Some class-IIS restriction endonucleases can cleave across a three-way DNA junction.

Three-way DNA junctions were synthesized with class-IIS restriction endonuclease (ENase) recognition sites and potential cleavage sites located on separate arms. Cleavage was investigated with junctions labeled in each of the three strands. BpmI and BsaI failed to cleave either strand of either arm, whereas BsmAI cleaved one strand. FokI and HphI cleaved both strands of both arms at the expected nucleotide positions. FokI cleavage was independent of the spacing between the recognition site and the junction. This new activity of class-IIS ENases may be useful for investigating branched DNA structures.