Exonuclease III (XthA) enforces in vivo DNA cloning of Escherichia coli to create cohesive ends

Abstract

Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15-40 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 recombinase, but is dependent on XthA, a 3 to 5 exonuclease. Here, in vivo cloning of E. coli by XthA is referred to as iVEC (in vivo E. coli cloning). Next, we show that the 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.\n\nImportanceCloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, in vitro recombination of DNA fragments effectively joins multiple DNA fragments in place of the canonical method. Interestingly, E. coli can take up linear double-stranded vectors, insert DNA fragments and assemble them in vivo. The in vivo cloning have realized a high level of usability comparable to that by in vitro recombination reaction, since now it is only necessary to introduce PCR products into E. coli for the in vivo cloning. However, the mechanism of in vivo cloning is highly controversial. Here we clarified the fundamental mechanism underlying in vivo cloning of E. coli and also constructed an E. coli strain that was optimized for in vivo cloning.