Endonuclease activity of RecJ from extremely alkaliphilic Bacillus alcalophilus

ABSTRACT At present, all documented RecJs are exonucleases, degrading single-stranded nucleic acids. Here, we report a novel RecJ, from the extremely alkaliphilic bacterium Bacillus alcalophilus (BaRecJ), which possesses endonuclease activity and can cleave supercoiled DNA. BaRecJ contains the typical DHH and DHHA1 domains, which are conserved in all RecJs, and a functionally unknown PIWI-like domain at the C-terminus. The endonuclease activity originates from the C-terminal domain of BaRecJ which contains PIWI-like domain, and the exonuclease activity from the DHH and DHHA1 domains. Mutational analysis reveals that several important residues affect the endonuclease activity of BaRecJ. Moreover, BaRecJ cleaves specific target sequences at moderate temperature when directed by a phosphorothioate-modified single-stranded DNA (S-modified ssDNA) guide. These findings suggest that BaRecJ is substantially different from any reported RecJs and has the potential to be developed as a new gene editing tool.