Molecular mechanisms of DNA replication

摘要 DNA复制是所有生物精确复制遗传物质之前的一个基本过程 细胞分裂。这个过程的中心是一种解旋酶，它使用ATP水解酶来分离碱基对 DNA允许聚合酶获得合成互补链的途径，并驱动 DNA上的复制机制。在人类和其他真核细胞中，解旋酶引擎是6- 蛋白质MCM复合体。MCM介导的DNA操纵的机制目前还不太清楚 分子水平上由于长期缺乏结构信息，对几个离散的官能团 各州。拟议中的研究将通过提供MCM蛋白质的详细图片来填补知识空白 与ATP化合物和不同形式的DNA相互作用。这些将在分子实验室进行研究。 水平的协调方法，涉及结构研究的X射线结晶学和体外方法 研究它们的功能和相互作用。已经为这个项目获得了大量的初步数据， 包括MCM：ATP共晶结构，重要的人类MCM的初步衍射晶体 单链DNA，以及几种纯化的具有生化活性的蛋白质。

ABSTRACT DNA replication is a fundamental process for all organisms to precisely duplicate genetic material prior to cell division. Central to the process is a helicase enzyme that uses ATP-hydrolysis to separate base-paired DNA to allow polymerases to gain access to synthesize complementary strands and also to drive the replication machinery along the DNA. In human and other eukaryotic cells, the helicase engine is the 6- protein MCM complex. The mechanism for MCM-mediated manipulation of DNA is poorly understood at the molecular level because of a long-standing lack of structural information for several discrete functional states. The proposed research will fill a knowledge gap by providing detailed pictures of MCM proteins interacting with ATP compounds and with different forms of DNA. These will be studied at the molecular level by a coordinated approach involving structural studies by X-ray crystallography and in vitro methods to study their functions and interactions. A large body of preliminary data has been obtained for this project, including MCM:ATP co-crystal structures, important preliminary diffracting crystals of human MCMs with single-stranded DNA, and several purified proteins with confirmed biochemical activity.