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首页> 美国卫生研究院文献>Nucleic Acids Research >Identification of factors influencing strand bias in oligonucleotide-mediated recombination in Escherichia coli
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Identification of factors influencing strand bias in oligonucleotide-mediated recombination in Escherichia coli

机译：鉴定影响大肠杆菌寡核苷酸介导的重组中链偏向的因素

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Recombinogenic engineering methodology, also known as recombineering, utilizes homologous recombination to create targeted changes in cellular DNA with great specificity and flexibility. In Escherichia coli, the Red recombination system from bacteriophage lambda has been used successfully to modify both plasmid and chromosomal DNA in a highly efficient manner, using either a linear double-stranded DNA fragment or a synthetic single-stranded oligonucleotide (SSO). The current model for Red/SSO-mediated recombination involves the SSO first annealing to a transient, single-stranded region of DNA before being incorporated into the chromosome or plasmid target. It has been observed previously, in both eukaryotes and prokaryotes, that mutations in the two strands of the DNA double helix are ‘corrected’ by complementary SSOs with differing efficiencies. Here we investigate further the factors that influence the strand bias as well as the overall efficiency of Red/SSO-mediated recombination in E.coli. We show that the direction of DNA replication and the nature of the SSO-encoded mismatch are the main factors dictating the recombinational strand bias. However, the influence that the SSO-encoded mismatch exerts upon the recombinational strand bias is abolished in E.coli strains that are defective in mismatch repair (MMR). This reflects the fact that different base–base mispairs are corrected by the mutS/H/L-dependent MMR pathway with differing efficiencies. Furthermore, our data indicate that transcription has negligible influence on the strand bias. These results demonstrate for the first time that the interplay between DNA replication and MMR has a major effect on the efficiency and strand bias of Red/SSO-mediated recombination in E.coli.
机译：重组基因工程方法学，也称为重组，利用同源重组以极高的特异性和灵活性在细胞DNA中产生靶向变化。在大肠杆菌中，已成功使用来自噬菌体λ的Red重组系统，使用线性双链DNA片段或合成的单链寡核苷酸（SSO），以高效的方式修饰质粒和染色体DNA。 Red / SSO介导的重组的当前模型涉及SSO首先退火到DNA的瞬时单链区域，然后再掺入染色体或质粒靶中。以前，在真核生物和原核生物中都观察到，DNA双螺旋的两条链中的突变被效率不同的互补SSO所“纠正”。在这里，我们进一步调查影响链偏向的因素以及大肠杆菌中Red / SSO介导的重组的整体效率。我们表明DNA复制的方向和SSO编码的错配的性质是决定重组链偏向的主要因素。但是，在错配修复（MMR）有缺陷的大肠杆菌菌株中，消除了SSO编码的错配对重组链偏向的影响。这反映了一个事实，即不同的碱基错配可以通过mutS / H / L依赖的MMR途径以不同的效率进行校正。此外，我们的数据表明转录对链偏向的影响可忽略不计。这些结果首次证明，DNA复制和MMR之间的相互作用对大肠杆菌中Red / SSO介导的重组的效率和链偏向具有重大影响。

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