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)

Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endonuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80%. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the alpha NH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.
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PMID:Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities. Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis. 189 43

RNA-polymerase of E. coli was bound in vitro under physiological conditions to a recombinant plasmid pBR322 carrying two identical segments of bacteriophage T4 DNA, each containing a complete gene coding for T4 DNA ligase. After fixation of the complex with formaldehyde it was analysed by electron microscopy. A map of binding sites of the enzyme to DNA was obtained after a statistical assessment of micrographs. The inserted repeat revealed itself in the map as two regions of identical binding patterns, thus proving the adequacy of the preparation procedure. In pBR322 the strong binding sites correlate with the position of promoters. Also, apart from this, there are other strong binding sites within the T4 sequences which correlate strongly with the regions of abnormally high AT content. That means that under physiological conditions the RNA polymerase forms strong binding complexes with any AT rich DNA regions, as well as with real promoters.
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PMID:[Study of the binding of RNA polymerase by a recombinant plasmid using electron microscopy]. 266 82

Lipid peroxidation aldehydes of the 4-hydroxy-alpha, beta-unsaturated type, as well as the tobacco-smoke related alpha, beta-unsaturated aldehyde, acrolein, were highly cytotoxic and decreased the intracellular thiol content in cultured human bronchial fibroblasts after treatment with micromolar concentrations. In comparison, formaldehyde and acetaldehyde were less toxic and 100- to 300-fold higher doses were required to affect cell survival or thiol levels. The unsaturated aldehydes also markedly inhibited the DNA repair enzyme O6-methylguanine-DNA methyltransferase known to have a cysteine residue in its active site, but had no effect on the activity of uracil-DNA glycosylase. Our results indicate that reactive aldehydes of either exogenous or endogenous origin have direct cytotoxic effects and may also make cells more susceptible to other toxic chemicals due to an impairment in cellular defense mechanisms, e.g., DNA repair and detoxification by systems requiring glutathione.
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PMID:Cytotoxicity, thiol depletion and inhibition of O6-methylguanine-DNA methyltransferase by various aldehydes in cultured human bronchial fibroblasts. 406 50

A recent study reported that exposure of student embalmers in Cincinnati to high concentrations of formaldehyde (2 mg/m3) reduced the activity of the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). Reduction in a DNA repair enzyme may strongly increase the cancer risk not only with respect to the repair-enzyme causing agent but with respect to all carcinogens causing lesions subject to repair by the enzyme in question. Thus, we examined whether formaldehyde exposure of 57 medical students during their anatomy course at two different Universities in Germany influenced MGMT activity in mononuclear blood cells. Mean formaldehyde exposure of 41 students was 0.2 +/- 0.05 mg/m3 for 6 h per week. MGMT activity was 133.2 +/- 14.9 fmol MGMT/10(6) cells before the beginning of the formaldehyde exposure, 131.1 +/- 15.8 fmol MGMT/10(6) cells after 50 days (P = 0.56) and 128.2 +/- 19.0 fmol MGMT/10(6) cells after 111 days of exposure (P = 0.92). Similarly, no significant influence of formaldehyde exposure was observed, when smoking habits, alcohol consumption, allergic disease and sex of students were considered. In addition no significant difference was obtained in MGMT activity between 16 students with mean formaldehyde exposure of 0.8 +/- 0.6 mg/m3 and students without formaldehyde exposure (n = 51; P = 0.37). In conclusion, exposure of the medical students in western Europe to formaldehyde did not decrease MGMT activity in mononuclear blood cells.
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PMID:Activity of O6-methylguanine DNA methyltransferase in mononuclear blood cells of formaldehyde-exposed medical students. 1020 10

Recent work has uncovered a novel DNA repair enzyme: the AlkB protein of Escherichia coli, which oxidises the methyl groups of 1-methyladenine and 3-methylcytosine to hydroxymethyl moieties; the oxidised groups are subsequently released as formaldehyde, regenerating the unmodified bases.
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PMID:DNA repair: bioinformatics helps reverse methylation damage. 1249 4

E. coli AlkB is a DNA repair enzyme that catalyzes the de-methylation of DNA by means of a non-heme iron and alpha-keto glutarate as a co-factor. The proposed reaction mechanism can be separated in four stages. The first stage involves the binding of the co-factor and molecular oxygen to the Fe in the active site. This is followed by the formation of a ferryl intermediate in a high-spin state, along with CO(2) and succinate. Subsequently, the O atom on the Fe center is reoriented. The last stage comprises the oxidative de-methylation of the base to produce the native DNA base and formaldehyde. This stage also includes the rate limiting step in the reaction. Here, the last stage of the proposed reaction mechanism of AlkB has been studied for a model of the active site with DFT methods. Minimum structures have been calculated for all intermediates along the path in triplet and quintet spin states. Our results point to the quintet states as more stable, in agreement with previously reported calculations. Potential energy barriers have been obtained for all the steps along this last stage in the quintet state. In the first step the oxygen bound to the Fe center of the ferryl intermediate abstracts a hydrogen atom from the methyl moiety. This first step corresponds to the rate limiting step in the reaction. The calculated barrier for this step is 26.7 kcal/mol. The subsequent steps are highly exoergic. This energetic picture is in qualitative agreement with previously reported results. The calculated energy difference between the ferryl intermediate and the final product is -75.7 kcal/mol for a model with succinate in the active site and -49.3 kcal/mol for a model where the succinate is replaced by water. Our calculated mechanism is slightly different than the previously reported one. These results suggest the possibility of more than one mechanism. This is currently under investigation by ab initio QM/MM methods.
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PMID:DFT study of a model system for the dealkylation step catalyzed by AlkB. 2064 Jul 98