Only a few naturally occurring DNA nicking endonu- cleases (NEases) have been described from bacterial and viral sources. Two strand-speciﬁc and sequence-speciﬁc DNA NEases were found in the lysates of Chlorella viruses NYs-1 and NY2A (17,18). These are Nt.CviPII (previously named CviNYSI nickase, #CCD, D = A, G or T) and Nt.CviQII (previously named CviNY2A nickase, R#AG, R = A or G). The CviPII and CviQII N-M systems have been cloned and expressed in heterologous hosts (19,20). Nt.BstSEI and isoschizomer Nt.BstNBI (GAGTCN4#) were found in two strains of B. stearothermophilus (21,22). Nt.BspD6I, an isoschizomer of Nt.BstNBI with identical amino acid sequence has also been cloned (23). Nt.BstNBI was thought to be a nicking enzyme that had lost the ability to dimerize because the enzyme puriﬁed from the native strain was a NEase. Recently, however, the gene encoding its small subunit partner has been identiﬁed adjacent to the gene coding for the nicking subunit (Heiter,D. and G.G.W., unpublished data). Presumably, the small subunit of the BstNBI endonuclease was dissociated from the large subunit and was separated from the large subunit during puriﬁcation, resulting in a natural top-strand nicking enzyme. Similarly, a gene encoding a small subunit of 186 aa residues had been found downstream of the Nt.BspD6I gene. BspD6I restriction activity was reconstituted by mixing Nt.BspD6I with the puriﬁed small subunit (24). Based on the mutagenesis results of BsrDI and BtsI, it should be possible to engineer bottom-strand nicking variants from BstNBI/BstSEI/BspD6I REases by mixing the catalytic-deﬁcient variants of Nt.BstNBI/Nt.BstSEI/ Nt.BspD6I with their wt small subunit, assuming that two independent catalytic sites reside in two subunits. Strand-speciﬁc NEases have been used in DNA strand displacement ampliﬁcation (SDA), EXPAR DNA ampli- ﬁcation (25), in DNA fragment assembly and cloning, and in preparation of nicked-duplex or gapped DNA for studying DNA repair and DNA base stacking (26,27). Recently, a nicking endonuclease-mediated DNA ampli- ﬁcation (NEMDA) using Nt.CviPII and Bst DNA polymerase in the absence of input primers has also been described (19). Nt.CviPII-digested genomic DNA into small partial duplex DNA that serves as the templates and primers for primer extension. Recently, nicking enzymes have been used to nick and label DNA for single-molecule barcoding system (28,29). The relatively small number of NEases identiﬁed in nature has prompted eﬀorts to engineer NEases from existing Type IIA/Type IIS/Type IIT REases by domain swapping or mutagenesis (Type IIA, REases that cleave asymmetric sites; Type IIT, heterodimeric REases). Strand-speciﬁc nicking variants have been created from AlwI, Bpu10I, BbvCI, BsaI, BsmBI, BsmAI, BsmI, BspQI, MlyI, Mva1269I and SapI (REBASE) (30). For Mva1269I, mutating the critical Asp, Glu, and Lys residues of Ct produced enzymes that nicked the bottom strand of the recognition sequence. Mutating Cb of Mva1269I produced enzymes that nicked the top strand speciﬁcally, but at very low eﬃciency unless the bottom strand was already nicked (15). The catalytic sites of Mva1269I appear to act sequentially, with Cb cleaving the Nucleic Acids Research, 2007, Vol. 35, No. 14 4617 bottom strand ﬁrst, and in so-doing creating the substrate upon which Ct can act to cleave the top strand (15). Comparable results were observed with BsmI: bottom- strand nicking derivatives (Nb.BsmI, E546V) have been generated when the Glu residue of Ct was substituted for Val or Ala (Z.Z., Meixsell,T. and S.-Y.X., unpublished data). Mutating the Arg residue of the PD-Xn-E-Xn-QR motif in Cb produces a variant R123D with low nicking activity. Although the native BsrDI and BtsI prefer to nick the bottom strand ﬁrst and then break the top strand in restriction of ds DNA (data not shown), the isolation of Nt.BsrDI and Nt.BtsI implies that there is no stringent requirement for nicking the bottom strand ﬁrst, i.e. top-strand hydrolysis can take place without a pre- nicked bottom strand. Possible DNA cleavage mechanism There are a few other nucleases whose catalytic sites are held on the same polypeptide chain, on separate chains or formed by dimerization. For example, homing endo- nuclease PI-SceI has two catalytic sites within one polypeptide chain (one for cleavage of each strand) (31). The Tn7 transposase consists of two diﬀerent subunits, TnsA and B, each of which cleaves a separate strand (32). In contrast, some recombinases (Tn10 and Tn5) use a single active site to cleave both strands. BﬁI requires homodimerization to form the catalytic site located on the dimer interface. A homodimer of BﬁI cleaves the bottom strand ﬁrst, generating a charged group that helps the enzyme attack the top strand (1). Previously we have generated top-strand or bottom- strand speciﬁc nicking variants from BsaI, BsmBI, BsmAI, SapI (10,11) and BspQI (P.Z. and S.-Y.X., unpublished data). Based on these results and previous biochemical studies, it was proposed that some Type IIS REases operate as monomers and carry two separate catalytic sites for top and bottom strand scission (9,10). Here we provide experimental evidence that BsrDI and BtsI possess separate catalytic sites for top-strand and bottom-strand nicking. This group of Type IIS enzymes cleaves DNA close to the recognition sequence (N0–5), suggesting a mechanism that diﬀers from the well-studied FokI where the reach is N9–13. FokI consists of a DNA- binding domain and a catalytic domain within the same polypeptide. FokI binds to a cognate DNA site as a monomer and then forms a dimer transiently with a second molecule in the presence of divalent cation. DNA cleavage takes place at the deﬁned position downstream of the recognition site by the non-speciﬁc nuclease domain (5,8). In FokI, dimerization activates the ds cleavage domain (6,7). In BsrDI and BtsI, however, dimerization is required for top strand cleavage only. ACKNOWLEDGEMENTS We thank Richard Roberts for critical comments; Iain Murray for discussion, Laurie Mazzola, Beth Ann Catin and Barton Slatko for run-oﬀ sequencing; Organic Synthesis Division of NEB for the synthesis of oligo- nucleotides; Donald Comb for his support and