Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chemiluminescent hybridization-ligation assays were devised to detect the delta F508 and delta I507 cystic fibrosis mutations in samples of human DNA that had been amplified by PCR. Two synthetic DNA oligomers were used in each assay. One of the oligomers was labeled with an acridinium ester and the other was immobilized on paramagnetic particles. The oligomers were hybridized to the samples and the target sequences discriminated by ligation with T4 or a thermostable DNA ligase. The performance of the assay was evaluated in a blind study of 30 samples. There was complete correspondence between the assignments based on the chemiluminescent assay and those made previously by gel electrophoresis, with one exception. The assignment of this discrepant sample by the chemiluminescent assay as a delta I507/normal heterozygote rather than a delta F508/normal heterozygote was confirmed by sequencing. The chemiluminescent hybridization-ligation assay provides a rapid and convenient means of discriminating DNA sequences differing by a single nucleotide.
 ...
PMID:Chemiluminescent hybridization-ligation assays for delta F508 and delta I507 cystic fibrosis mutations. 856 16

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.
 ...
PMID:Engineering hyperactive variants of human deoxyribonuclease I by altering its functional mechanism. 918 42

The ABC transporters are ubiquitous membrane proteins that couple adenosine triphosphate (ATP) hydrolysis to the translocation of diverse substrates across cell membranes. Clinically relevant examples are associated with cystic fibrosis and with multidrug resistance of pathogenic bacteria and cancer cells. Here, we report the crystal structure at 3.2 angstrom resolution of the Escherichia coli BtuCD protein, an ABC transporter mediating vitamin B12 uptake. The two ATP-binding cassettes (BtuD) are in close contact with each other, as are the two membrane-spanning subunits (BtuC); this arrangement is distinct from that observed for the E. coli lipid flippase MsbA. The BtuC subunits provide 20 transmembrane helices grouped around a translocation pathway that is closed to the cytoplasm by a gate region whereas the dimer arrangement of the BtuD subunits resembles the ATP-bound form of the Rad50 DNA repair enzyme. A prominent cytoplasmic loop of BtuC forms the contact region with the ATP-binding cassette and appears to represent a conserved motif among the ABC transporters.
 ...
PMID:The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. 1200 8