Speck Christian, Stillman Bruce
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
J Biol Chem. 2007 Apr 20;282(16):11705-14. doi: 10.1074/jbc.M700399200. Epub 2007 Feb 21.
DNA replication, as with all macromolecular synthesis steps, is controlled in part at the level of initiation. Although the origin recognition complex (ORC) binds to origins of DNA replication, it does not solely determine their location. To initiate DNA replication ORC requires Cdc6 to target initiation to specific DNA sequences in chromosomes and with Cdt1 loads the ring-shaped mini-chromosome maintenance (MCM) 2-7 DNA helicase component onto DNA. ORC and Cdc6 combine to form a ring-shaped complex that contains six AAA+ subunits. ORC and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPase mutants have reduced activity in ORC x Cdc6 x DNA complex formation. Here we analyzed the role of the Cdc6 ATPase on ORC x Cdc6 complex stability in the presence or absence of specific DNA sequences. Cdc6 ATPase is activated by ORC, regulates ORC x Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the conserved origin A element, and to a lesser extent mutations in the B1 and B2 elements, induce Cdc6 ATPase activity and prevent stable ORC x Cdc6 formation. By analyzing ORC x Cdc6 complex stability on various DNAs, we demonstrated that specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls the rate of Cdc6 dissociation from the ORC x Cdc6 x DNA complex. We propose a mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in a stable ORC x Cdc6 x DNA complex, which can then promote MCM loading. This model has relevance for origin specificity in higher eukaryotes.
与所有大分子合成步骤一样，DNA复制部分是在起始水平受到控制的。尽管起源识别复合物（ORC）会结合到DNA复制起点，但它并不能单独决定其位置。为了启动DNA复制，ORC需要Cdc6将起始过程靶向到染色体中的特定DNA序列，并与Cdt1一起将环状的微型染色体维持（MCM）2 - 7 DNA解旋酶组件加载到DNA上。ORC和Cdc6结合形成一个包含六个AAA +亚基的环状复合物。ORC和Cdc6 ATP酶突变体在MCM加载方面存在缺陷，并且ORC ATP酶突变体在ORC x Cdc6 x DNA复合物形成中的活性降低。在这里，我们分析了在存在或不存在特定DNA序列的情况下，Cdc6 ATP酶对ORC x Cdc6复合物稳定性的作用。Cdc6 ATP酶被ORC激活，调节ORC x Cdc6复合物的稳定性，并受到起始DNA的抑制。保守的起始A元件中的突变，以及在较小程度上B1和B2元件中的突变，会诱导Cdc6 ATP酶活性并阻止稳定的ORC x Cdc6形成。通过分析ORC x Cdc6复合物在各种DNA上的稳定性，我们证明特定的DNA序列控制Cdc6 ATP酶的速率，进而控制Cdc6从ORC x Cdc6 x DNA复合物中解离的速率。我们提出了一种机制，解释Cdc6 ATP酶活性如何促进起始DNA序列特异性；在缺乏起始活性的DNA上，Cdc6 ATP酶促进Cdc6的解离，而起始DNA下调Cdc6 ATP酶，导致形成稳定的ORC x Cdc6 x DNA复合物，然后该复合物可以促进MCM加载。该模型与高等真核生物中的起始特异性相关。
