DNA fragments with overlapping homologous sequences at each end are joined as recombinant DNA molecules when introduced into Escherichia coli. However, it has long been unknown how the DNA fragments are joined in the cells. In this study, we demonstrated that single-stranded overhangs generated by 3’ to 5’ exonuclease activity of Exonuclease III (XthA) contribute to hybridization between the overlapping homologous sequences within the cells. Insights into the mechanism of in vivo DNA cloning, in combination with a simple transformation method, improve the ability of E. coli as a host for DNA manipulation.

Figure:
(A) Model of the mechanism of iVEC (in vivo E. coli cloning)
(B) Work flow of “one-tube” transformation. Preparation of competent cells and introduction of DNA can be done with a single tube.