Nucleic Acids Research, 2007, Vol. 35, No. 14 4613 Figure 3. Characterization of BtsI A/B subunits. (A) Lane 1, protein size marker; puriﬁed BtsI B subunit (lane 2) and A subunit (lane 3). The predicted molecular masses of A and B subunits are 18.6 and 37.6 kDa, respectively. (B) DNA nicking activity of the BtsI large subunit (Nb.BtsI). Lane 1, 1-kb marker; lane 2, linearized DNA by XhoI; lane 3, fX174 DNA; lanes 4–12, fX174 DNA digested with BtsI B subunit. (C) Recombinant BtsI double-strand cleavage activity reconstituted by combining puriﬁed BtsI A and B subunits. Lane 1, DNA marker; lane 2, fX174 DNA digested with BtsI B subunit; lanes 3–7, fX174 digested with BtsI A and B subunits; lane 8, fX174 treated with A subunit; lane 9, fX174 DNA; lane 10, Nt.SapI nicked fX174; lane 11, linearized fX174 by XhoI. Figure 4. Recognition sequences and cleavage sites of REases that share aa sequence similarity with BsrDI and BtsI. The recognition sequences are aligned with respect to the invariant RNTG/CANY sequence (shown in bold) and the consistent 2-base 30-overhang. Positions of cleavage are indicated by arrows; unspeciﬁed nucleotides are shown as N. sequence: PD-Xn-E-Xn-QR, whereas the second catalytic site (Ct) in each is canonical: PD-Xn-EXK (Figure 5) (16). One site (Cb, catalytic site for bottom strand) is located in the large subunit of BsrDI and BtsI, or near the N- terminus of the monomeric proteins (BsmI/Mva1269I, BsrI) and catalyzes the cleavage of the bottom strand of the recognition sequence. The other site (Ct, catalytic site for top strand), is located near the C-terminus in the monomeric enzymes, and within the small subunit in the heterodimeric ones, and probably catalyzes hydrolysis of the top-strand (see mutagenesis results section). The region between the catalytic sites is variable and perhaps comprises the DNA target-recognition domain (TRD) of each protein. To a ﬁrst approximation, the domain/subunit architecture of the heterodimers BsrDI and BtsI can be described as Cb TRD:Ct, where ‘ ’ represents covalent polypeptide linkage and ‘:’ represents non-covalent association. Likewise, the architecture of monomers BsmI/Mva1269I, and BsrI can be described as Cb TRD Ct. As described above, the isolated large subunits of BsrDI and BtsI (composition Cb TRD), in the absence of their Ct-containing small subunit, nick only the bottom strand of the target sequence. Site-directed mutagenesis of two catalytic sites in A/B subunits of BsrDI and BtsI endonucleases The Asp, Glu, Gln and Arg in Cb the putative catalytic site for bottom-strand nicking in BsrDI B subunit (Nb.BsrDI) were mutated to Ala. The mutant cell extracts were assayed for DNA nicking or ds cleavage activity. Figure 6A shows that [B]-D83A, [B]-E99A and the double mutant [B]-Q112A/R113A lack DNA nicking activity as anticipated. Similarly, the Asp, Glu and Lys