| Title: |
Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI |
| Authors: |
Xu, Shuang-Yong; Zhu, Zhenyu; Zhang, Penghua; Chan, Siu-Hong; Samuelson, James C.; Xiao, Jianping; Ingalls, Debra; Wilson, Geoffrey G. |
| Publisher Information: |
Oxford University Press |
| Publication Year: |
2007 |
| Collection: |
HighWire Press (Stanford University) |
| Subject Terms: |
Nucleic Acid Enzymes |
| Description: |
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-X 12 -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. |
| Document Type: |
text |
| File Description: |
text/html |
| Language: |
English |
| Relation: |
http://nar.oxfordjournals.org/cgi/content/short/gkm481v1; http://dx.doi.org/10.1093/nar/gkm481 |
| DOI: |
10.1093/nar/gkm481 |
| Availability: |
http://nar.oxfordjournals.org/cgi/content/short/gkm481v1; https://doi.org/10.1093/nar/gkm481 |
| Rights: |
Copyright (C) 2007, Oxford University Press |
| Accession Number: |
edsbas.277564B6 |
| Database: |
BASE |