沙门氏菌DNA磷硫酰化修饰蛋白复合物的功能研究

DNA phosphorothioate modification, which modifies on the DNA backbone is another revolutionary contribution to the research of DNA modification after the discovery of DNA methylation. The dnd cluster is responsible for the DNA phosphorothioation modification, which has selectivity for special DNA sequences and confirmations, while the mechanisms for the recognition of DNA sequences as well as the process of phosphorothioation are still unclear. With employment of the well established in vitro phosphorothioation platform that uses cell extracts, we will perform an in-depth study of the function of complex DptCDE, which is responsible for DNA phosphorothioation in Salmonella, during the process of DNA recognition. Meanwhile, key amino acid residues in the complex will be mutated to study the transmission process of sulphur during phosphorothioation as well as the roles the complex plays. Finally, we hope to delineate the moelcular mechanism of DNA phosphorothioation, which may also provide guidance for the study of the physiological function of DNA phosphorothioation and other useful applications, e.g. in DNA manipulation.

DNA磷硫酰化修饰是由dnd基因簇负责催化的DNA碳骨架修饰，该修饰具有序列专一性和空间构象特异性, 是继发现DNA甲基化之后对研究DNA修饰的又一突破性贡献，但是到目前为止负责特异性识别的机制以及磷硫酰化修饰的反应过程都还不清楚。本项目拟以沙门氏菌中负责DNA磷硫酰化修饰的DptCDE蛋白复合体为研究对象，利用本实验室已经成功建立的细胞裂解液体外磷硫酰化平台，来深入研究DptCDE特异识别的DNA序列及其特异性识别的分子机制；同时，采用分子酶学等手段，结合关键氨基酸的点突变分析来深入研究DNA磷硫酰化过程中硫的传递途径以及DptCDE蛋白复合体所发挥的功能，进而阐释DNA磷硫酰化修饰的分子机理。此外，该研究也为揭示DNA磷硫酰化的生理功能及其在DNA操作等方面的研究奠定了基础。

DNA磷硫酰化是硫原子取代DNA骨架非桥联氧原子的一种新型表观遗传学修饰，这种修饰广泛存在于沙门氏菌等原核生物中。磷硫酰化修饰是继发现DNA甲基化之后对DNA修饰研究的又一突破性贡献，科学家们对其表现出浓厚的研究兴趣，但其特异性识别的机制以及磷硫酰化修饰的反应过程一直不清楚。本研究利用构建的沙门氏菌全细胞裂解液催化体系以及dptCDE基因敲除突变株细胞裂解液添加纯化的DptCDE反应体系，在体外再现了DNA磷硫酰化修饰反应。发现GAAC/GTTC为修饰反应的保守序列，其两侧序列对修饰没有影响，双链DNA可以在体外被修饰，但单链DNA不能被修饰。发现参与修饰反应的DptC、DptD和DptE 3个蛋白形成了稳定的600 kDa复合体，用于底物识别和催化。发现DNA磷硫酰化修饰dptB-E共转录，并且敲除dptB基因的沙门氏菌的DNA磷硫酰化修饰水平明显增强。一系列转录水平实验揭示了沙门氏菌中dptB是DNA磷硫酰化修饰的负调控基因。通过鸭疫里默士杆菌磷硫酰化修饰的研究，发现DNA磷硫酰化修饰蛋白DndEi具有解旋酶和ATP酶活性。这些研究不仅拓展了对DNA磷硫酰化修饰分子机制的理解，也为阐明DNA磷硫酰化修饰的生理功能建立了基础。