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)

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.
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PMID:A new system for studying molecular mechanisms of mutation by carcinogens. 22 5

An examination of x-ray structures of single-cluster [4Fe-4S] proteins in the Protein Data Bank has revealed that all redox proteins and the glutamine 5-phosphoribosyl-l-pyrophosphate amidotransferase from Bacillus subtilis have a topological configuration arbitrarily designated as D, whereas the DNA repair enzyme endonuclease III from Escherichia coli has the opposite topological configuration L. This is the first example in which both senses of topological chirality have been observed in a class of proteins.
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PMID:Topological chirality of iron-sulfur proteins. 928 90

ATP-dependent DNA ligases, NAD(+)-dependent DNA ligases, and GTP-dependent RNA capping enzymes are members of a covalent nucleotidyl transferase superfamily defined by a common fold and a set of conserved peptide motifs. Here we examined the role of nucleotidyl transferase motif V ((184)LLKMKQFKDAEAT(196)) in the nick joining reaction of Chlorella virus DNA ligase, an exemplary ATP-dependent enzyme. We found that alanine substitutions at Lys(186), Lys(188), Asp(192), and Glu(194) reduced ligase specific activity by at least an order of magnitude, whereas substitutions at Lys(191) and Thr(196) were benign. The K186A, D192A, and E194A changes had no effect on the rate of single-turnover nick joining by preformed ligase-adenylate but affected subsequent rounds of nick joining at the ligase adenylation step. Conservative substitutions K186R, D192E, and E194D partially restored activity, whereas K186Q, D192N, and E194Q substitutions did not. Alanine mutation of Lys(188) elicited distinctive catalytic defects, whereby single-turnover nick joining by K188A-adenylate was slowed by an order of magnitude, and high levels of the DNA-adenylate intermediate accumulated. The rate of phosphodiester bond formation at a pre-adenylated nick (step 3 of the ligation pathway) was slowed by the K188A change. Replacement of Lys(188) by arginine reversed the step 3 arrest, whereas glutamine substitution was ineffective. Gel-shift analysis showed that the Lys(188) mutants bound stably to DNA-adenylate. We infer that Lys(188) is involved in the chemical step of phosphodiester bond formation.
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PMID:Role of nucleotidyl transferase motif V in strand joining by chlorella virus DNA ligase. 1175 16