Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs.
Type IIS restriction endonuclease BtsCI (GGATG 2/0) is a neoschizomer of FokI (GGATG 9/13) and cleaves closer to the recognition sequence. Although M.BtsCI shows 62% amino acid sequence identity to M.FokI, BtsCI and FokI restriction endonucleases do not share significant amino acid sequence similarity. BtsCI belongs to a group of Type IIS restriction endonucleases, BsmI, Mva1269I and BsrI, that carry two different catalytic sites in a single polypeptide. By inactivating one of the catalytic sites through mutagenesis, we have generated nicking variants of BtsCI that specifically nick the bottom-strand or the top-strand of the target site. By treating target DNA sequentially with the appropriate combinations of FokI and BtsCI nicking variants, we are able to generate long overhangs suitable for fluorescent labeling through end-filling or other techniques based on annealing of complementary DNA sequences.
Too
Priscilla Hiu-Mei
PH
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Zhu
Zhenyu
Z
Chan
Siu-Hong
SH
Xu
Shuang-yong
SY
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015280:DNA Restriction-Modification Enzymes / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering; D016415:Sequence Alignment
D015280:DNA Restriction-Modification Enzymes; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific