Mechanism of DNA gyrase-mediated illegitimate recombination: characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype.
To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we isolated temperature-sensitive gyrA mutants that confer spontaneous illegitimate recombination and spontaneous induction of lambda prophage at higher frequencies than that in the wild-type. After reconstruction of single mutations by targeted mutagenesis, we confirmed that two single mutations, gyrAL492P and gyrAL488P, and a double mutation, gyrAI203V+gyrAI205V, show the same properties as those described above. With respect to the phenotypes of hyper-recombination and higher induction of lambda prophage, these mutations were dominant over the wild-type. Analysis of recombination junctions of lambdabio transducing phages formed spontaneously in these mutants showed that the parental E. coli bio and lambda recombination sites have a homologous sequence of only 0. 7 base-pair on average, indicating that homology is not required for this illegitimate recombination. Analysis of nucleotide sequences of mutant gyrA genes revealed that the gyrAL492P and gyrAL488P mutations contain amino acid substitutions of Leu492-->Pro and Leu488-->Pro, respectively, which correspond to the alpha18 helix in the breakage-reunion domain of DNA gyrase A subunit. The gyrAI203V and gyrAI205V mutations contain Ile203-->Val and Ile205-->Val, respectively, which correspond to the alpha10' helix, also in the breakage-reunion domain of DNA gyrase A subunit. Biochemical analysis indicated that the GyrA63 protein that contains the L492P mutation has an apparently normal supercoiling activity, but it also produces a small amount of linear DNA in the absence of DNA gyrase inhibitor during the supercoiling reaction, suggesting that the mutant DNA gyrase may have a defect at the step of religation or a defect in the subunit interaction. These results suggest that the recombination is induced by defects of religation and/or dimer formation in the mutant DNA gyrases, implying that two alpha helices, alpha10' and alpha18, of DNA gyrase A subunit have crucial roles in subunit interaction and/or resealing of DNA.
Ashizawa
Y
Y
Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan.
Yokochi
T
T
Ogata
Y
Y
Shobuike
Y
Y
Kato
J
J
Ikeda
H
H
D000483:Alleles; D010582:Bacteriophage lambda / Q000235:genetics; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013696:Temperature
D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II