Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function
Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function
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DOI：
10.1093/nar/gkaa117
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发表时间：
2020-02
影响因子：
14.9
通讯作者：
Crystal M Vander Zanden-Crystal-M-Vander Zanden-2127635681;R. S. Czarny;Ethan N Ho;A. Robertson;P. S. Ho
中科院分区：
文献类型：
--
作者：
Crystal M Vander Zanden-Crystal-M-Vander Zanden-2127635681;R. S. Czarny;Ethan N Ho;A. Robertson;P. S. Ho
摘要修饰的DNA碱基在功能上区分了生命的分类形式--5-甲基胞嘧啶将原核生物与真核生物区分开来，5-羟甲基胞嘧啶（5 hmC）将无脊椎动物与脊椎动物区分开来。我们在这里证明，小鼠核酸内切酶G（mEndoG）显示5 hmC和霍利迪交界处的特异性。该酶对接头的亲和力高于双链DNA（>50倍）。5 hmC修饰改变了切割位点的位置，并增加了修饰与未修饰连接处的DNA切割速率。mEndoG的晶体结构显示，半胱氨酸（Cys 69）通过巯基-羟基氢键定位以识别5 hmC。尽管这种Cys从蠕虫到哺乳动物都是保守的，但脊椎动物中相对于无脊椎动物序列的两个氨基酸缺失使α-螺旋解旋，将Cys 69的巯基置于mEndoG活性位点。具有丙氨酸或丝氨酸的Cys 69的突变显示5 hmC特异性，其反映侧链的氢键结合潜力（C-H < S-H < O-H）。在mEndoG结构中鉴定的第二正交DNA结合位点容纳接头的第二臂。因此，mEndoG对5 hmC和接头的特异性来源于将脊椎动物与无脊椎动物酶区分开的结构适应，从而支持5 hmC在重组过程中的作用。
Abstract Modified DNA bases functionally distinguish the taxonomic forms of life—5-methylcytosine separates prokaryotes from eukaryotes and 5-hydroxymethylcytosine (5hmC) invertebrates from vertebrates. We demonstrate here that mouse endonuclease G (mEndoG) shows specificity for both 5hmC and Holliday junctions. The enzyme has higher affinity (>50-fold) for junctions over duplex DNAs. A 5hmC-modification shifts the position of the cut site and increases the rate of DNA cleavage in modified versus unmodified junctions. The crystal structure of mEndoG shows that a cysteine (Cys69) is positioned to recognize 5hmC through a thiol-hydroxyl hydrogen bond. Although this Cys is conserved from worms to mammals, a two amino acid deletion in the vertebrate relative to the invertebrate sequence unwinds an α-helix, placing the thiol of Cys69 into the mEndoG active site. Mutations of Cys69 with alanine or serine show 5hmC-specificity that mirrors the hydrogen bonding potential of the side chain (C–H < S–H < O–H). A second orthogonal DNA binding site identified in the mEndoG structure accommodates a second arm of a junction. Thus, the specificity of mEndoG for 5hmC and junctions derives from structural adaptations that distinguish the vertebrate from the invertebrate enzyme, thereby thereby supporting a role for 5hmC in recombination processes.