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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)

Tumor cells resistant to chloroethylnitrosourea (CENU) therapy contain high levels of O6-alkylguanine DNA-alkyltransferase (GATase), a DNA repair enzyme that aborts DNA interstrand cross-linking by removing CENU-induced O6-alkylguanine adducts. Because the transferase binds covalently to CENU-treated oligonucleotides, we reacted partially purified GATase from cultured human lymphoblasts with a BCNU-treated, 35S-5'-end-labeled, synthetic oligonucleotide designed to have a polyadenylated 3' terminus. Immunoprobing Western blots of this reaction mixture with GATase-specific monoclonal antibody indicated that 25-30% of the transferase became complexed. We purified this complex by affinity chromatography with oligo(dT) cellulose, recovering homogenous material that appeared as a discrete 35-kDa Coomassie blue or silver-stained band after SDS-polyacrylamide gel electrophoresis. Autoradiography and Western immunoblotting confirmed that this band contained both the radiolabeled oligonucleotide and the GATase protein.
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PMID:Affinity purification and characterization of human O6-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide. 278 Feb 88

Using specific antibodies against calf thymus DNA ligases I and II (EC 6.5.1.1), we have investigated the polypeptide structures of DNA ligases I and II present in the impure enzyme preparations, and estimated the polypeptides of DNA ligases I and II present in vivo. Immunoblot analysis of DNA ligase I after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 130-kDa polypeptide as a major one in the enzyme preparations from calf thymus throughout the purification. In addition to the 130-kDa polypeptide, a 200-kDa polypeptide was detected in the enzyme preparations at the earlier steps of the purification, and a 90-kDa polypeptide was observed as a minor one in the enzyme preparations at the later steps of the purification. The polypeptides with molecular weight of 130 000 and 90 000 were detected by SDS-polyacrylamide gel electrophoresis of DNA ligase I-[3H]AMP complex. These results suggest that a 200-kDa polypeptide of DNA ligase I present in vivo is degraded to a 130-kDa polypeptide and then to a 90-kDa polypeptide during the isolation and purification procedures. On the other hand, the monospecific antibody against calf thymus DNA ligase II cross-reacted with only a 68 kDa polypeptide in the enzyme preparations throughout the purification, suggesting that the 68-kDa polypeptide is a single form of calf thymus DNA ligase II present in vivo as well as in vitro.
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PMID:Immunochemical analysis of molecular forms of mammalian DNA ligases I and II. 353 Mar 31

Endonuclease VIII, a novel presumptive DNA repair enzyme, was isolated from Escherichia coli by FPLC1 purification. The enzyme was found in strains that contained or lacked endonuclease III and was purified by radial flow S-Sepharose, Mono S, phenyl-Superose, and Superose 12 FPLC. Examination of the properties of endonuclease VIII showed it to have many similarities to endonuclease III. DNA containing thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, urea residues, or AP sites was incised by the enzyme; however, DNA containing reduced AP sites was not. HPLC analysis of the products formed by exhaustive enzymatic digestion of damage-containing DNA showed that endonuclease VIII released thymine glycol and dihydrothymine as free bases. Taken together, these data suggest that endonuclease VIII contains both N-glycosylase and AP lyase activities. Consistent with this idea, DNA containing AP sites or thymine glycols, that was enzymatically nicked by endonuclease VIII was not a good substrate for E. coli DNA polymerase I, suggesting that endonuclease VIII nicks damage-containing DNA on the 3' side of the lesion. Also, since monophosphates were not released after treating thymine glycol-containing DNA with endonuclease VIII, the enzyme does not appear to have exonuclease activity. The enzyme activity was maximal in 75 mM NaCl or 5 mM MgCl2. Analysis of endonuclease VIII by both Superose FPLC and Sephadex yielded native molecular masses of 28,000 and 30,000 Da, respectively. SDS-PAGE, in conjunction with activity gel analysis, gave a molecular mass of about 29,000 Da. Furthermore, renaturation of the putative active band from SDS-PAGE gave rise to an active enzyme.
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PMID:Isolation and characterization of endonuclease VIII from Escherichia coli. 811 Jul 59

A recombinant plasmid for expression of full-length human DNA ligase I (phLig-I) was constructed in a plasmid/phage chimeric vector, pTD-T7N, which was derived from pUC118 by oligonucleotide-directed mutagenesis. The insert contained a 2757-base pair coding sequence for a whole human DNA ligase I and an extra ACC codon adjacent to the ATG initiation codon. This ACC codon was required for achieving high levels of expression of full-length DNA ligase I in Escherichia coli strain BL21. The recombinant plasmid, which was designed to exploit the T7 late promoter and the ATG initiation codon for beta-galactosidase was transfected into E. coli BL21 cells that express T7 RNA polymerase. The recombinant clone produced relatively high levels of DNA ligase I with a molecular mass of 130 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. The DNA ligase was purified to near-homogeneity by the two-step column chromatographic procedure from BLphLig-I cells that had been induced with isopropyl beta-D-thiogalactoside. The specific activity, chromatographic behavior, kinetic properties, molecular mass, and antigenicity of the recombinant human DNA ligase I were indistinguishable from those of purified mammalian DNA ligase I. Metabolically labeling experiments with 32P(i) indicate that the recombinant DNA ligase I was present as an enzyme-AMP reaction intermediate, but not as a phosphoprotein, in the E. coli cells.
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PMID:Expression of active human DNA ligase I in Escherichia coli cells that harbor a full-length DNA ligase I cDNA construct. 822 62

Sequencing of the EcoRI N' fragment of African swine fever virus (ASFV) DNA revealed an open reading frame encoding a protein similar to ATP-dependent DNA ligases. When the gene encoding this protein was expressed in Escherichia coli, a protein of the expected molecular mass was labeled in bacterial extracts upon incubation with [alpha-32P]ATP. The recombinant protein comigrated in SDS-PAGE with the putative viral DNA ligase detected in extracts of infected cells. We demonstrate that the recombinant protein is a DNA ligase by dissociation of the protein-[32P]AMP adduct with pyrophosphate and nicked DNA. The putatively adenylylated lysine in ASFV is surrounded by two arginine residues, instead of by two hydrophobic amino acids as in the other ATP-dependent DNA ligases. This might explain the high concentration of pyrophosphate necessary to revert the DNA ligase--AMP adduct in ASFV, 10- to 100-fold higher than that required for other DNA ligases. A comparison of the amino acid sequences reported for ATP-dependent DNA ligases disclosed three new amino acid motifs around the adenylylation site of these enzymes. ASFV DNA ligase has little similarity to the other enzymes at the ends of the molecule, but conserves the amino acid motifs of the central region.
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PMID:African swine fever virus encodes a DNA ligase. 843 92

Purification and characterization of a DNA repair enzyme having 5' apurinic/apyrimidinic (AP) endonuclease activity are reported. The enzyme extracted from mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time) and single-stranded DNA cellulose, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme has an apparent molecular mass of 30 kDa as determined by SDS-PAGE. It was shown to have nicking activity on acid-depurinated DNA but not on intact DNA, and to have priming activities for DNA polymerase on acid-depurinated DNA and bleomycin-treated DNA. The results indicate that it is a multifunctional DNA repair enzyme having 5' AP endonuclease and DNA 3' repair diesterase activities. The enzyme activity is dependent upon the presence of a divalent cation such as Mg2+. Its amino-terminal amino acid and internal amino acid sequences are determined.
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PMID:Purification and characterization of an AP endonuclease/DNA 3' repair diesterase from mouse ascites sarcoma cells. 854 4

We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny.
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PMID:Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide. 861 53

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

An endonuclease was extracted from intact rat liver mitochondria with 0.4 M NaCl, and partially purified. A zymographic assay in SDS-polyacrylamide gel containing single-stranded DNA revealed that the enzyme has an apparent molecular mass of 55 kDa. It was different from the molecular mass of the major endonuclease of bovine heart mitochondria (a homodimer of a 29-kDa peptide), that was recently shown to be identical to the endonuclease G. The purified 55-kDa enzyme degraded both DNA and RNA, preferring RNA and single-stranded DNA at a weak alkaline pH, required Mg(2+) and Mn(2+) but not Ca(2+) for activity, and was strongly inhibited by monovalent cations. Nicks generated by the enzyme were resealable with T4 DNA ligase, indicating that the enzyme produces 5'-p and 3'-OH ends. The 55-kDa enzyme, like endonuclease G, displayed a strong preference to nick within a (dG)n.(dC)n sequence tract.
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PMID:Identification of a 55-KDA endonuclease in rat liver mitochondria with nucleolytic properties similar to endonuclease G. 913 52

A novel nucleolar component has been identified and cloned using a human autoimmune serum. This antigen, as inferred from the cDNA sequence, is an Mr 55000 protein. Immuno blot analysis, however, of both the native protein and the in vitro translation products of the cDNA showed that they migrate on SDS-PAGE at an apparent molecular mass of 90000 A BLAST search using the cDNA sequence indicated that it is in an antisense orientation to and overlaps the gene of the DNA repair enzyme ERCC-1. An open reading frame, without a translational start site, had been observed by others in this region of the chromosome 19 (19q13.3) and the putative protein was termed ASE-1 (Anti-Sense to ERCC-1). Our cDNA is a full-length equivalent of that open reading frame. ASE-1 was found to contain two domains that are present in a number of nucleolar specific proteins originating from a variety of organisms: a glycine-, arginine- and phenylalanine-rich putative nucleotide interaction domain and an alternating basic/acidic region. Indirect immunofluorescence analysis using antibodies generated to cloned regions of ASE-1 indicated that this protein occurs at the fibrillar centres of the nucleolus in interphase, the putative sites of rDNA transcription, and during cell division it is localized to the nucleolus organizer regions of the chromosomes. ASE-1 co-localises with the RNA polymerase I transcription initiation factor UBF/NOR-90 throughout all stages of the cell cycle and these two proteins associate with each other in vitro.
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PMID:ASE-1: a novel protein of the fibrillar centres of the nucleolus and nucleolus organizer region of mitotic chromosomes. 942 81


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