The BisI family of restriction endonucleases is unique in requiring multiple methylated or hydroxymethylated cytosine residues within a short recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of two members of the BisI family, NhoI and Eco15I_Ntd (N-terminal domain of Eco15I), in the absence of DNA and in specific complexes with tetra-methylated GCNGC target DNA. The structures show that NhoI and Eco15I_Ntd sense modified cytosine bases in the context of double-stranded DNA (dsDNA) without base flipping. In the co-crystal structures of NhoI and Eco15I_Ntd with DNA, the internal methyl groups (G5mCNGC) interact with the side chains of an (H/R)(V/I/T/M) di-amino acid motif near the C-terminus of the distal enzyme subunit and arginine residue from the proximal subunit. The external methyl groups (GCNG5mC) interact with the proximal enzyme subunit, mostly through main chain contacts. Surface plasmon resonance analysis for Eco15I_Ntd shows that the internal and external methyl binding pockets contribute about equally to sensing of cytosine methyl groups.

中文翻译：

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修饰依赖性限制性内切酶 BisI 家族的结构分析


BisI 限制性内切核酸酶家族的独特之处在于，需要短识别序列 (GCNGC) 内的多个甲基化或羟甲基化胞嘧啶残基，并且直接在该序列内而不是远处进行切割。在这里，我们报告切割所需的修饰胞嘧啶的数量可以通过盐浓度来调节。我们展示了 BisI 家族两个成员 NhoI 和 Eco15I_Ntd（Eco15I 的 N 端结构域）在不存在 DNA 的情况下以及与四甲基化 GCNGC 靶 DNA 形成特定复合物的晶体结构。结构表明，NhoI 和 Eco15I_Ntd 在双链 DNA (dsDNA) 背景下感知修饰的胞嘧啶碱基，而无需碱基翻转。在 NhoI 和 Eco15I_Ntd 与 DNA 的共晶结构中，内部甲基 (G5mCNGC) 与 C-附近的 (H/R)(V/I/T/M) 二氨基酸基序的侧链相互作用。远端酶亚基的末端和近端亚基的精氨酸残基。外部甲基 (GCNG5mC) 与近端酶亚基相互作用，主要通过主链接触。 Eco15I_Ntd 的表面等离振子共振分析表明，内部和外部甲基结合口袋对胞嘧啶甲基的感测贡献大致相同。