DNA磷硫修饰蛋白DndE与DNA复合物结构研究

项目名称： DNA磷硫修饰蛋白DndE与DNA复合物结构研究

项目编号： No.21272261

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 数理科学和化学

项目作者： 曹春阳

作者单位： 中国科学院上海有机化学研究所

项目金额： 80万元

中文摘要： DNA骨架磷硫修饰是我国科学家邓子新院士团队于2007年在细菌中发现的第一种DNA骨架生理性修饰，具有立体选择性（Rp）和分布普遍性，针对不同菌株还具有DNA碱基序列专一性。DNA磷硫修饰受dndABCDE五个基因所编码的蛋白(DndA,DndB,DndC,DndD和DndE)控制，但5个蛋白具体如何发挥作用以完成DNA磷硫修饰没有任何文献报道。我们前期利用NMR和X-衍射等技术确定了DndE蛋白三维结构，表明DndE是一个具有新型骨架的nicked dsDNA结合蛋白，而不是基于生物信息学分析所得的硫转移蛋白，或者NCAIR合成酶类似蛋白。本项目计划利用X-ray、NMR技术、荧光极化技术等技术，开展DndE与dsDNA复合物三维结构研究，同时结合DndA、DndB、DndC和DndD蛋白预测的功能，以及体内体外实验，探讨DndE蛋白在DNA磷硫修饰过程中发挥生理功能时的结构生物学基础。

中文关键词： DNA磷硫修饰；DnDE；dsDNA；结合模式；晶体结构

英文摘要： DNA backbone phosphorothioation modification, in which sulfur replaces a nonbridging phosphate oxygen, found by prof. Zixin Deng and his members, and colaborators in 2007, was discovered that the dnd gene products incorporate sulfur into the DNA backbone in a sequence- and stereo-specific manner, and DNA phosphorothioation has also been found widespread and quantized in bacterial genomes.It was reported that the five-gene dnd cluster (dndA-E) caused DNA degradation during electrophoresis. However,the mechanism about how DNA backbone phosphorothioation is produced, and its biological function remain extremely unclear. Moreover, how DNA phosphorothioation is controled by five proteins (DndA,DndB,DndC,DndD and DndE encoded by dnd gene cluster) is also kept unknown. Previously, in a project funded by NSFC, we determined three dimensional structure of DndE by using X-ray technique, two dimensional NMR mothod and fluorescence anistrospy assay, revealing that DndE is neither a sulphur transferase, nor a NCAIR synthase analogue, but a nicked dsDNA binding protein with an unrecognized folding. In this project, to investigate the detailed structural basis for DndE functioning in the pathway of DNA phosphorothioation, we plan to determine the structure of the complex of DndE bound with dsDNA or nicked dsDNA by using X-ray

英文关键词： DNA phosphorathioation；DndE；dsDNA；binding model；crystal structure