Exonuclease III (XthA) enforcesin vivoDNA cloning ofEscherichia colito create cohesive ends

AbstractEscherichia colihas 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 whichE. coliaccomplishes such cloning is still unknown. In this study, we provide evidence that thein vivocloning ofE. coliis independent of both RecA and RecET recombinase, but is dependent on XthA, a 3’ to 5’ exonuclease. Here, in vivocloning ofE. coliby XthA is referred to as iVEC (in vivo E. colicloning). 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 intoE. colicells, 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 ofin vivoDNA-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.ImportanceCloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently,in vitrorecombination of DNA fragments effectively joins multiple DNA fragments in place of the canonical method. Interestingly,E. colican take up linear double-stranded vectors, insert DNA fragments and assemble themin vivo.Thein vivocloning have realized a high level of usability comparable to that byin vitrorecombination reaction, since now it is only necessary to introduce PCR products intoE. colifor thein vivocloning. However, the mechanism ofin vivocloning is highly controversial. Here we clarified the fundamental mechanism underlyingin vivocloning of E. coli and also constructed anE. colistrain that was optimized forin vivocloning.