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 conf er spontaneous illegitimate recombination and spontaneous induction of lamb da prophage at higher frequencies than that in the wild-type. After reconst ruction of single mutations by targeted mutagenesis, we confirmed that two single mutations, gyrAL492P and gyrAL488P, and a double mutation, gyuAI203V + gyrAI205V, show the same properties as those described above. With respe ct to the phenotypes of hyper-recombination and higher induction of lambda prophage, these mutations were dominant over the wild-type. Analysis of rec ombination junctions of hbio transducing phages formed spontaneously in the se mutants showed that the parental E. coli bio and lambda recombination si tes have a homologous sequence of only 0.7 base-pair on average, indicating that homology is not required for this illegitimate recombination. Analysi s of nucleotide sequences of mutant gyrA genes revealed that the gyrAL492P and gyrwAL488P mutations contain amino acid substitutions of Leu492 --> Pro and Leu488 --> Pro, respectively, which correspond to the alpha 18 helix i n the breakage-reunion domain of DNA gyrase A subunit. The gyrAI203V and gy rAI205V mutations contain Ile203 --> Val and Ile205 --> Val, respectively, which correspond to the alpha 10' helix, also in the breakage-reunion domai n of DNA gyrase A subunit. Biochemical analysis indicated that the GyrA63 p rotein that contains the L492P mutation has an apparently normal supercoili ng activity, but it also produces a small amount of linear DNA in the absen ce of DNA gyrase inhibitor during the supercoiling reaction, suggesting tha t the mutant DNA gyrase may have a defect at the step of religation or a de fect in the subunit interaction. These results suggest that the recombinati on is induced by defects of religation and/or dimer formation in the mutant DNA gyrases, implying that two oc helices, alpha 10' and alpha 18, of DNA gyrase A subunit have crucial roles in subunit interaction and/or resealing of DNA. (C) 1999 Academic Press.