4614 Nucleic Acids Research, 2007, Vol. 35, No. 14 Figure 5. Amino acid sequence alignments of two catalytic sites. Top amino acid sequences: top-strand catalytic site (Ct) residues of BsrDI and BtsI A subunits, C-terminal regions of BsmI/Mva1269I, and BsrI. Bottom amino acid sequences: bottom-strand catalytic site (Cb) residues of BsrDI and BtsI B subunits, N-terminal regions of BsmI/Mva1269I, and BsrI. BsrDI and BtsI are heterodimeric while BsmI, Mva1269I and BsrI are monomeric. The Genbank accession numbers for BsrDI, BtsI, BsmI and Mva1269I are DQ367879, DQ355163, AY079085 and DQ074451, respectively. in Ct, the putative catalytic site for top-strand nicking in the A subunit were changed to Ala by site-directed mutagenesis. BsrDI A subunit mutants [A]-D67A, [A]-E82A/K84A, [A]-K86A, as well as the wt A subunit do not show DNA nicking or restriction activity. Surprisingly, when the catalytic-deﬁcient B subunit mutants were combined with wt A subunit, DNA nicking activity was reconstituted. To determine the nicking strand speciﬁcity of the newly restored nicking activity, the nicked circular DNA was gel-puriﬁed and subjected to run-oﬀ sequencing. Figure 6B shows that [wt]-A/[B]-D83A or [wt-A]/[B]-E99A are top-strand nicking enzymes with substrate speciﬁcity of GCAATGNN#. [wt-A]/[B]- Q112A/R113A gave rise to the same result (data not shown). Combining BsrDI wt B subunit with A subunit mutants [A]-D67A or [A]-E82A/K84A resulted in bottom- strand nicking activity only, further conﬁrming that [A]-Asp67, [A]-Glu82 or [A]-Lys84 are involved in top- strand catalysis. Mixing BsrDI wt B subunit with [A]-K86A produced BsrDI restriction activity, indicating [A]-Lys86 is not a catalytic residue although this Lys residue is conserved in four of the ﬁve endonucleases (see amino acid alignment in Figure 5). Alanine scanning was also applied to BtsI A/B subunits. The Asp, Glu, Gln and Arg in Cb, the putative catalytic site for bottom-strand nicking in BtsI B subunit (Nb.BtsI) were changed to Ala by site-directed mutagenesis. The mutant cell extracts were assayed for DNA nicking or restriction activity. Figure 7A shows that [B]-D58A, [B]-E72A and the double mutant [B]-Q85A/R86A lost DNA nicking activity as expected. Similarly, the Asp, Glu and Lys in Ct, the putative catalytic site for top-strand nicking in the A subunit were substituted by Ala. BtsI A subunit mutants [A]-D65A, [A]-E78A/K80A, [A]-K82A as well as the wt A subunit do not show nicking or restriction activity. However, when the catalytic-deﬁcient B subunit mutants were combined with wt A subunit, DNA nicking activity was restored. Run-oﬀ sequencing of the gel-puriﬁed nicked-circular DNA products showed that [wt]-A/[B]-D58A or [wt-A]/[B]-E72A are top-strand nick- ing enzymes with substrate speciﬁcity of GCAGTGNN# (Figure 7B). [wt-A]/[B]-Q85A/R86A is also a top-strand nicking enzyme (data not shown). Combining BtsI wt B subunit with A subunit mutants [A]-D65A or [A]-E78A/ K80A produced only bottom-strand nicking activity, further conﬁrming that [A]-Asp65, [A]-Glu78 or [A]-Lys80 are involved in top-strand hydrolysis. Similar to [A]-Lys86 of BsrDI, [A]-Lys82 of BtsI is not a critical residue for top-strand cleavage because mixing BtsI wt B subunit with [A]-K82A generated BtsI restriction activity (Figure 7A). DISCUSSION We describe here cloning and expression of BsrDI and BtsI R-M systems, and the discovery of two natural nicking enzymes Nb.BsrDI and Nb.BtsI. In addition, top- strand nicking variants, Nt.BsrDI and Nt.BtsI, were created by mixing catalytic-deﬁcient B subunits with the respective wt A subunit. Two independent catalytic sites were implicated in BsrDI/BtsI large and small subunits for top-strand and bottom-strand cleavage.