Mapping subunit location on the Saccharomyces cerevisiae origin recognition complex free and bound to DNA using a novel nanoscale biopointer
Mapping subunit location on the Saccharomyces cerevisiae origin recognition complex free and bound to DNA using a novel nanoscale biopointer
复制标题
DOI：
10.1074/jbc.m403501200
复制
发表时间：
2004-08-27
影响因子：
4.8
通讯作者：
Griffith, JD
中科院分区：
文献类型：
--
作者：
Chastain, PD;Bowers, JL;Griffith, JD
酿酒酵母起始识别复合物（ORC）由六个亚基组成，是复制装置组装中的重要组成部分。为了通过电子显微镜探测这种多蛋白复合物的组织，每个亚基在其C或N末端用生物素标记，并与其他五个未修饰的亚基组装成复合物。一个纳米级的生物指针组成的短DNA双链霉亲和素在一端被用来映射的N和C末端的每个亚基的位置。这些观察结果是使用溶液中游离的ORC并与ARS 1复制起点结合进行的。这一映射证实并扩展了以前的研究映射与起源DNA的亚基相互作用的网站。特别是，我们提供了新的信息有关的ORC-ARS 1复合物的化学计量和构象的变化，与DNA结合ORC。这种多功能的蛋白质结构绘图新方法具有许多应用潜力。
The Saccharomyces cerevisiae origin recognition complex (ORC) is composed of six subunits and is an essential component in the assembly of the replication apparatus. To probe the organization of this multiprotein complex by electron microscopy, each subunit was tagged on either its C or N terminus with biotin and assembled into a complex with the five other unmodified subunits. A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N and C termini of each subunit. These observations were made using ORC free in solution and bound to the ARS1 origin of replication. This mapping confirms and extends previous studies mapping the sites of subunit interaction with origin DNA. In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 complex and the changes in conformation that are associated with DNA binding by ORC. This versatile, new approach to mapping protein structure has potential for many applications.