Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI.
Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI.
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DOI：
10.1093/nar/gkm481
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发表时间：
2007
影响因子：
14.9
通讯作者：
Wilson, Geoffrey G
中科院分区：
文献类型：
--
作者：
Xu, Shuang-Yong;Zhu, Zhenyu;Zhang, Penghua;Chan, Siu-Hong;Samuelson, James C;Xiao, Jianping;Ingalls, Debra;Wilson, Geoffrey G
BsrDI和BtsI限制性内切酶分别识别和切割GCAATG（2/0）和GCAGTG（2/0）的双链DNA。我们对这两种酶进行了纯化和部分表征，并分析了编码它们的基因。BsrDI和BtsI在两个方面是不寻常的：它们都将DNA切割成一个大亚基（B亚基）和一个小亚基（a亚基）的异源二聚体；并且，在缺少它们的小亚基的情况下，大亚基表现为序列特异性DNA刻痕酶，并且仅在序列的这些相应位置上刻痕：GCAATG（−/0）和GCAGTG（−/0）。我们将单个亚基，即底链切口形式称为“半聚体”。氨基酸序列比较表明，BsrDI和BtsI属于一个具有两个催化位点的限制性内切酶家族：一个典型的PD-Xn-EXK和一个非典型的PD-Xn-E-X12-QR。有趣的是，其他家族成员，包括BsrI （ACTGG 1/−1）和BsmI/Mva1269I （GAATGC 1/−1）是单肽链，即单体，而不是异源二聚体。在BsrDI和BtsI中，两个催化位点位于两个不同的亚基上。位点导向突变证实，位于大亚基n端的规范催化位点负责底部链的裂解，而位于小亚基的非规范催化位点负责顶部链的水解。通过将催化缺陷B亚基与野生型A亚基结合，成功构建了顶链特异性缺口变异Nt.BsrDI和Nt.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-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.