Grainge Ian, Gaudier Martin, Schuwirth Barbara S, Westcott Sarah L, Sandall Jane, Atanassova Neli, Wigley Dale B
Cancer Research UK, Clare Hall Laboratories, The London Research Institute, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3LD, UK.
J Mol Biol. 2006 Oct 20;363(2):355-69. doi: 10.1016/j.jmb.2006.07.076. Epub 2006 Aug 1.
We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple origin recognition box (ORB) sites that we show to be a replication origin. At this origin, there are four ORB elements disposed either side of an A+T-rich region. An ORC1 protein dimer binds at each of these ORB sites. Once all four ORB sites have bound ORC1 protein, there is a transition to a higher-order assembly with a defined alteration in topology and superhelicity. Furthermore, after this transition, the A+T-rich region becomes sensitive to digestion by DNase I and P1 nuclease, revealing that the transition promotes distortion of the DNA in this region, presumably as a prelude to loading of MCM helicase.
我们利用DNA酶I足迹法对嗜热栖热菌起始识别复合物蛋白（ORC1和ORC2）与DNA的相互作用进行了表征。每种蛋白都结合在其各自基因的上游。然而，单独的ORC1蛋白与一个包含多个起始识别框（ORB）位点的额外区域结合得更紧密，我们证明该区域是一个复制起点。在这个起点处，有四个ORB元件分布在富含A+T的区域两侧。一个ORC1蛋白二聚体结合在每个这些ORB位点上。一旦所有四个ORB位点都结合了ORC1蛋白，就会转变为具有确定拓扑结构和超螺旋变化的高阶组装体。此外，在这种转变之后，富含A+T的区域对DNA酶I和P1核酸酶的消化变得敏感，这表明这种转变促进了该区域DNA的扭曲，推测这是加载MCM解旋酶的前奏。
