Snm1家族蛋白参与DNA损伤修复及其活性转化机制的研究

Deinococcus radiodurans is a popular model organism to study DNA repair due to its famous DNA damage tolerance. SNM1 family genes, which encode nuclease enzymes, have been found widespread in most organisms. It has been reported that SNM1 family proteins play key roles in DNA replication and DNA repair. It has been shown that the snm1 gene from D. radiodurans (drsnm1), a new member of SNM1 family genes, is critical for its stalled DNA replication forks restart and non-homologous end joining (NHEJ) repair pathway. Meanwhile, drSnm1 protein showed conserved activities as the eukaryotic homologous protein hsSnm1C, which could recognize and cleave hairpin structure DNA. However, most results are based on re-folding protein, which was insoluble during the expression in vitro. The major aims of this study are: (1) Construct mutant drsnm1 strains using new method based on CRISPR/CAS9 system. Study the mechanism of the stalled DNA replication forks restart by different assays including growth curve measurement, stress sensitivity, pulsed field gel electrophoresis and genome mutation rate measurement. (2) Express and purified drSnm1 protein and the interactive proteins DnaK, SSB and PprA. Study and evaluate the role of NHEJ contributing to DNA damaging resistance in D. radiodurans by in vitro NHEJ efficiency measurement assay. (3) Biochemical and structural analysis of drSnm1-DNA binary complex, revealing the mechanism of the exo/endonuclease activity switching. The results of this study will add valuable information about the mechanisms of the powerful DNA repair capability of D. radiodurans under different stresses. Moreover, considering the mutations in SNM1 family genes has been linked to diseases such as small-cell lung cancer and RS-SCID, these results could also have possible implication in several areas of clinical medicine.

耐辐射球菌拥有强大的DNA损伤修复能力，是研究DNA损伤修复的一种理想模式生物。SNM1家族基因广泛的分布于不同生物体内，在DNA复制和损伤修复过程中起到了关键作用。研究表明耐辐射球菌Snm1（drSnm1）是SNM1家族的新成员，具有与人源蛋白hsSnm1C相同的核酸酶活性，参与了耐辐射球菌复制叉重启和非同源末端连接（NHEJ）的过程。本项目将对该基因及其编码的蛋白开展以下几个方面的研究：（1）使用CRISPR/CAS9系统构建耐辐射球菌突变株，研究drSnm1介导的复制叉重启机制。（2）体外重建drSnm1介导的NHEJ修复途径，研究其修复损伤DNA的机制。（3）Snm1-DNA复合物晶体结构的解析，揭示该家族蛋白活性转化的机制。研究成果将进一步的阐明耐辐射球菌所具有的极端抗性机制。同时，鉴于SNM1家族基因的缺陷与多种疾病的发生相关，在临床医学上具有潜在的应用价值。

经过3年的研究，本项目研究完成并达到了原申请书的项目计划和任务书的要求和指标。我们的研究表明耐辐射球菌Snm1（drSnm1，也称为drRNase J）是一个保守的核酸酶，其属于β-CASP家族蛋白，同时具有核酸外切酶以及核酸内切酶的活性；通过CRISPR/CAS9对编码该蛋白的基因进行原位敲除，获得了杂合突变株，其生长速率较野生型大大降低；对该蛋白的核酸酶进行了鉴定，发现其核酸外切/内切酶活性的切换受到锰金属离子的调控；对该蛋白与底物DNA的晶体结构进行了解析，鉴定了其所具有的保守催化位点及相应的双金属离子催化机制；发现了一个5’磷酸的结合位点，与该蛋白的底物识别相关。这些研究成果系统的阐明了该蛋白的作用机制，为后续的研究提供了新的思路。