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)

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight fragments by altering its functional mechanism from the native single-stranded nicking pathway to a much more efficient one which results in increased double-stranded scission. By introducing positively charged amino acids at DNase I positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA, we have created a hyperactive variant with approximately 35-fold higher DNA-degrading activity relative to wild type. This enhancement can be attributed to both a decrease in Km and an increase in Vmax. Furthermore, unlike wild-type DNase I, the hyperactive variants are no longer inhibited by physiological saline. Replacement of the same positions with negatively charged amino acids greatly reduced DNA cleavage activity, consistent with a repulsive effect with the neighboring DNA phosphates. In addition, these variants displayed similar activities toward a small synthetic substrate, p-nitrophenyl phenylphosphonate, suggesting that the difference in DNA cleavage activity is due to the interaction of the engineered charged residues with the DNA phosphate backbone rather than any change in catalytic machinery. Finally, experiments involving the repair of DNase I digested DNA with T4 DNA ligase and the Klenow fragment of DNA polymerase I suggest that single-stranded gaps are introduced by the hyperactive variants. Thus, the increased functional activity of the hyperactive variants may be explained in part by a shift toward a processive DNA nicking mechanism, which leads to a higher frequency of double-stranded breaks.
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PMID:Engineering hyperactive variants of human deoxyribonuclease I by altering its functional mechanism. 918 42

This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.
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PMID:Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker. 1821 46