磷硫酰化修饰保护DNA免于切割的分子机理研究

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.

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。

本项目基本按照原计划执行。我们经过艰苦努力，取得了以下结果。我们发现DndF、DndG和DndH三个蛋白可以形成一个复合物， 我们称为DndFGH复合物，表现出特异性地针对非磷硫酰化修饰DNA的缺刻酶活性，限制外源非磷硫酰化修饰DNA的侵染。接着，我们发现DndF蛋白表现出ATP水解酶（ATPase）活性，如果突变DndF上对ATPase活性起关键作用的Walker A motif，就会破坏DndFGH复合物的DNA缺刻酶活性。然后，我们通过解析DndG蛋白的X射线晶体结构，发现DndG是一个含有helix-turn-helix (HTH) motif的DNA结合蛋白，并通过非变性凝胶电泳实验证实了其确实可以结合DNA。随后，我们通过晶体结构解析和生化功能实验分析，发现DndH亚基为DndFGH复合物提供缺刻DNA以及在DNA上移位（translocation）的活性。而且，DndH的C端结构域也有ATPase活性，与DNA的结合会激活DndH的ATPase活性来水解ATP，DndH利用水解ATP所释放的能量在DNA上移位。最后，我们通过荧光共振能量转移（简称FRET）实验发现DndFGH复合物在溶液中可以采取多种构象，与DNA和ATP的结合会促使DndFGH复合物的构象转换。有趣的是，DNA磷硫酰化修饰可以下调DndFGH复合物的活性，与非磷硫酰化DNA相比，磷硫酰化修饰DNA存在时，DndH的ATPase活力以及转位酶活力都大大降低，从而使得DndFGH复合物不会缺刻细菌自身的磷硫酰化修饰DNA，实现对“非我”的防御和对“自我”的保护。本研究成果以“The functional coupling between restriction and DNA phosphorothioate modification systems underlying the DndFGH restriction complex”为题发表在《Nature Catalysis》期刊上。吴更教授为并列通讯作者。