Structural basis for the substrate selectivity of PvuRts1I, a 5-hydroxymethylcytosine DNA restriction endonuclease.
Structural basis for the substrate selectivity of PvuRts1I, a 5-hydroxymethylcytosine DNA restriction endonuclease.
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5-羟甲基胞嘧啶 DNA 限制性内切酶 PvuRts1I 底物选择性的结构基础
DOI：
10.1107/s139900471401606x
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发表时间：
2014-09
期刊：
影响因子：
--
通讯作者：
Zang J
中科院分区：
文献类型：
--
作者：
Shao C;Wang C;Zang J
PvuRts 1 I的晶体结构进行了测定，并确定了5-羟甲基胞嘧啶结合口袋中的SRA样结构域。基于PvuRts 1 I的晶体结构，酶变体被工程化以帮助羟甲基化组作图。5-羟甲基化是胞嘧啶的一种奇怪的修饰，几十年前就被发现了，但它在真核生物中的功能作用仍有待阐明。5-羟甲基胞嘧啶是一种表观遗传标记，对多种生物过程至关重要。在某些疾病如癌症、亨廷顿氏病和阿尔茨海默氏病的情况下，该谱发生改变。使用DNA修饰依赖性限制性内切酶AbaSI与测序（Aba-seq）偶联，可以在单个碱基的分辨率下破译羟甲基化组。该方法基于AbaSI的酶性质，AbaSI是核酸内切酶PvuRts 1 I家族的成员。PvuRts 1 I是一种修饰依赖性核酸内切酶，对5-羟甲基胞嘧啶的选择性高于5-甲基胞嘧啶和胞嘧啶。在这项研究中，PvuRts 1 I的晶体结构进行了测定，以了解和提高基板的选择性。核酸酶结构域和SRA样结构域分别位于N末端和C末端。通过与其他SRA结构域结构的比较，提出类SRA结构域作为5-hmC的识别模块。基于结构分析产生了几种酶活性仅限于5-羟甲基胞嘧啶的PvuRts 1 I突变体，这些酶变体适合于从更宽的甲基化组中分离羟甲基化组。
The crystal structure of PvuRts1I was determined and a 5-hydroxymethylcytosine-binding pocket was identified in the SRA-like domain. Enzyme variants were engineered to assist in hydroxymethylome mapping based on the crystal structure of PvuRts1I. 5-Hydroxymethylation is a curious modification of cytosine that was discovered some decades ago, but its functional role in eukaryotes still awaits elucidation. 5-Hydroxymethylcytosine is an epigenetic marker that is crucial for multiple biological processes. The profile is altered under certain disease conditions such as cancer, Huntington’s disease and Alzheimer’s disease. Using the DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq), the hydroxymethylome can be deciphered at the resolution of individual bases. The method is based on the enzymatic properties of AbaSI, a member of the PvuRts1I family of endonucleases. PvuRts1I is a modification-dependent endonuclease with high selectivity for 5-hydroxymethylcytosine over 5-methylcytosine and cytosine. In this study, the crystal structure of PvuRts1I was determined in order to understand and improve the substrate selectivity. A nuclease domain and an SRA-like domain are located at the N- and C-termini, respectively. Through comparison with other SRA-domain structures, the SRA-like domain was proposed to be the 5-hmC recognition module. Several mutants of PvuRts1I with enzymatic activity restricted to 5-hydroxymethylcytosine only were generated based on the structural analysis, and these enzyme variants are appropriate for separating the hydroxymethylome from the wider methylome.