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Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI

Author:
Xu, Shuang-Yong, Zhu, Zhenyu, Zhang, Penghua, Chan, Siu-Hong, Samuelson, James C., Xiao, Jianping, Ingalls, Debra, Wilson, Geoffrey G.
Source:
Nucleic acids research 2007 v.35 no.14 pp. 4608-4618
ISSN:
0305-1048
Subject:
DNA, active sites, amino acid sequences, engineering, genes, hydrolysis, polypeptides, restriction endonucleases, sequence analysis, site-directed mutagenesis
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 'hemidimers'. 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 polypeptide chains, i.e. monomers, rather than heterodimers. 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.
Agid:
1964344