DNA磷硫酰化修饰体系中dndE的功能分析及在细菌基因组修饰选择性的分子机制研究

DNA phosphorothioate (PT) modification is a new type of DNA epigenetic physiological modification for the first time in recent years,which occurs when non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This phenomenon has been discovered recently and only widely diverse existed in prokaryotes. The discovery of PT modification may expand the traditional theory of DNA structures and also cause widely attention in the world. However, the biological function of related genes in this DNA physilogical modification, the progress of modification, and mechanism of selectivity have noot been fully revealed. In this study, dndE gene will be used as the research object to elucidate the biological function and the mode of action of the dndE gene in the phosphorothioation system. The compatibility and interaction between DNA phosphorothioate modification and methylation will be analyzed by RNA-seq, SMRT single molecule real-time sequencing and LC-MS/MS. The effects of DNA phosphorothioatie modification and methylation on the transcription of global gene will also be analyzed to elucidate the molecular mechanism of the specific selection of the modified locus, and to reveal its distribution at the level of bacterial genome. This project is conducive to broaden the field of vision and direction of DNA physiological modification, laying the foundation for further clarifying the biological significance of DNA phosphorothioate modification.

DNA磷硫酰化修饰是近年来首次发现的一种新型DNA表观遗传学修饰，由硫原子取代DNA磷酸骨架上非桥联氧原子而形成具有序列特异性和空间构象专一性的修饰，仅发现广泛存在于原核生物中。该发现拓展了经典的DNA结构组成理论，具有重大基础理论意义。但这种生理修饰中相关基因的生物学功能，修饰位点发生的选择性机制，一直未完全揭示。本项目以dndE基因为研究对象，通过分析单个基因可能的生理多效性功能，阐明dndE基因在磷硫酰化修饰体系中的生物学功能以及作用方式。利用RNA-seq、SMRT单分子测序及LC-MS/MS等技术，通过比较分析DNA磷硫酰化修饰与甲基化修饰之间兼容性、相互作用方式，系统分析硫化修饰对细菌全局性基因转录的影响，阐明其在细菌基因组水平上的分布规律，揭示修饰位点选择性的分子机制。这些工作，为进一步明确DNA磷硫酰化修饰的生物学意义奠定基础，也有利于拓宽对DNA生理修饰的研究视野。

DNA磷硫酰化修饰是一种新型的硫原子取代DNA骨架磷原子上非桥联氧原子的微生物表观遗传修饰。该修饰广泛存在于细菌中并具有序列和手性特异性，但这种新型修饰相关基因的生物学功能及在修饰体系中的必要性，该修饰体系在细菌基因组中分布规律及生理功能等问题依然尚未完全明晰。本研究通过综合多学科的研究手段和技术，明确了dndE基因在DNA磷硫酰化修饰系统中的必需性。通过测定S. enterica serovar Cerro 87 全基因组中的DNA磷硫酰化修饰位点及在细菌基因组中的分布情况，发现DNA磷硫酰化修饰位点的个数及在基因组中所在位置表现为不均匀的随机性分布状况。进一步明确了Dam甲基化与DNA磷硫酰化修饰的相互作用关系。同时对来自弧菌的II型DNA硫化修饰系统中的半胱氨酸脱硫酶SspA与底物半胱氨酸的复合物晶体结构及功能进行了解析。通过构建的异源MVA途径在基因工程大肠杆菌中的表达，首次实现了T-杜松醇、β-桉叶醇以及二萜类化合物西松烷酚醇的生物合成。