Department of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19807-12. doi: 10.1073/pnas.0905281106. Epub 2009 Nov 11.
All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.
所有生物体都需要确保在单个细胞周期内不会重新复制 DNA 片段。真核生物通过一种称为原点许可的过程来实现这一点，该过程涉及到将预复制复合物（pre-RC）组装到染色质上的严格时空控制。Cdt1 是 pre-RC 组装的关键组成部分和关键调节因子。在高等真核生物中，Geminin 对 Cdt1 的及时抑制对于防止 DNA 重复复制至关重要。在这里，我们通过结合 X 射线晶体学、小角度 X 射线散射和 Xenopus 和哺乳动物细胞中的功能研究，解决了 Geminin 抑制 DNA 许可的机制。我们的发现表明，Cdt1:Geminin 复合物可以存在两种不同的形式，一种是“允许”的三聚体和一种是“抑制”的异六聚体。位于异六聚体中的特定 Cdt1 残基对于许可很重要。我们推测，三聚体和异六聚体之间的转换代表了许可能力和许可缺陷状态之间的分子开关。
