The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study

The recombination of short homologous ends in Escherichia coli has been known for 30 years, and it is often used for both site-directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3MODIFIER LETTER PRIME-5MODIFIER LETTER PRIME exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5MODIFIER LETTER PRIME single-strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5MODIFIER LETTER PRIME-3MODIFIER LETTER PRIME exonuclease activity of Pol I were also required, likely to generate 5MODIFIER LETTER PRIME phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5MODIFIER LETTER PRIME single-stranded overhangs is different from established recombination pathways that all generate 3MODIFIER LETTER PRIME single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.

Automated summary

The recombination of short homologous ends in Escherichia coli involves the digestion of linear dsDNA by exonucleases ExoIII and ExoX, followed by annealing and extension of single-stranded overhangs, ultimately joined by Ligase A. This model, requiring Pol I polymerase activity, differs from established recombination pathways and is likely common in bacteria.