DNA磷硫酰化修饰，即一个硫原子替代DNA磷酯骨架上的一个氧原子，是迄今发现的唯一一种DNA骨架上的生理修饰。DNA磷硫酰化由dnd基因簇编码的四个蛋白所完成，有着保护DNA不被断裂的功能。与dnd基因簇相邻，还有四个与DNA磷硫酰化相关的基因dndF、dndG、dndH、dndI。敲除dnd基因簇造成的DNA磷硫酰化丧失会导致DNA损伤断裂，而同时对dndFGH基因做同框敲除则使细菌恢复正常表型，不再出现DNA断裂现象。过表达DndF、DndG、DndH蛋白对细菌有致死作用。这表明DndF、DndG、DndH蛋白对非磷硫酰化DNA有切割作用，而磷硫酰化则保护DNA不受切割。本项目中，我们拟解析DndF、DndG、DndH、DndI蛋白的晶体结构以及它们的复合物结构，从而揭示它们是怎样协同作用，识别并切割非磷硫酰化DNA的，并探讨磷硫酰化修饰保护DNA不被切割的分子机理。

DNA phosphorothioation, in which an oxygen atom on the phosphorodiester backbone of DNA is replaced with a sulfur atom, is the only discovered physiological modification on the DNA backbone. The DNA phosphorothioation process is accomplished by four proteins, DndA, DndC, DndD, and DndE, which are encoded by genes on the dnd gene cluster. Functions of DNA phosphorothioation include protecting DNA from cleavage and oxidation. Recently, it was found that next to the dnd gene cluster, there are another four genes related to DNA phosphorothioation, dndF, dndG, dndH, and dndI. If the dnd gene cluster is knocked out, DNA phosphorothioation in the bacterial cells would be lost, and it will lead to DNA damage and breakage. On the other hand, when the dndF, dndG, and dndH genes are in-frame deleted at the same time with the dnd gene cluster, the bacterial cells return to the normal phenotype and DNA breakage no longer happens. In addition, overexpression of DndF, DndG, and DndH proteins also has lethal effects on bacteria. This indicates that DndF, DndG, and DndH proteins can cleave non-phosphorothioated DNA, whereas phosphorothioation is able to protect DNA from being cleaved by the DndF, DndG, and DndH proteins. In this project, we are going to determine the crystal structures of DndF, DndG, DndH, and the closely related DndI proteins. We plan to investigate whether DndF, DndG, DndH, and DndI form a protein-protein complex, and will set out to determine the crystal structure of this protein-protein complex. By combining structural biology together with enzymological and microbiological assays, we are going to reveal how the DndF, DndG, DndH, and DndI proteins recognize and cleave non-phosphorothioated DNA, and elucidate the molecular mechanism of how the phosphorothioation modification protects DNA from cleavage by DndF, DndG, DndH, and DndI.