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)

Hershey circles and linear tandem aggregated forms of DNA have been obtained in vitro and treated with polynucleotide ligase to form phosphodiester bond. Using zone centrifugation in glycerol gradient covalently closed circles and linear dimers have been purified and their biological activity investigated. It was found that closed circular molecules lost most, if not all, of their activity in CaCl2-dependent system. In order to investigate the biological activity of tandem dimer molecules, hybrid dimers consisting of DNA's from lambda C1857 and lambda 1434 have been obtained. In plaque assay with the appropriate non-permissive strains of E. coli the efficiency of infectivity of hybrid dimers was measured. Biological activity of dimer molecules sealed with ligase was about 5% of the activity of linear monomers. Ig has been suggested that tandem dimers of lambda DNA joined by phosphodiester bond are able to penetrate into the CaCl2-treated host cells and both components of dimers are active during subsequent multiplication.
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PMID:[Biological activity of different forms of bacteriophage lambda DNA]. 121 77

A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.
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PMID:A 21S enzyme complex from HeLa cells that functions in simian virus 40 DNA replication in vitro. 216 68

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids.
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PMID:Purification and characterization of primer recognition proteins from HeLa cells. 236 57

Ultraviolet irradiation (520 ergs/mm(2) at 254 nm) causes the respiration of Escherichia coli B/r cells to cease after about 90 min postirradiation incubation in a minimal medium containing glycerol as the sole source of carbon. The cessation of respiration is associated with loss of pyridine nucleotides. Agents which interfere with postirradiation transcription and translation prevent cessation of respiration. We have studied the effects of one of these agents, 5-fluorouracil (FU), on respiration, pyridine nucleotide levels, viability, capacity to support phage growth, and the repair of irradiated deoxyribonucleic acid (DNA). Addition of FU to cells immediately after irradiation results in the continuance of respiration at a linear rate and the maintenance of high levels of pyridine nucleotides. Cellular viability increases dramatically during the first 60 min of postirradiation incubation in the presence of FU. The ability of irradiated cells to support the growth of phage T4 is also greatly increased. FU treatment has no effect on the kinetics of pyrimidine dimer excision or the degradation of DNA. However, treated cells repair single-strand breaks resulting from early steps in excision repair slightly more efficiently than do untreated cells. The results support the hypothesis that one of the causes of death in these irradiated cells is the disappearance of pyridine nucleotides, coenzymes of certain respiratory dehydrogenases, and, in the case of nicotinamide adenine dinucleotide, for polynucleotide ligase, the enzyme responsible for the final step in the repair of DNA.
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PMID:Role of pyridine nucleotides in 5-fluorouracil-mediated reactivation of ultraviolet radiation damage. 493 68

In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to X rays. This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action. In an effort to learn more of the relationship between X-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out. The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis. However, I-, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.
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PMID:The effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and X-irradiated Escherichia coli. 634 66

DNA ligase was purified about 2,000-fold from blastulae of sea urchin, Hemicentrotus pulcherrimus, by means of 1 M KCl-extraction, phosphocellulose, DEAE-cellulose, Sepharose CL-6B, and double-stranded DNA cellulose column chromatography. The purified DNA ligase had a molecular weight of 80,000 (determined by Sephadex G-150) and a sedimentation coefficient of 4.1S (by glycerol gradient centrifugation). The purified enzyme required ATP and Mg2+ (or Mn2+) as cofactors for activity, and was inhibited by N-ethylmaleimide. Apparent Km values for ATP, Mg2+, and Mn2+ were 4 microM, 2.7 mM, and 0.3 mM, respectively.
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PMID:Purification and properties of a DNA ligase from sea urchin embryos. 674 96

A DNA ligase has been purified from a subnuclear soluble replication complex isolated from adenovirus type 2-infected human KB cells. DNA ligase activity could not be demonstrated using an exogenous template until the complex was dissociated, suggesting that the ligase activity may be a component of the complex. The purified enzyme was free of endonuclease, exonuclease, 5'-nucleotidase, and phosphatase activities, and had a molecular weight of 105 000, as estimated by sedimentation in a glycerol gradient. The ligase requires ATP and a divalent cation for activity. The optimum of the reaction is at pH 7.8 in 50--100 mM Tris-HCl buffer and 10--20 mM MgCl2. Monovalent salts greatly stimulate ligase activity and the optimum was found at 150 mM. The reaction is very sensitive to high temperature; maximum activity was observed at 25--30 degrees C. ATP is the sole required cofactor and NAD, dATP and GTP could not replace the requirement for ATP. The Km for ATP is 60 microM. The Km for DNA is 250 microgram/ml or 1.6 nmol of terminal phosphate/ml and thus the enzyme shows relatively weak affinity for exogenous DNA. The maximum conversion of 32P into a phosphatase-resistant form is approximately 1.3% of the total, whereas T4 ligase, under the same conditions, can convert more than 25% of phosphate into a resistant form.
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PMID:Purification and properties of a DNA ligase from a soluble DNA replication complex. 735 2

It was possible to obtain high-efficiency transformation of E. coli MC1061 by the following modifications of the standard procedure: cells were harvested at A600 of 550-650, washed with 1, 1/2, and 1/40, and were resuspended in 1/500 culture vol of 1 mM Hepes, pH 7.0, to a cell concentration of 6 x 10(10)-6 x 10(11) cells/ml. Electrocompetent cells were used immediately for electroporation to yield 1.3 +/- 0.5 x 10(9) (mean +/- SD) transformants micrograms of plasmid DNA, which is comparable to the efficiency of bacteriophage lambda infection. Alternatively, cells can be stored frozen in 10% glycerol, although glycerol reduced transformation efficiency to approximately 30% (data not shown). Freezing and thawing of glycerol-treated cells did not result in any further loss of transformation efficiency (data not shown). This study showed that it is crucial to inactivate the T4 DNA ligase prior to electrotransformation of ligated DNA, which can be ensured by the introduction of a simple heat inactivation step, increasing the number of transformants by 260-fold. Although this paper focuses on the use of E. coli MC1061/p3, the experiments were repeated with a different plasmid in the parental strain E. coli MC1061 and showed the same result (data not shown.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transformation of Escherichia coli increases 260-fold upon inactivation of T4 DNA ligase. 777 74

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results.
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PMID:Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited. 1009 Feb 82

We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid MTH: ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K(m) 1.1 microM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD(+) cannot. MTH: ligase activity is thermophilic in vitro, with optimal nick-joining at 60 degrees C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase-adenylate intermediate (step 1) and hence cannot seal a 3'-OH/5'-PO(4) nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase-adenylate formation, but defective in activating the nick via formation of the DNA-adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA-adenylate, could be elicited for the wild-type MTH: ligase by inclusion of calcium as the divalent cation cofactor. MTH: ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that MTH: ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.
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PMID:Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum. 1087 42


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