(CCTNAGC 5/ 2), BbvCI (CCTCAGC 5/ 2) and BspD6I (GAGTC 4/?), but they are relatively rare. Bpu10I and BbvCI each comprise two similar but non-identical subunits. Both subunits contain a single catalytic site, and both are required for ds cleavage activity. Mutating either catalytic site produces ‘half-active’ heterodimers that cleave one strand of the recognition sequence only; that is, ‘nick’ the DNA instead of cleaving it (Janulaitis,A. et al., 2005, US patent 6867028) (4). Another example of a heterodimeric REase is BseYI (CCCAGC 5/ 1) con- sisting of two non-identical subunits that can be expressed separately in Escherichia coli. BseYI restriction activity can be reconstituted by mixing the two subunits (Nkenfou,C., Morgan,R. and S.-Y.X., unpublished data). An alternative mode of homodimerization is exempli- ﬁed by FokI (GGATG 9/13), the archetypal Type IIS REase. FokI binds to DNA as a monomer but cleaves cooperatively with another monomer (5–8) because its recognition sequence is asymmetric, it binds in one orientation only and cleaves asymmetrically, to one particular side of the recognition sequence. FokI com- prises an N-terminal DNA-binding domain and a discrete C-terminal catalytic domain with a single catalytic site. Cleavage is accomplished by transient homodimerization between the catalytic domains of neighboring DNA- bound monomers (6). At low enzyme concentrations, solitary DNA-bound FokI molecules neither nick nor cleave the DNA. The truncated catalytic domain of FokI can dimerize transiently with such solitary molecules in vitro to stimulate ds cleavage, although this domain on its own is inactive (7). Cooperativity implies that the extent of cleavage increases sigmoidally with increasing enzyme concentration rather than monotonically, and that DNA molecules with a single recognition sequence are cleaved poorly compared with those containing multiple recognition sequences. These two mechanistic character- istics have been shown experimentally for FokI, suggest- ing that transient dimerization is a strategy commonly adapted by these REases. Steric considerations suggest that this form of dimerization is feasible only for enzymes in which the amino acid residues responsible for catalysis and DNA recognition are distinct and well separated; for enzymes that cleave well outside of their recognition sequences. A number of REases cleave within asymmetric sequences or very close to the recognition sequences. These enzymes do not appear to engage in transient homodimerization. Instead these enzymes use two diﬀer- ent catalytic sites for cleavage (9–11). We describe here four enzymes of this kind from thermophilic isolates of Bacillus: BsrDI, BtsI, BsmI and BsrI. The four appear to be distantly related: they exhibit limited amino acid sequence similarities; they recognize similar asymmetric ﬁve- and six-base sequences in ds DNA; and they all cleave very close to their recognition sequences and produce fragments with 2-base, 30-overhangs. Of interest is our ﬁnding that these enzymes possess two oligomeric organizations: BsrDI and BtsI endonucleases are heterodimers; BsmI, Mva1269I (a BsmI isoschizomer) and BsrI are monomers that might have evolved from heterodimeric ancestors by gene fusion. Of interest, too, is Nucleic Acids Research, 2007, Vol. 35, No. 14 4609 our ﬁnding that BsrDI and BtsI act as sequence-speciﬁc DNA nicking enzymes, or DNA cleaving enzymes, depending on their subunit compositions. Each enzyme comprises one large subunit and one small subunit. The large subunits, reminiscent of Type IIS enzymes, contain one catalytic site and all the elements for DNA recogni- tion. On their own, they bind to DNA and speciﬁcally nick the bottom strand of their recognition sequence. We found that the small subunit of BsrDI and BtsI contain a catalytic site for top-strand cleavage. The small subunit is inactive on its own but in combination with the large subunit the heterodimer becomes a REase that cleaves both strands. We refer to the large subunits of BsrDI (Nb.BsrDI) and BtsI (Nb.BtsI)—natural, as opposed to mutationally engineered, DNA nicking enzymes—as ‘hemidimers’. By combining the catalytic-deﬁcient large subunit of BsrDI and BtsI with its small subunit partner, we have successfully created top-strand-speciﬁc nicking enzymes Nt.BsrDI and Nt.BtsI. MATERIALS AND METHODS Strains and plasmid vectors The BsrDI- and BtsI-producing strains were from NEB’s strain collection. Escherichia coli strains ER2502, ER2566, ER2683 (KmR, lacIq) and ER2848 (TcR, lacIq) were provided by M. Sibley and E. Raleigh (NEB). Plasmids R pRRS (Ap , a pUC19 derivative), pLG339 [pSC101 origin, KmR, TcR (12)], pAGR3 [ApR, a pBR322 R derivative with Ptac promoter, (13)] and pAII17 (Ap , a pBR322 derivative with T7 promoter, Bill Jack, NEB) were used as the expression vectors. Genomic DNA library construction and methylase selection Bacillus stearothermophilus D70 genomic DNA was digested with REases and ligated into pRRS with compatible ends. Plasmid library was challenged with BsrDI and the digested DNA was used to transform ER2683 competent cells. Plasmids from cultures of individual transformants were screened for resistance to BsrDI digestion. The inserts in the methylase-positive clone and subclones were sequenced using a kit from ABI. A DNA fragment carrying bsrDIM1M2 genes were ampliﬁed in PCR, digested with BamHI and SphI and ligated to pLG339 with compatible ends. Inverse PCR amplification, sequencing and expression of the REase genes Inverse PCR was carried out to amplify the DNA adjacent to the M genes. Inverse PCR products were puriﬁed and directly sequenced by primer walking. The pre-modiﬁed host ER2848 (pLG-bsrDIM1M2) was used for endonu- clease expression. Cells were cultured to late-log phase at 378C and IPTG induction (0.5 mM) was carried out for 3 h. Cell extracts were prepared by sonication. In order to improve the expression level, bsrDIA and bsrDIB genes were ampliﬁed in PCR and independently cloned into the expression vector pAII17. Recombinant BsrDI activity was reconstituted by mixing cell extracts containing BsrDI