Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI.
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-X(n)-EXK and a second non-canonical PD-X(n)-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.
Xu
Shuang-Yong
SY
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA. xus@neb.com
Zhu
Zhenyu
Z
Zhang
Penghua
P
Chan
Siu-Hong
SH
Samuelson
James C
JC
Xiao
Jianping
J
Ingalls
Debra
D
Wilson
Geoffrey G
GG
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D019281:Dimerization; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D021122:Protein Subunits; D015254:DNA Modification Methylases; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific