Chastain Paul D, Bowers Jayson L, Lee Daniel G, Bell Stephen P, Griffith Jack D
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA.
J Biol Chem. 2004 Aug 27;279(35):36354-62. doi: 10.1074/jbc.M403501200. Epub 2004 Jun 16.
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.
酿酒酵母起源识别复合体（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究这种多蛋白复合体的组织结构，每个亚基的C端或N端都用生物素进行了标记，并与其他五个未修饰的亚基组装成一个复合体。一种由一端带有链霉亲和素的短DNA双链体组成的纳米级生物指针被用于绘制每个亚基的N端和C端的位置。这些观察是在溶液中游离的ORC以及与ARS1复制起点结合的ORC上进行的。这种定位证实并扩展了先前关于亚基与起点DNA相互作用位点的研究。特别是，我们提供了关于ORC-ARS1复合体化学计量以及与ORC结合DNA相关的构象变化的新信息。这种用于绘制蛋白质结构的通用新方法具有许多应用潜力。
