4608–4618 Nucleic Acids Research, 2007, Vol. 35, No. 14 doi:10.1093/nar/gkm481 Published online 22 June 2007 Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI Shuang-Yong Xu*, Zhenyu Zhu, Penghua Zhang, Siu-Hong Chan, James C. Samuelson, Jianping Xiao, Debra Ingalls and Geoffrey G. Wilson New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA Received May 10, 2007; Revised May 30, 2007; Accepted June 1, 2007 ABSTRACT BsrDI and BtsI restriction endonucleases recognize and cleave double-strand DNA at the sequences GCAATG (2/0) and GCAGTG (2/0), respectively. We have purified and partially characterized these two enzymes, and analyzed the genes that encode them. BsrDI and BtsI are unusual in two respects: each cleaves DNA as a heterodimer of one large subunit (B subunit) and one small subunit (A subunit); and, in the absence of their small subunits, the large subunits behave as sequence-specific DNA nicking enzymes and only nick the bottom strand of the sequences at these respective positions: GCAATG ( /0) and GCAGTG ( /0). We refer to the single subunit, the bottom-strand nicking forms as ‘hemi- dimers’. Amino acid sequence comparisons reveal that BsrDI and BtsI belong to a family of restriction enzymes that possess two catalytic sites: a canonical PD-Xn-EXK and a second non-canonical PD-Xn-E-X12-QR. Interestingly, the other family members, which include BsrI (ACTGG 1/ 1) and BsmI/Mva1269I (GAATGC 1/ 1) are single polypep- tide chains, i.e. monomers, rather than heterodi- mers. In BsrDI and BtsI, the two catalytic sites are found in two separate subunits. Site-directed mutagenesis confirmed that the canonical catalytic site located at the N-terminus of the large subunit is responsible for the bottom-strand cleavage, whereas the non-canonical catalytic site located in the small subunit is responsible for hydrolysis of the top strand. Top-strand specific nicking variants, Nt.BsrDI and Nt.BtsI, were successfully engineered by combining the catalytic-deficient B subunit with wild-type A subunit. INTRODUCTION Many restriction endonucleases (REases) bind to their target sequences and hydrolyze both strands of the duplex DNA simultaneously, cleaving the molecule in a single catalytic cycle. In general, hydrolysis reactions for the two strands proceed simultaneously, thus requiring the presence of two catalytic sites. Evolutionarily, the simplest way for a protein with one catalytic site to acquire the second one is to dimerize with one another—as a homodimer—and this is the strategy that many REases appear to have adopted. As a result, and in spite of their considerable diversity, REases usually accomplish double- stranded (ds) cleavage of DNA using two identical catalytic sites. Not all do so, however. Some enzymes use a single catalytic site to cleave the two strands of DNA sequentially in a single binding event (e.g. BﬁI) or in consecutive binding events (e.g. MspI and HinP1I) (1–3). Here we describe a family of unusual REases from the bacterial genus Bacillus that accomplish ds cleavage using two diﬀerent catalytic sites. Some members of this family are heterodimers with the two catalytic sites located in separate polypeptide chains. Other members are monomeric with the two catalytic sites located in a single polypeptide chain. One common mode of homodimerization is exhibited by the familiar ‘Type IIP’ REases such as EcoRI (GAATTC 5/ 1) and BglI (GCCNNNNNGGC 4/ 7) that recognize palindromic sequences. These enzymes bind to ds DNA as homodimers and recognize their target sequences by a concerted process in which both subunits participate in equal and opposite measure: one subunit recognizes certain features of the sequence in one orientation, while the other subunit recognizes the same features but in the other orientation. In consequence, the overall recognition sequence is symmetric, and so are the cleavage positions within the sequence. Heterodimeric REases have been discovered in the forms of Bpu10I *To whom correspondence should be addressed. Tel: +1 978 380 7287; Fax: +1 978 921 1350; Email: xus@neb.com 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.