Nucleic Acids Research, 2007, Vol. 35, No. 14 4611 Figure 1. Gene organization of BsrDI and BtsI R-M systems. ORF3 upstream of BsrDI R-M genes encodes a putative transposase. The ORF lengths are not drawn to scale. ORF1 = bsrDIRB, encoding BsrDI B subunit (Nb.BsrDI, large subunit); ORF2 = bsrDIRA, encoding BsrDI A subunit (small subunit). ORF S = btsIRA, encoding BtsI A subunit (small subunit); ORF L = btsIRB; encoding BtsI B subunit (Nb.BtsI, large subunit). Figure 2. Characterization of BsrDI A/B subunits. (A) Puriﬁed recombinant BsrDI A and B subunits. The calculated molecular weight of BsrDI A and B subunits are 25 and 56 kDa, respectively. Lane 1, protein size marker; lane 2, BsrDI B subunit (Nb.BsrDI); lane 3, BsrDI A subunit. (B) Reconstituted BsrDI double-strand cleavage activity by mixing puriﬁed BsrDI A and B subunits. Lane 1, 1-kb DNA marker; lane 2, pUC19; lane 3, 8 nM pUC19 treated with A subunit; lanes 4–8, 8 nM pUC19 digested with A and B subunits (linear pUC19 = 2.7 kb); sc, supercoiled DNA. (C) Nb.BsrDI DNA nicking activity on 8-nM pUC19. Lane 1, 1-kb DNA marker; lane 2, pUC19; lanes 3–9, pUC19 digested with BsrDI B subunit, enzyme concentration as indicated on the top of each lane. A subunit was incubated with 8 nM pUC19 (lane 3) there was no change in DNA mobility. However, when 0.1 nM of the large subunit (B subunit) was added to the same reaction containing A subunit, complete cleavage of pUC19 was achieved (lane 4). The data from Figure 2B demonstrate that restriction activity requires the presence of both subunits. Thus, BsrDI is a heterodimeric REase encoded by bsrDIRB (ORF1) and bsrDIRA (ORF2). We further demonstrated that BsrDI large subunit (B subunit) is a bottom-strand speciﬁc DNA nicking endonuclease. The BsrDI large subunit (B subunit) was puriﬁed by chromatography such that 495% homogeneity