Mechanism of DNA gyrase-mediated illegitimate recombination:: Characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype
Mechanism of DNA gyrase-mediated illegitimate recombination:: Characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype
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DOI：
10.1006/jmbi.1999.2758
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发表时间：
1999-06-11
影响因子：
5.6
通讯作者：
Ikeda, H
中科院分区：
文献类型：
--
作者：
Ashizawa, Y;Yokochi, T;Ikeda, H
为了研究DNA旋转酶在大肠杆菌中介导的非法重组的机制，我们分离了温度敏感的gyrA突变体，这些突变体具有自发的非法重组和自发诱导Lambda原噬菌体的频率高于野生型。通过定点突变重建单突变后，我们证实了两个单突变gyrAL492P和gyrAL488P以及一个双突变gyuAI203V+gyrAI205V表现出与上述相同的性质。关于Lambda原噬菌体的超重组和更高诱导的表型，这些突变在野生型中占优势。对这些突变体中自发形成的hbio转导噬菌体的重组连接的分析表明，亲本大肠杆菌的bio和lambda重组位点的同源序列平均只有0.7b个碱基对，表明这种非法重组不需要同源性。对突变的gyrA基因的核苷酸序列分析表明，gyrAL492P和gyrwAL488P突变分别含有Leu492--&Gt；Pro和Leu488--&Gt；Pro的氨基酸替换，对应于DNA旋转酶A亚单位断裂-重组区的α18螺旋。GyrAI203V和gyrAI205V突变分别含有Ile203-->；Val和Ile205-->；Val，它们对应于α10‘螺旋，也位于DNA旋转酶A亚单位的断裂-重组域。生化分析表明，含有L492P突变的GyrA63蛋白具有明显的正常超螺旋活性，但在超螺旋反应中，在没有DNA旋转酶抑制剂的情况下也能产生少量的线状DNA，提示突变的DNA旋转酶可能存在宗教步骤的缺陷或亚基相互作用的缺陷。这些结果表明，这种重组是由突变的DNA旋转酶的宗教和/或二聚体形成缺陷引起的，这意味着DNA旋转酶A亚单位的两个α10‘和α18螺旋在亚基相互作用和/或DNA的再密封中起着关键作用。(C)1999年学术出版社。
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, gyuAI203V + 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 hbio 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 gyrwAL488P mutations contain amino acid substitutions of Leu492 --> Pro and Leu488 --> Pro, respectively, which correspond to the alpha 18 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 alpha 10' 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 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.