Structural mechanisms of Mcm10 in DNA replication

描述(由申请人提供)：在真核细胞中启动DNA复制是一个高度调控的过程，对基因组完整性至关重要。在细胞周期中，如果不在适当的时间只复制一次基因组，可能会导致突变率上升、染色体不稳定和癌症的发展。因此，了解复制启动的机制对人类健康至关重要。复制启动涉及几个动态多蛋白复合体的精心设计的组装，这些复合体必须识别和解开复制起始处的DNA，解释检查点信号，并最终建立复制分叉。尽管许多启动因子的招募顺序已经确定，但它们组装DNA解离/合成机制或复制体的机制尚不清楚。该研究计划的长期目标是了解Mcm10的分子机制，Mcm10是一种中央复制因子，负责激活复制前复合体和DNA合成的早期步骤。Mcm10是第一个在S相开始时负载染色质的蛋白质，它是起始解离、将必要的复制因子(如CDC45、RPA)加载到染色质上以及稳定DNA聚合酶a催化亚单位(Pola)所必需的。我们实验室的初步结果已经确定了非洲爪哇Mcm10的整体结构，并确定了与DNA和pola相互作用的区域。在当前的提议中，我们将使用结构生物学和生物化学的工具来确定Mcm10与DNA(目标1)和pola(目标2)相互作用的物理基础。X射线结晶学和核磁共振波谱将联合用于表征Mcm10结构域及其蛋白质/DNA复合体的结构。然后，这些信息将使用基于结构的突变方法，利用与战略选择的合作者的互动，被置于更广泛的功能背景中。对Mcm10进行的这种强大的结构-功能分析是从机制上理解起源解离如何与复制体组装和功能相关联的重要的第一步。对关键复制因子及其分子间相互作用的更高分辨率描述将为可能开发针对癌症和其他人类疾病的治疗剂奠定基础。

DESCRIPTION (provided by applicant): Initiation of DNA replication in eukaryotic cells is a highly regulated process that is essential for genome integrity. Failure to copy the genome only once and at the proper time during the cell cycle can lead to elevated mutation rates, chromosome instability, and the development of cancer. It is therefore of paramount importance for human health to understand the mechanism of replication initiation. Replication initiation involves a choreographed assembly of several dynamic multiprotein complexes, which must recognize and unwind DNA at origins of replication, interpret checkpoint signals, and ultimately establish the replication fork. Although the order of recruitment of many initiation factors has been defined, the mechanisms by which they assemble the DNA unwinding/synthesis machinery, or replisome, are unknown. The long-range goals of this research program are to understand the molecular mechanism of Mcm10, a central replication factor responsible for activation of pre-replicative complexes and early steps of DNA synthesis. Mcm10 is the first protein to load chromatin at the onset of S-phase, is required for origin unwinding, loading essential replication factors (e.g., Cdc45, RPA) onto chromatin, and stabilizing the catalytic subunit of DNA polymerase a (pol a). Preliminary results from our laboratory have defined the overall domain architecture of Mcm10 from Xenopus laevis, and have identified the regions that interact with DNA and pol a. In the current proposal, we will use the tools of structural biology and biochemistry to determine the physical basis for Mcm10 interaction with DNA (Aim 1) and pol a (Aim 2). X-ray crystallography and NMR spectroscopy will be used jointly to characterize the structures of Mcm10 domains and their protein/DNA complexes. This information will then be placed into a broader functional context using a structure-based mutagenesis approach, taking advantage of interactions with strategically chosen collaborators. This powerful structure-function analysis of Mcm10 is an essential first step toward a mechanistic understanding of how origin unwinding is coupled to replisome assembly and function. A higher resolution description of key replication factors and their intermolecular interactions will lay the foundation for the possible development of therapeutic agents against cancer and other human disease.