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

Abstract

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 3ʹ-5ʹ exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5ʹ 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 5ʹ-3ʹ exonuclease activity of Pol I were also required, likely to generate 5ʹ phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5ʹ single-stranded overhangs is different from established recombination pathways that all generate 3ʹ single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.