DNA修饰依赖型HNH核酸内切酶切割活性机制及应用研究

Versatile defense systems including CRISPR-Cas are developed in bacteria to prevent the invasion of phages and mobile elements, they generally use endonucleases as weapon to destroy the alien DNA. Discrimination of incoming DNA from its own is not dependent on nuclease catalytic domain, but on the cognate DNA recognition domain, which has variable specificity in the selection of DNA target sequence, and different affinity to diverse DNA modifications. HNH nuclease domain is widely distributed in all three kingdoms of life with as small as 25 to 50 amino acids residues in length. We previously identified three functional DNA modification-dependent HNH nucleases in Streptomyces, namely, ScoMcrA, SprMcrA and Sco5333. The first two proteins displayed low DNA cleavage activity in vitro, usually with 20% of DNA digested. Moreover, both of them made multiple cleavages in the vicinity of DNA modification site, in a similar way to non-stringent cleavage by Cas9-HNH. By contrast, Sco5333 only showed weak single-strand nicking activity, another modification-dependent HNH endonuclease EcoMcrA didn't show any DNA cleavage activity at all. Based on the comparative analysis of above four endonuclease homologs, this project aims to improve their in vitro DNA cleavage activity and specificity through systematic mutations, truncations and combination with other well-studied catalytic or recognition domains. The engineered proteins with enhanced efficiency and specificity will not only help to uncover underlined new mechanism in enzyme catalysis, but also be of potential tools for epigenetic study.

微生物使用多样化的防御系统来抵挡噬菌体和可移动元件的入侵，绝大部分使用核酸内切酶直接降解外来DNA，核酸酶切割结构域无法区分自身和外来DNA，依靠偶联的DNA识别结构域实现靶DNA序列的选择性，决定核酸酶DNA修饰的敏感性或依赖性。HNH核酸酶是广泛存在于三界生物中小而保守的蛋白家族。本项目前期从链霉菌中鉴定了三个体内高活性体外低活性的DNA修饰依赖型HNH核酸酶ScoMcrA，SprMcrA和Sco5333，类似于Cas9-HNH，前两个蛋白体外DNA切割位点不专一，Sco5333仅有DNA单链切割活性；而大肠杆菌中修饰依赖型HNH核酸酶EcoMcrA至今也无法实现其体外DNA切割活性。本项目将对这四个同源HNH蛋白进行比较研究，通过系统突变和杂合，来提高它们的DNA切割效率和专一性，阐明金属离子，识别结构域以及HNH基序中潜在的关键氨基酸残基调控DNA切割效率和专一性的机理。

本研究中的SBD是靶向DNA硫修饰的一种识别结构域，自然界中SBD与HNH核酸酶结构域偶联构成硫修饰依赖型的限制酶。我们筛选和合成了77个同源SBD-HNH核酸酶，测试了它们体内和体外的切割活性；表达和纯化了所有的SBD蛋白，对其进行了硫修饰DNA序列特异性，结合力，单双链结合特性和机理的研究；阐明了SBD-HNH体外切割硫修饰DNA效率低下的机制：酶结合在硫修饰DNA产物上，导致酶分子不能周转；没有发现添加RecA，polymerase，helicase可以将SBD-HNH切割硫修饰DNA的活性提高10%。我们将耐热的SBD和耐热的BclI进行偶联形成人工杂合核酸酶，应用于核酸恒温检测,大大提升了检测的时间和灵敏度，将SBDmmo与RNA碱基编辑器进行了偶联，提高了RNA单碱基编辑的效率，并申请了专利。发表第论文7篇，申请专利2项，培养3名博士，出站1名博士后。

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