4612 Nucleic Acids Research, 2007, Vol. 35, No. 14 was achieved (Figure 2A, lane 2). Most striking is the nicking activity displayed by the large subunit (Figure 2C, lanes 3–9). Complete conversion of 8 nM pUC19 to open circular form can be achieved with 4 nM of the large subunit in 30 min at 658C. To determine the strand and sequence speciﬁcity, the plasmid pGPS2.1 with a single BsrDI site was treated with the large subunit. The nicked DNA product was puriﬁed and subjected to run-oﬀ sequencing. The sequencing peaks drop oﬀ at 50 GCAATG(a) 30, indicating a nick at 50#CATTGC 30. The sequence was continuous on the opposite strand, indicating that no nicks have been introduced to the top-strand template (data not shown). The gene product of ORF1 was thus named Nb.BsrDI (Nb for bottom-strand nicking) with the speciﬁcity of 50 #CATTGC 30 (# indicating the nicking site). These results indicate that in addition to residues responsible for binding speciﬁcally to the recognition sequence, all catalytic residues necessary for bottom-strand cleavage (Cb) are located in the BsrDI large subunit (B subunit). In contrast, top-strand nicking and thus ds cleavage activity requires the assembly of A and B subunits. Amino acid sequence alignment of BsrDI large subunit with the N-terminus of BsmI/Mva1269I, and BsrI revealed a non- canonical catalytic site Cb with conserved residues P/A/F D-X13–17-E-X12–QR, whereas the BsrDI small subunit contains a canonical Type II catalytic site Ct with conserved amino acid residues PD-Xn-EXK. The BsmI/ Mva1269I and BsrI REases are monomeric proteins with large molecular weights (78.1, 78.4 and 69.4 kDa, respectively) (15) (S.-Y.X., Z.Z. and G.G.W., unpublished data). Cloning and expression of BtsI R-M system The BtsI M genes were successfully cloned by the methylase selection procedure. The inserts of the M+ clones were sequenced and the assembled sequence generated a 4986-bp sequence with four potential ORFs. The predicted aa sequences of two ORFs contain conserved motifs of amino-methyltransferases and were named M1.BtsI and M2.BtsI. The two M genes were ampliﬁed separately in PCR and cloned in two steps into the plasmid vector pACYC184 to generate a pre-modiﬁed expression host ER2683 (pACYC-btsIM1M2). We sur- mised that the third ORF (ORF S, 495 bp) ﬂanked by the two M genes might encode a regulatory protein or one subunit of the BtsI REase. A partial ORF of 657 bp was also found adjacent to the btsIM2 gene. Following inverse PCR and direct sequencing, a complete ORF of 987 bp was derived (ORF L, Figure 1B). Expression of ORF S (btsIRA) and ORF L (btsIRB) Similar to BsrDI, we found that the BtsI large subunit (B subunit, gpORF L) is a strand-speciﬁc DNA nicking endonuclease. Both the large and small subunits are required for BtsI restriction activity. The four genes in the BtsI R-M system are organized in the order of btsIM1, ORF S, btsIM2 and ORF L (Figure 1B). ORF S and ORF L are oriented in the opposite direction and are separated by the btsIM2 gene. The ORF S and ORF L were independently expressed in E. coli. No speciﬁc endonu- clease activity was detected on or supercoiled fX174 DNA with cell extracts of gpORF S. However, cell extracts containing the large subunit prepared from ER2683 (pACYC-btsIM1M2, pUC19-ORF L) displayed DNA nicking activity when fX174 RF was used as the substrate (data not shown). Figure 3A shows the puriﬁed BtsI A/B subunits. The A subunit displayed aberrant migration on the protein gel. The apparent molecular mass is 27 kDa, while the predicted size is 18.6 kDa. Puriﬁed BtsI A subunit forms oligomers with three distinct species in native PAG electrophoresis (data not shown). The signiﬁcance of this self-assembly is not known. The apparent molecular mass of the B subunit on the protein gel was as predicted (38 kDa). DNA nicking activity was detected when puriﬁed BtsI B subunit was incubated with fX174 ds DNA (Figure 3B, lanes 4–12). Ds cleavage activity of BtsI was reconstituted by mixing the two puriﬁed subunits (Figure 3C, lanes 3–7). The A subunit alone did not show nicking or ds DNA cleavage activity (Figure 3C, lane 8). To determine the nicking strand and sequence speciﬁcity of the BtsI large subunit (B subunit), the nicked-circular product was puriﬁed and sequenced. The run-oﬀ sequencing results show sequencing peaks drop oﬀ after the sequence 50 GCAGTG(a) 30, indicating that BtsI large subunit (B subunit) is a bottom-strand NEase (Nb.BtsI) that speciﬁcally nicks the 50 #CACTGC 30 site. DNA sequence from the top-strand template is continuous, indicating no nicks are introduced on the top strand (data not shown). We also found that Nb.BtsI nicks a miscognate site (star site) 50 #CACTGG 30 that is one base (underlined G) diﬀerent from the cognate site when 10-fold over-digestion was carried out. BtsI endonuclease also displays strong star activity in over-digestion. However, the entire spectrum of BtsI star sites has not been fully characterized (data not shown). Amino acid sequence alignment of catalytic sites of related restriction enzymes BsrDI GCAATG (2/0) and BtsI GCAGTG (2/0) share limited amino acid sequence similarity with several other Type IIS REases that cleave DNA in the same general manner, that is, close to their recognition sequences with a 2-base 30-cohesive end. These related enzymes include BsmI/Mva1269I (GAATGC 1/-1), and BsrI (ACTGG 1/-1). Aligning the recognition sequences of these enzymes reveals that each of them cleaves DNA two bases downstream from an invariant ANTG tetranucleotide in the top strand of the recognition sequence and immedi- ately before the complementary CANT tetranucleotide in the bottom strand (Figure 4). Interestingly, while BsrDI and BtsI are heterodimeric, the other three enzymes appear to be monomeric. These latter might have arisen by gene fusion between ancestral subunits that remain separated in BsrDI and BtsI. Amino acid sequence analysis and experimental observations suggest that all of these enzymes harbor two catalytic sites. The ﬁrst, N-terminal, catalytic site (Cb) in each enzyme is somewhat non-canonical in