New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Nucleic Acids Res. 2010 Mar;38(4):1294-303. doi: 10.1093/nar/gkp1092. Epub 2009 Dec 2.
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.
II 型限制内切酶 BtsCI（GGATG 2/0）是 FokI（GGATG 9/13）的新型同裂酶，其切割位点更接近识别序列。尽管 M.BtsCI 与 M.FokI 的氨基酸序列同一性为 62%，但 BtsCI 和 FokI 限制内切酶并没有显著的氨基酸序列相似性。BtsCI 属于 II 型限制内切酶组，包括 BsmI、Mva1269I 和 BsrI，它们在单个多肽中携带两个不同的催化位点。通过突变使其中一个催化位点失活，我们生成了 BtsCI 的切口变异体，这些变异体可以特异性地切割靶位点的下链或上链。通过用适当的 FokI 和 BtsCI 切口变异体组合处理靶 DNA，我们能够通过末端填充或基于互补 DNA 序列退火的其他技术生成适合荧光标记的长突出端。
