Bielinsky A K, Blitzblau H, Beall E L, Ezrokhi M, Smith H S, Botchan M R, Gerbi S A
Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA.
Curr Biol. 2001 Sep 18;11(18):1427-31. doi: 10.1016/s0960-9822(01)00444-4.
The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.
真核细胞在S期开始时启动DNA复制需要起始识别复合物（ORC）[1]。这个由六个亚基组成的复合物最初是在酿酒酵母中分离出来的[2]，在进化上是保守的[1]。ORC参与前复制复合物的形成[3]，这对于建立复制能力是必需的。在酵母中，ORC与DNA的相互作用在自主复制序列（ARS）元件中已得到充分证实，其中ARS共有序列[4]（ACS）构成ORC结合位点的一部分[2,5]。对于后生动物中ORC与DNA的相互作用了解甚少。对于果蝇绒毛膜基因座，有人提出ORC结合是分散的[6]。我们分析了果蝇嗜粪Sciara coprophila的扩增起始点（ori）II/9A。我们鉴定出一个独特的80碱基对（bp）的ORC结合位点，并绘制了与其相邻的复制起始位点。ORC与这个80 bp核心区域的结合是ATP依赖性的，并且对于与另外65 bp的DNA建立进一步相互作用是必需的。这是首次在多细胞后生动物的扩增起始点中同时鉴定出ORC结合位点和复制起始位点。因此，我们的发现将范例从酵母ARS1扩展到多细胞真核生物，表明ORC是复制起始位置的决定因素。
