. A sedimentation coefficient of 4.2 S was observed for DNA ligase II. Consequently, Drosophila DNA ligase I and II are quite similar to mammalian DNA ligase I and II. Drosophila DNA ligase I and a DNA ligase by B.A. Rabin et al. [(1986) J. Biol. Chem. 261, 10637-10645] seem to be the same enzyme.
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PMID:Two distinct DNA ligases from Drosophila melanogaster embryos. 310 89

In the presence of high concentrations of the nonspecific polymer polyethylene glycol (PEG), intermolecular cohesive-end ligation with the DNA ligase from Escherichia coli was stimulated by high salt concentrations: 200 mM NaCl or 300 mM KCl in 10% (w/v) PEG 6000 solutions, and 100-200 mM NaCl or 150-300 mM KCl in 15% PEG 6000 solutions. Intermolecular blunt-end ligation with this ligase was also stimulated at 100-150 mM NaCl or 150-250 mM KCl in 15% PEG 6000 solutions. The extent of such intermolecular ligation increased and the salt concentrations at which ligation was stimulated extended to lower concentrations when we raised the temperature from 10 to 37 degrees C.
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PMID:Stimulation of intermolecular ligation with E. coli DNA ligase by high concentrations of monovalent cations in polyethylene glycol solutions. 390 65

A progressive accumulation of DNA breaks has been reported to occur in nuclear DNA obtained from putative premalignant hepatic lesions induced by carcinogens. To determine if this alteration resulted from a defect in the level of, or functional activity of DNA ligases, we compared these enzymes in normal rat liver, 24-h regenerating liver, and hepatic nodules at intervals after cessation of N-2-acetylaminofluorene (AAF) treatment. Nuclear extracts of hepatocytes were separated into soluble and chromatin fractions, and multiple forms of DNA ligase activity were obtained by AcA34 gel filtration chromatography. In activities of the two largest species, DNA ligase Ia (480 kd) and DNA ligase Ib (240 kd), were present exclusively in soluble, nuclear fractions and were increased 4-fold and 2-fold, respectively, in 24-h regenerating livers. In AAF-induced nodules, these species were increased 3-fold and 1.5-fold, respectively, above those of normal rat liver, somewhat higher than predicted from the rate of cell division. In all of the test tissues, these ligase species demonstrated identical sensitivity to inhibition with 0.1 M NaCl or heating at 50 degrees C. DNA ligase II (80 kd) was found in both soluble nuclear fractions and chromatin at approximately identical levels in all tissues tested. Ligase II from all tissues also demonstrated identical responses to salt and heat. These data support the concept that DNA ligases Ia and Ib are related to DNA replication and suggest that ligase II may be a repair enzyme. The failure to detect significant alterations from expected values in the hepatic nodules and the lack of alteration in sensitivity to salt and heat indicate that the accumulation of DNA damage (presumably breaks) previously observed in carcinogen-induced altered hepatocytes is not due to an alteration in the level or the biochemical properties of DNA ligase.
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PMID:DNA ligase activities during hepatocarcinogenesis induced by N-2-acetylaminofluorene. 402 24

The ability of DNA repair enzymes to carry out excision repair of pyrimidine dimers in SV40 minichromosomes irradiated with 16 to 64 J/m2 of UV light was examined. Half of the dimers were substrate for the DNA glycosylase activity of phage T4 UV endonuclease immediately after irradiation, but this limit decreased to 27% after 2 h at 0 degrees C. Moreover, the apyrimidinic (AP) endonuclease activity of the enzyme did not incise all of the AP sites created by glycosylase activity, although all AP sites were substrate for HeLa AP endonuclease II. The initial rate of the glycosylase was 40% that upon DNA. After incision by the T4 enzyme, excision was mediated by HeLa DNase V (acting with an exonuclease present in the chromatin preparation). Under physiological salt conditions, excision did not proceed appreciably beyond the damaged nucleotides in DNA or chromatin. With chromatin, about 70% of the accessible dimers were removed, but at a rate slower than for DNA. Finally, HeLa DNA polymerase beta was able to fill the short gaps created after dimer excision, and these patches were sealed by T4 DNA ligase. Overall, roughly 30% of the sites incised by the endonuclease were ultimately sealed by the ligase. The resistance of some sites was due to interference with the ligase by the chromatin structure, as only 30-40% of the nicks created in chromatin by pancreatic DNase could be sealed by T4 or HeLa DNA ligases. The overall excision repair process did not detectably disrupt the chromatin structure, since the repair label was recovered in Form I DNA present in 75 S condensed minichromosomes. Although other factors might stimulate the rate of this repair process, it appears that the enzymes utilized could carry out excision repair of chromatin to a limit near that observed at the initial rate in mammalian cells in vivo.
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PMID:Excision repair of pyrimidine dimers from simian virus 40 minichromosomes in vitro. 608 90

A procedure for simultaneous large-scale purification of the bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non-permissive Escherichia coli B-23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essential lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.
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PMID:A new procedure for the simultaneous large-scale purification of bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase. 626 Apr 93

A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simultaneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive or false-negative reactions due to variations in these factors. Because the assay is simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory.
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PMID:Robust nonradioactive oligonucleotide ligation assay to detect a common point mutation in the CYP2D6 gene causing abnormal drug metabolism. 788 17

Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG. Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT). This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases. The enzyme requires high ATP concentrations with a Km for the overall ligation of a ssDNA substrate of 0.8 mM. The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described.
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PMID:Ligation of double-stranded and single-stranded [oligo(dT)] DNA by vaccinia virus DNA ligase. 866 20

Schizosaccharomyces pombe mitochondria were isolated from the cells treated with Novozyme 234, and purified in a Percoll gradient. A zymographic assay in a SDS-polyacrylamide gel containing single-stranded DNA revealed that an endonuclease of 32 kDa is associated with the mitochondria. The endonuclease was extracted from the mitochondria with 0.5 M KCl and was partially purified. The 32-kDa enzyme degraded both DNA and RNA at a weak alkaline pH, but preferred single-stranded DNA. The enzyme required Mg2+ or Mn2+, but not Ca2+ or Zn2+ for activity, and was inhibited by 50% with a 150 mM salt solution. Nicks generated by the enzyme could be resealed with T4 DNA ligase, indicating that the enzyme produces 5'-P and 3'-OH ends.
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PMID:Identification and characterization of a mitochondrial endonuclease from yeast, Schizosaccharomyces pombe. 895 92

NAD+-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 98%. Thermus species AK16D ligase, the most divergent of the seven Thermus isolates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity. This Thermus ligase is similar to Thermus thermophilus HB8 ligase with respect to pH, salt, NAD+, divalent cation profiles and steady-state kinetics.However, the former is more discriminative toward T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates. The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1-2 orders of magnitude higher fidelity than viral T4 DNA ligase. Both Thermus ligases are active with either the metal cofactor Mg2+, Mn2+or Ca2+but not with Co2+, Ni2+, Cu2+or Zn2+. While the nick closure step with Ca2+becomes rate-limiting which results in the accumulation of DNA-adenylate intermediate, Ni2+only supports intermediate formation to a limited extent. Both Thermus ligases exhibit enhanced mismatch ligation when Mn2+is substituted for Mg2+, but the Tsp. AK16D ligase remains more specific toward perfectly matched substrate.
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PMID:Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D. 988 74


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