International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
Institute of Biochemistry and Biophysics PAS, Pawinskiego 5a, 02-106 Warsaw, Poland.
Nucleic Acids Res. 2018 Nov 2;46(19):10489-10503. doi: 10.1093/nar/gky781.
TagI belongs to the recently characterized SRA-HNH family of modification-dependent restriction endonucleases (REases) that also includes ScoA3IV (Sco5333) and TbiR51I (Tbis1). Here, we present a crystal structure of dimeric TagI, which exhibits a DNA binding site formed jointly by the nuclease domains, and separate binding sites for modified DNA bases in the two protomers. The nuclease domains have characteristic features of HNH/ββα-Me REases, and catalyze nicks or double strand breaks, with preference for /RY and RYN/RY sites, respectively. The SRA domains have the canonical fold. Their pockets for the flipped bases are spacious enough to accommodate 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC), but not glucosyl-5-hydroxymethylcytosine (g5hmC). Such preference is in agreement with the biochemical determination of the TagI modification dependence and the results of phage restriction assays. The ability of TagI to digest plasmids methylated by Dcm (C5mCWGG), M.Fnu4HI (G5mCNGC) or M.HpyCH4IV (A5mCGT) suggests that the SRA domains of the enzyme are tolerant to different sequence contexts of the modified base.
TagI 属于最近被描述的 SRA-HNH 家族的修饰依赖性限制内切酶（REases），其中还包括 ScoA3IV（Sco5333）和 TbiR51I（Tbis1）。在这里，我们展示了二聚体 TagI 的晶体结构，它表现出由核酸酶结构域共同形成的 DNA 结合位点，以及两个亚基中修饰的 DNA 碱基的单独结合位点。核酸酶结构域具有 HNH/ββα-Me REases 的特征，并且分别优先催化切口或双链断裂，分别为 /RY 和 RYN/RY 位点。SRA 结构域具有典型的折叠。它们用于翻转碱基的口袋足够宽敞，可以容纳 5-甲基胞嘧啶（5mC）或 5-羟甲基胞嘧啶（5hmC），但不能容纳葡萄糖基-5-羟甲基胞嘧啶（g5hmC）。这种偏好与 TagI 修饰依赖性的生化测定结果和噬菌体限制测定结果一致。TagI 能够消化由 Dcm（C5mCWGG）、M.Fnu4HI（G5mCNGC）或 M.HpyCH4IV（A5mCGT）甲基化的质粒，这表明该酶的 SRA 结构域能够容忍修饰碱基的不同序列背景。
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