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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)

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

DNA ligase was partially purified from normal and regenerating rat liver. Its structure was studied using the activity gel procedure that identifies the functional polypeptides. Two slightly different purification procedures were followed leading to the isolation of one or two peaks (fractions A and B) of DNA ligase by hydroxyapatite chromatography. When analyzed on activity gels, all these enzyme fractions corresponded to a single active 130-kDa polypeptide both in normal and regenerating liver. A limited trypsin digestion of ligase fractions A and B gave rise to an identical pattern of smaller polypeptides of 110 kDa, 100 kDa and 75 kDa. Also storage at 4 degrees C of fractions A and B produced smaller polypeptides of 110 kDa, 100 kDa, 85 kDa and 60 kDa, which were identical for the two fractions. Our results indicate that the same ligase polypeptide of 130 kDa can be isolated from stationary or regenerating rat liver cells. However, physiological or artifactual proteolysis during various purification procedures can lead to the isolation of two enzyme fractions with different chromatographic behaviour but with the same molecular mass.
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PMID:Mammalian DNA ligase. Structure and function in rat-liver tissues. 380 89

The inducible resistance to alkylation mutagenesis and killing in Escherichia coli (the adaptive response) is controlled by the ada gene. The Ada protein acts both as a positive regulator of the response and as a DNA repair enzyme, correcting premutagenic O6-alkylguanine in DNA by suicidal transfer of the alkyl group to one of its own cysteine residues. We have determined the DNA sequence of the cloned ada+ gene and its regulatory region. The data reveal potential sites of ada autoregulation. Amino acid sequence determinations show that the active center for the O6-methylguanine-DNA methyltransferase is located close to the polypeptide COOH terminus and has the unusual sequence -Pro-Cys-His-, preceded by a very hydrophobic region. These same structural features are present at the active site of thymidylate synthase, suggesting a common chemical mechanism for activation of the cysteine.
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PMID:Active site and complete sequence of the suicidal methyltransferase that counters alkylation mutagenesis. 388 9

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [alpha-32P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.
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PMID:DNA ligase-AMP adducts: identification of yeast DNA ligase polypeptides. 390 11

Using a method that detects catalytically active DNA ligase in NaDodSO4-polyacrylamide gels (activity gels) we have characterized ligase produced in CV1-P monkey kidney cells infected with SV40 or treated with mitomycin C. Purification on hydroxylapatite columns of DNA ligase from control cells results in two peaks of activity called ligases I and II, respectively. Analysis of ligase I on activity gels revealed major catalytic peptides with Mr of 120, 110, 70 and 58 kDa, while analysis of ligase II revealed two major peptides of 65 and 58 kDa. Infecting CV1-P cells with SV40 produced a significant increase in the 120, 110, 70 and 58 kDa peptides while treating them with mitomycin C produced a significant increase in the 70 and 58 kDa peptides and a decrease in the 120 and 110 kDa ones. Autoproteolysis of partially purified ligase under several conditions resulted in an increase in the 58 kDa peptide and in the disappearance of other peptides. These results suggest that at least one active polypeptide is common to ligases I and II.
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PMID:Variation in DNA ligase structure during repair and replication processes in monkey kidney cells. 393 8

A monospecific antibody against calf thymus DNA ligase composed of a single polypeptide with Mr = 130,000 cross-reacts with rodent and calf thymus DNA ligases. The antibody precipitates a single Mr = 200,000 polypeptide from detergent lysates of [3H] leucine-labeled mouse Ehrlich tumor cells and calf thymocytes. Pulse-chase experiments show that the Mr = 200,000 polypeptide in Ehrlich tumor cells has a half-life of about 0.5 h. In addition to the Mr = 200,000 polypeptide, a Mr = 130,000 polypeptide is detected in the partially purified enzyme preparations from radiolabeled Ehrlich tumor cells. These results suggest that DNA ligase is synthesized in mammalian cells as a Mr = 200,000 polypeptide and that the Mr = 200,000 polypeptide is degraded to a Mr = 130,000 polypeptide by a limited proteolysis in vitro.
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PMID:Biosynthesis of mammalian DNA ligase. 397 11

We have recently obtained a monospecific antibody against calf thymus DNA ligase composed of a single Mr = 130,000 polypeptide. Immunohistochemical studies using the antibody and immunoperoxidase detection methods indicated that DNA ligase in a rat liver parenchymal cell line (BB) is localized essentially in nucleus. The specific activity of DNA ligase from growing BB cells was more than 10-fold higher than that from rat hepatocytes. The molecular forms of DNA ligase in these cell-free extracts were also analyzed.
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PMID:Immunohistochemical and biochemical studies on DNA ligase from a rat liver parenchymal cell line. 399 97

DNA ligase of E. coli is a polypeptide of molecular weight 75,000. The comparable T4-induced enzyme is somewhat smaller (63,000 to 68,000). Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA, coupled to the cleavage of the pyrophosphate bond of DPN (E. coli) or ATP (T4). Phosphodiester bond synthesis catalyzed by both enzymes occurs in a series of these discrete steps and involves the participation of two covalent intermediates (Fig. 1). A steady state kinetic analysis of the reaction-catalyzed E. coli ligase supports this mechanism, and further demonstrates that enzyme-adenylate and DNA-adenylate are kinetically significant intermediates on the direct path of phosphodiester bond synthesis. A strain of E. coli with a mutation in the structural gene for DNA ligase which results in the synthesis of an abnormally thermolabile enzyme is inviable at 42 degrees C. Although able to grow at 30 degrees C, the mutant is still defective at this temperature in its ability to repair damage to its DNA caused by ultraviolet irradiation and by alkylating agents. At 42 degrees C, all the newly replicated DNA is in the form of short 10S "Okazaki fragments," an indication that the reason for the mutant's failure to survive under these conditions is its inability to sustain the ligation step that is essential for the discontinuous synthesis of the E. coli chromosome. DNA ligase is therefore an essential enzyme required for normal DNA replication and repair in E. coli. Purified DNA ligases have proved to be useful reagents in the construction in vitro of recombinant DNA molecules.
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PMID:DNA ligase: structure, mechanism, and function. 437 58

Escherichia coli photolyase is a DNA repair enzyme which monomerizes pyrimidine dimers, the major UV photoproducts in DNA, to pyrimidines in a light-dependent reaction. We recently described the construction of a tac-phr plasmid that greatly overproduces the enzyme (Sancar, G. B., Smith, F. W., and Sancar, A. (1983) Nucleic Acids Res. 11, 6667-6678). Using a strain carrying the overproducing plasmid as the starting material, we have developed a purification procedure that yields several milligrams of apparently homogeneous enzyme. The purified protein is a single polypeptide that has an apparent Mr of 49,000 under both denaturing and nondenaturing conditions. The enzyme has no requirement for divalent cations and it restores the biological activity of irradiated DNA only in the presence of photoreactivating light. The purified photolyase has a turnover number of 2.4 dimers/molecule/min; this value agrees well with the in vivo rate of photoreactivation in E. coli.
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PMID:Purification of Escherichia coli DNA photolyase. 632 59

The unc operon of Escherichia coli was split into two fragments by the restriction endonuclease HindIII. The operator-proximal portion was cloned into plasmid pACYC184, forming plasmid pAN51, which included the genes uncB, uncE, and uncA. When plasmid pAN51 was used as template in an in vitro transcription/translation system, the alpha subunit (from the uncA gene) and delta subunit of the F(1) adenosine triphosphatase (ATPase) were formed. In addition, three polypeptides of molecular weights 18,000, 17,000, and 14,000 were formed, and the significance of these polypeptides is discussed. The operator-distal portion of the unc operon was also cloned into plasmid pACYC184, forming plasmid pAN36, which included the uncD and uncC genes. When this plasmid was used as template in an in vitro transcription/translation system, the beta subunit (from the uncD gene) and the epsilon subunit (from the uncC gene) of the F(1) ATPase were formed. A polypeptide of a molecular weight similar to the epsilon subunit but of different net charge was also formed. Plasmid pAN45, carrying the complete unc operon, was isolated after digestion of a mixture of plasmids pAN51 and pAN36 with the restriction endonuclease HindIII and then religation with T4 deoxyribonucleic acid ligase. It was concluded that a HindIII restriction site occurred within the newly described uncG gene, which was shown, by complementation studies with Mu-induced mutants, to be located between the uncA and uncD genes to give the gene order uncBEAGDC. The uncG gene appears to code for the gamma subunit of the F(1) ATPase.
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PMID:Subunits of the adenosine triphosphatase complex translated in vitro from the Escherichia coli unc operon. 644 44


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