耐辐射球菌PprI-Rddr介导的DNA损伤响应途径机制研究

To maintain the stability of their genomes, organisms have evolved various elegant DNA damage response pathways and networks. In bacteria, RecA-LexA-dependent SOS pathway is a classical DNA damage response system. Recently, we found a novel DNA damage response pathway that is dependent on the DNA damage response switch PprI (also called IrrE) and its lately identified substrate Rddr (also called DdrO) in Deinococcus radiodurans. This project is established to investigate the upstream activator of the PprI-Rddr pathway through RT-PCR and Western Blotting, to define the active form of PprI through Mass Spectrum identification, modification sites analysis, and point mutation construction, to investigate the coordination control of PprI, Rddr and other proteins and targeted promoters over the downstream genes’ expression through Real-time Quantative PCR, EMSA and Northern Blotting. The research will illuminate the mechanism of specific PprI-Rddr-mediated DNA damage response pathway in stress resistances of Deinococcus radiodurans, so as to deepen the knowledge of micorganisms’ adaption to various environmental stresses, and provide theoretical basis for the development and utilization of the extreme microorganism.

细菌中经典的DNA损伤响应系统是一种依赖于LexA和RecA蛋白的SOS途径。最近在耐辐射球菌中发现了一条独特的应急响应途径，依赖于新型的DNA损伤响应修复开关PprI和新发现的底物蛋白Rddr。本项目拟通过RT-PCR和Western印迹等技术，研究PprI-Rddr途径上游激活因素；运用质谱鉴定、蛋白修饰位点分析和点突变株构建等技术，研究PprI-Rddr途径关键蛋白酶的活化方式；利用荧光定量PCR、EMSA和Northern杂交等技术，研究PprI等蛋白和所作用启动子对下游基因的协调控制作用。通过本研究，旨在阐明以PprI-Rddr为中心的新型DNA损伤响应途径作用机理，加深对微生物的环境胁迫适应性机制的认识，并为极端微生物资源的开发利用提供理论依据。

细菌中经典的DNA损伤响应系统是一种依赖于LexA和RecA蛋白的SOS途径。最近在耐辐射球菌中发现了一条独特的应急响应途径，依赖于新型的DNA损伤响应修复开关PprI和新发现的底物蛋白Rddr。本项目通过蛋白表达纯化、Western印迹、质谱鉴定、蛋白修饰位点分析、点突变株构建、EMSA、蛋白质结晶及分析等技术手段，优化了信号通路重要的蛋白表达和纯化、分析了信号传导途径重要蛋白PprI和Rddr的体外生化活性、研究了PprI蛋白酶活性启动和提高方法、建立了DNA-蛋白质特异性结合位点的新方法、探索了电离辐照对PprI蛋白翻译后修饰的影响、研究了PprI重要位点突变对耐辐射球菌辐射抗性的影响、检测了PprI蛋白的重要磷酸化位点突变对信号途径活性的影响、分析了辐射抗性相关磷酸化位点突变对PprI体外活性的影响、探讨了PprI蛋白通过与RNA polymerase等蛋白互作促进下游基因转录、并初步解析了PprI、Rddr及其复合物的晶体结构。通过本研究，初步阐明了以PprI-Rddr为中心的新型DNA损伤响应途径作用机理，加深对微生物的环境胁迫适应性机制的认识，并为极端微生物资源的开发利用提供理论依据。