The nicking endonuclease N.BstNBI is closely related to Type IIs restriction endonucleases MlyI and PleI

N.BstNBI is a nicking endonuclease that recognizes the sequence GAGTC and nicks the top strand preferentially, The Type IIs restriction endonucleases Plel and Mlyl also recognize GAGTC, but cleave both DNA strands. Cloning and sequencing the genes encoding each of these three endonucleases discloses significant sequence similarities. Mutagenesis studies reveal a conserved set of catalytic residues among the three endonucleases, suggesting that they are closely related to each other. Furthermore, Plel and Mlyl contain a single active site for DNA cleavage. The results from cleavage assays show that the reactions catalyzed by Plel and Mlyl are sequential two step processes. The double-stranded DNA is first nicked on one DNA strand and then further cleaved on the second strand to form linear DNA. Gel filtration analysis shows that Mlyl dimerizes in the presence of a cognate DNA and Ca2+ whereas N.BstNBI remains a monomer, implicating dimerization as a requisite for the second strand cleavage. We suggest that N.BstNBI, Mlyl and Plel diverged from a common ancestor and propose that N.BstNBI differs from Mlyl and Plel in having an extremely limited second strand cleavage activity, resulting in a site-specific nicking endonuclease.