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 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 whichE. coliaccomplishes such cloning is still unknown. In this study, we provide evidence that thein vivocloning ofE. coliis independent of both RecA and RecET recombinases but is dependent on XthA, a 3′ to 5′ exonuclease. Here,in vivocloning ofE. coliby XthA is referred to asin vivoE. colicloning (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 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, anin vitrorecombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly,E. colipotentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for thisin vivocloning have realized a high level of usability, comparable to that byin vitrorecombination reactions. However, the mechanism ofin vivocloning is highly controversial. Here, we clarified the fundamental mechanism underlyingin vivocloning and also constructed a strain that was optimized forin vivocloning. Additionally, we streamlined the procedure ofin vivocloning by using a single microcentrifuge tube.