Structural mechanisms of Mcm10 in DNA replication

描述（由申请人提供）：真核细胞中DNA复制的起始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，如果不能在适当的时间复制一次基因组，就会导致突变率升高、染色体不稳定和癌症的发展。因此，了解复制起始机制对人类健康至关重要。复制起始涉及几个动态多蛋白复合物的编排组装，这些复合物必须在复制起点识别和解开DNA，解释检查点信号，并最终建立复制叉。虽然许多起始因子的招募顺序已经确定，但它们组装DNA解绕/合成机器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是负责激活复制前复合物和DNA合成早期步骤的中心复制因子。Mcm10是第一个在s期开始时装载染色质的蛋白质，是起始解绕、装载必要的复制因子（如Cdc45、RPA）到染色质以及稳定DNA聚合酶a （pol a）催化亚基所必需的。我们实验室的初步结果已经确定了非洲爪蟾Mcm10的整体结构域结构，并确定了与DNA和pol a相互作用的区域。在目前的提议中，我们将使用结构生物学和生物化学的工具来确定Mcm10与DNA （Aim 1）和pol a （Aim 2）相互作用的物理基础。x射线晶体学和核磁共振波谱学将共同用于表征Mcm10结构域及其蛋白质/DNA复合物的结构。然后使用基于结构的诱变方法将这些信息放入更广泛的功能环境中，利用与战略性选择的合作者的互动。这种对Mcm10的强大的结构-功能分析是了解起源解绕如何与复制体组装和功能耦合的机制的重要的第一步。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癌症和其他人类疾病治疗剂的可能发展奠定基础。

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.