Exonuclease III (XthA) Enforces DNA Cloning of Escherichia coli To Create Cohesive Ends.

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 accomplishes such cloning is still unknown. In this study, we provide evidence that the cloning of is independent of both RecA and RecET recombinases but is dependent on XthA, a 3' to 5' exonuclease. Here, cloning of by XthA is referred to as 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 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 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. Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, an recombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly, potentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for this cloning have realized a high level of usability, comparable to that by recombination reactions. However, the mechanism of cloning is highly controversial. Here, we clarified the fundamental mechanism underlying cloning and also constructed a strain that was optimized for cloning. Additionally, we streamlined the procedure of cloning by using a single microcentrifuge tube.