Fujita M, Hori Y, Shirahige K, Tsurimoto T, Yoshikawa H, Obuse C
Nara Institute of Science and Technology, Japan.
Genes Cells. 1998 Nov;3(11):737-49. doi: 10.1046/j.1365-2443.1998.00226.x.
The ORC (Origin Recognition Complex) of Saccharomyces cerevisiae is a protein complex for the initiation of replication which interacts with a cis-element, ACS (ARS Consensus Sequence), essential for DNA replication. The protein-DNA complex detected by the DNase I genomic footprinting method has been shown to vary depending on cell cycle progression. Further studies on topological changes of replication origin in vivo caused by ORC association are crucial for an understanding of chromosomal DNA replication in S. cerevisiae.
Topological changes in the replication origins of the S. cerevisiae chromosome were studied by an in vivo UV photofootprinting method which is capable of detecting the change in the flexibility of DNA caused by protein binding. The footprinting method detected the inhibition and enhancement of UV-induced pyrimidine dimer formation in A and B1 elements of a chromosomal origin, ARS1, depending on the activity of native ORC subunits. Furthermore, footprint patterns were reproduced in vitro with purified ORC. The inhibition regarding the A element was stronger during the S to late M phase than that during the progression through the G1 phase. Functional CDC6 and MCM5 were required for maintaining the weaker inhibition state in G1-arrested cells.
The application of in vivo UV photofootprinting in studies of topological changes of S. cerevisiae replication origins revealed the presence of two modes of topological ORC-ACS interaction. The weaker footprint in the G1 phase represents a specific topology of ACS, resulting from an alteration of the ORC-ACS interaction aided by CDC6 and MCM5, and this topological change may make the replication origin competent for initiating DNA replication.
酿酒酵母的ORC（复制起始识别复合体）是一种用于启动复制的蛋白质复合体，它与顺式元件ACS（ARS共有序列）相互作用，而ACS对DNA复制至关重要。通过DNA酶I基因组足迹法检测到的蛋白质-DNA复合体已被证明会随细胞周期进程而变化。进一步研究ORC结合在体内引起的复制起点拓扑变化对于理解酿酒酵母中的染色体DNA复制至关重要。
通过一种能够检测由蛋白质结合引起的DNA柔韧性变化的体内紫外光足迹法，研究了酿酒酵母染色体复制起点的拓扑变化。该足迹法检测到，根据天然ORC亚基的活性，染色体起点ARS1的A和B1元件中紫外线诱导的嘧啶二聚体形成受到抑制或增强。此外，用纯化的ORC在体外重现了足迹模式。A元件的抑制在S期到M期后期比在G1期进程中更强。功能性的CDC6和MCM5是维持G1期停滞细胞中较弱抑制状态所必需的。
体内紫外光足迹法在酿酒酵母复制起点拓扑变化研究中的应用揭示了拓扑ORC-ACS相互作用的两种模式。G1期较弱的足迹代表了ACS的一种特定拓扑结构，这是由CDC6和MCM5辅助的ORC-ACS相互作用改变导致的，这种拓扑变化可能使复制起点具备启动DNA复制的能力。
