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)

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA repair enzyme that catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3'-phosphate. The only known example of such a linkage in eukaryotic cells occurs normally as a transient link between a type IB topoisomerase and DNA. Thus human Tdp1 is thought to be responsible for repairing lesions that occur when topoisomerase I becomes stalled on the DNA in the cell. Tdp1 has also been shown to remove glycolate from single-stranded DNA containing a 3'-phosphoglycolate, suggesting a role for Tdp1 in repair of free-radical mediated DNA double-strand breaks. We report the three-dimensional structures of human Tdp1 bound to the phosphate transition state analogs vanadate and tungstate. Each structure shows the inhibitor covalently bound to His263, confirming that this residue is the nucleophile in the first step of the catalytic reaction. Vanadate in the Tdp1-vanadate structure has a trigonal bipyramidal geometry that mimics the transition state for hydrolysis of a phosphodiester bond, while Tdp1-tungstate displays unusual octahedral coordination. The presence of low-occupancy tungstate molecules along the narrow groove of the substrate binding cleft is suggestive evidence that this groove binds ssDNA. In both cases, glycerol from the cryoprotectant solution became liganded to the vanadate or tungstate inhibitor molecules in a bidentate 1,2-diol fashion. These structural models allow predictions to be made regarding the specific binding mode of the substrate and the mechanism of catalysis.
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PMID:Insights into substrate binding and catalytic mechanism of human tyrosyl-DNA phosphodiesterase (Tdp1) from vanadate and tungstate-inhibited structures. 1247 Sep 49

The Escherichia coli DNA repair enzyme endonuclease VIII (EndoVIII or Nei) excises oxidized pyrimidines from damaged DNA substrates. It overlaps in substrate specificity with endonuclease III and may serve as a back-up for this enzyme in E. coli. The three-dimensional structure of Nei covalently complexed with DNA has been recently determined, revealing the critical amino-acid residues required for DNA binding and catalytic activity. Based on this information, several site-specific mutants of the enzyme have been tested for activity against various substrates. Although the crystal structure of the DNA-bound enzyme has been fully determined, the important structure of the free enzyme has not previously been analyzed. In this report, the crystallization and preliminary crystallographic characterization of DNA-free Nei are described. Four different crystal habits are reported for wild-type Nei and two of its catalytic mutants. Despite being crystallized under different conditions, all habits belong to the same crystal form, with the same space group (I222) and a similar crystallographic unit cell (average parameters a = 57.7, b = 80.2, c = 169.7 A). Two of these crystal habits, I and IV, appear to be suitable for full crystallographic analysis. Crystal habit I was obtained by vapour diffusion using PEG 8000, glycerol and calcium acetate. Crystal habit IV was obtained by a similar method using PEG 400 and magnesium chloride. Both crystals are mechanically strong and stable in the X-ray beam once frozen under cold nitrogen gas. A full diffraction data set has recently been collected from a wild-type Nei crystal of habit I (2.6 A resolution, 85.2% completeness, Rmerge = 9.8%). Additional diffraction data were collected from an Nei-R252A crystal of habit IV (2.05 A resolution, 99.9% completeness, Rmerge = 6.0%) and an Nei-E2A crystal of habit IV (2.25 A resolution, 91.7% completeness, Rmerge = 6.2%). These diffraction data were collected at 95-100 K using a synchrotron X-ray source and a CCD area detector. All three data sets are currently being used to obtain crystallographic phasing via molecular-replacement techniques.
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PMID:Crystallization and preliminary crystallographic analysis of endonuclease VIII in its uncomplexed form. 1527 82

The effect of changes in the bulk dielectric constant on the DNA torsional properties was evaluated from plasmid circularization reactions. In these reactions, pUC18 previously linearized by EcoRI digestion was recircularized with T4 DNA ligase. The bulk dielectric constant of the reaction medium was decreased by the addition of different concentrations of neutral solutes: ethylene glycol, glycerol, sorbitol, and sucrose, or increased by the addition of glycine. The topoisomers generated by the ligase reaction were resolved by agarose-gel electrophoresis. The DNA twist energy parameter (kappa), which is an apparent torsional constant, was determined by linearization of the Gaussian topoisomers' distribution. It was observed that the twist energy parameter for the given solutes is almost linearly dependent on the bulk dielectric constant. In the reaction buffer, the twist energy parameter was determined to be 1100 +/- 100. By decreasing the dielectric constant to 74 with the addition of sorbitol, the value of the parameter reaches kappa = 900 +/- 100, whereas the addition of ethylene glycol leads to kappa = 400 +/- 50. Upon addition of glycine, which resulted in a dielectric constant equal to 91, the value of the twist energy parameter increased to kappa = 1750 +/- 100.
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PMID:The effect of changes in the bulk dielectric constant on the DNA torsional properties. 1787 31

Studies of RNA-protein interactions often require assembly of the RNA-protein complex using in vitro synthesized RNA or recombinant protein. Here, we describe a protocol to assemble a functional spliceosome in yeast extracts using transcribed or synthetic RNAs. The in vitro assembled spliceosome is stable and can be isolated by sedimentation through glycerol gradients for subsequent analysis. The protocols describe two procedures to prepare RNA: using bacteriophage RNA polymerases or ligation of RNA oligos using T4 DNA ligase. We also describe the preparation of splicing competent yeast extracts, the assembly of the spliceosome, and the isolation of the spliceosome by glycerol gradient sedimentation. To allow exogenously added U6 RNA to be incorporated into the spliceosome, the endogenous U6 small nuclear RNA (snRNA) in the extract is eliminated by an antisense U6 DNA oligo and ribonuclease H; a "neutralizing" U6 DNA oligo was then added to protect the incoming U6 RNA. This protocol allows study of the role individual bases or the phosphate backbone of U6 plays in splicing and of the interaction between U6 snRNA and the spliceosomal proteins.
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PMID:Assembly and glycerol gradient isolation of yeast spliceosomes containing transcribed or synthetic U6 snRNA. 1898 83

There is growing interest in biodiesel and this results in the accumulation of glycerol. The exploitation and application of glycerol has attracted more and more attention. In the current study, glycerol was biotransformed to produce 3-hydroxypropionaldehyde by genetic engineering bacteria. It is known that 3-hydroxypopionaldehyde has been widely used as an important intermediate for chemicals, effective antimicrobial agent, and fix agent for tissues. A pair of primers was designed on the basis of the sequence of both NH2-terminus and the amino acid sequence of glycerol dehydratase reported by NCBI, and a fragment about 1.6 kb was obtained by PCR amplification using the total genome DNA of Lactobacillus reuteri as template, then the fragment was cloned to the pMD18-T vector and sequenced. Two specific primers were designed according to the obtained sequence, and a fragment with length of 1674 bp was amplified using PCR with these two specific primers. Consequently, the resulting products were digested with EcoR I and Hind III and ligated using T4 DNA ligase to the pET28b vector digested with the same enzymes. The recombinant plasmid, named pET28b-dhaB, was transformed into E. coli BL21. The positive clones were induced with IPTG and the expression products were further analyzed by SDS-PAGE, indicating that protein with a molecule weight of around 65 kD was obtained. Furthermore, the glycerol dehydratase activity was evaluated and compared with the wild type strain as well.
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PMID:[Cloning and expression of Lactobaceillus reuteri glycerol dehydratase gene in Escherichia coil]. 2035 78

Bleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent that targets primarily the furanose rings of DNA and in the presence of ferrous ions produces oxidative damage and DNA strand breaks. Escherichia coli cells growing in broth medium and exposed to low concentrations of BLM contain double-strand breaks and require homologous recombination to survive. To a lesser extent, the cells also require the abasic (AP) endonucleases associated with base excision repair, presumably to repair oxidative damage. As expected, there is strong induction of the SOS system in treated cells. In contrast, E. coli cells growing in glucose or glycerol minimal medium are resistant to the lethal action of BLM and do not require either homologous recombination functions or AP-endonucleases for survival. DNA ligase activity, however, is needed for cells growing in minimal medium to resist the lethal effects of BLM. There is weak SOS induction in such treated cells.
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PMID:Bleomycin sensitivity in Escherichia coli is medium-dependent. 2243 5


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