Exonuclease III (XthA) Enforces In Vivo DNA Cloning of Escherichia coli To Create Cohesive Ends
Exonuclease III (XthA) Enforces In Vivo DNA Cloning of Escherichia coli To Create Cohesive Ends
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DOI：
10.1128/jb.00660-18
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发表时间：
2019-03-01
影响因子：
3.2
通讯作者：
Niki, Hironori
中科院分区：
文献类型：
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作者：
Nozaki, Shingo;Niki, Hironori
大肠杆菌具有组装两端同源重叠序列为15 ~ 40bp的DNA片段的能力。已经有几个改进的方案被报道来改进这种简单而有用的DNA克隆技术。然而，大肠杆菌完成这种克隆的分子机制尚不清楚。在这项研究中，我们提供了证据，证明大肠杆菌的体内克隆不依赖于RecA和RecET重组酶，但依赖于XthA，一种3‘到5’的外切酶。在这里，通过XthA在体内克隆大肠杆菌被称为体内大肠杆菌克隆（iVEC）。我们还发现DNA聚合酶I (PolA) c端结构域的缺失降低了iVEC的活性。综上所述，这些结果提示了iVEC的以下机制。首先，XthA切除引入大肠杆菌细胞的线性DNA片段的3‘端，从而暴露单链5’悬垂。然后，互补的单链DNA末端相互杂交，缺口由DNA聚合酶填补。在分子水平上阐明iVEC机制将进一步推动体内DNA克隆技术的发展。我们已经成功地展示了多片段组装，最多七个片段，结合使用改良的宿主菌株进行iVEC的轻松转化过程。将DNA片段克隆成载体是重组DNA技术的基础技术之一。最近，一种用于DNA克隆的体外重组系统被证明可以同时连接多个DNA片段。有趣的是，大肠杆菌可能会将多个线性DNA片段组装到细胞中。这种体内克隆的改进方案已经实现了高水平的可用性，可与体外重组反应相媲美。然而，体内克隆的机制存在很大争议。在此，我们明确了体内克隆的基本机制，并构建了一个优化的菌株进行体内克隆。此外，我们通过使用单个微离心管简化了体内克隆的程序。
Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA cloning technology. However, the molecular mechanism by which E. coli accomplishes such cloning is still unknown. In this study, we provide evidence that the in vivo cloning of E. coli is independent of both RecA and RecET recombinases but is dependent on XthA, a 3' to 5' exonuclease. Here, in vivo cloning of E. coli by XthA is referred to as in vivo E. coli cloning (iVEC). We also show that iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3' ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5' overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC.IMPORTANCE Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, an in vitro recombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly, E. coli potentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for this in vivo cloning have realized a high level of usability, comparable to that by in vitro recombination reactions. However, the mechanism of in vivo cloning is highly controversial. Here, we clarified the fundamental mechanism underlying in vivo cloning and also constructed a strain that was optimized for in vivo cloning. Additionally, we streamlined the procedure of in vivo cloning by using a single microcentrifuge tube.