Mechanisms of replication origin licensing studied by real-time single-molecule fluorescence

项目摘要 DNA复制对于维持所有生物体的基因组至关重要。在每一轮细胞分裂中， 真核细胞必须建立数百到数千个复制叉， 染色体这些事件开始于G1期，当两个拷贝的复制解旋酶Mcm 2 -7 复合物，装载在所有潜在的DNA复制起点。一旦加载，两个环形， 异六聚体Mcm 2 -7复合物包围DNA并通过其N-末端结构域紧密相互作用。 虽然不活跃，但所得的头对头Mcm 2 -7双六聚体许可每个起源用于随后的重组。 进入S期后双向启动。与它们的重要性相一致， 介导解旋酶加载的蛋白质的突变导致癌症和发育异常。因此，在本发明中， 了解这些过程的机制将提供有关维护的关键信息 基因组的完整性和潜在的治疗靶点。 使用芽殖酵母蛋白质的解旋酶加载的生化重建已经是一种强有力的方法。 然而，大量生物化学测定不太适合研究这些复杂的事件， 由于其经常不完全和异步的性质，涉及解旋酶加载的动力学。单身- 分子荧光显微镜实验通过监测个体上的事件来绕过这些问题。 真实的时间中的DNA分子，定义生化事件的顺序，检测短暂的中间体 和特定的蛋白质-蛋白质相互作用，并定义定量动力学机制。我们建议单身- 体外使用重组酵母蛋白进行解旋酶加载的分子实验，补充有 活细胞的分子遗传学实验。总之，这些研究将提供关键的见解， 解旋酶加载的动态机制，并将补充和帮助解释的静态 结构揭示在最近的冷冻电子显微镜研究。 拟议的研究主要集中在所有保守的解旋酶加载事件上。 真核生物酵母和后生动物ORC在结合后均诱导DNA中的强弯曲。在 具体目标一，我们调查的作用，这一活动的起源选择，并确定哪些步骤， 解旋酶加载需要该功能。MO复合物是一种关键的解旋酶加载中间体， 第二个补充的Mcm 2 -7与第一个形成头对头的相互作用。在目标二中，我们将确定 该复合物在关闭DNA周围的Mcm 2 -7环中的作用，并定义了ORC和Mcm 2- 七是形成这种复合体。在最后的目标，我们将确定如何核小体和序列非特异性ORC DNA结合改变解旋酶负载，这两个关键因素的起源选择在后生动物物种。

Project Summary DNA replication is essential to maintain the genome of all organisms. During each round of cell division, eukaryotic cells must establish hundreds to thousands of replication forks that coordinately replicate each chromosome. These events begin during G1, when two copies of the replicative helicase, the Mcm2-7 complex, are loaded at all potential origins of DNA replication. Once loaded, the two ring-shaped, heterohexameric Mcm2-7 complexes encircle the DNA and interact tightly via their N-terminal domains. Although inactive, the resulting head-to-head Mcm2-7 double hexamer licenses each origin for subsequent bidirectional initiation upon entry into S phase. Consistent with their importance, mutations in or misregulation of the proteins mediating helicase loading lead to cancer and developmental abnormalities. Thus, understanding the mechanism of these processes will provide critical information concerning the maintenance of genome integrity and potential targets for therapeutics. The biochemical reconstitution of helicase loading using budding yeast proteins has been a powerful tool to understand these events, however, bulk biochemical assays are poorly suited to study the complex dynamics involved in helicase loading due to their frequently incomplete and asynchronous nature. Single- molecule fluorescence microscopy experiments bypass these problems by monitoring events on individual DNA molecules in real time, defining the sequence of biochemical events, detecting short-lived intermediates and specific protein-protein interactions, and defining quantitative kinetic mechanisms. We propose single- molecule experiments on helicase loading using reconstituted yeast proteins in vitro, supplemented with molecular genetics experiments on live cells. Together, these studies will provide critical insights into the dynamic mechanisms of helicase loading and will complement and aid in the interpretation of the static structures revealed in recent cryoelectron microscopy studies. The proposed research primarily focuses on events of helicase loading that are conserved across all eukaryotic organisms. Both yeast and metazoan ORC induce a strong bend in the DNA upon binding. In Specific Aim one, we investigate the role of this activity in origin selection and determine which steps in helicase loading require this function. The MO complex is a key helicase-loading intermediate that ensures the second recruited Mcm2-7 forms head-to-head interactions with the first. In Aim two, we will determine the role of this complex in closing of the Mcm2-7 ring around DNA and define the pathways by which ORC and Mcm2- 7 form this complex. In the final Aim, we will determine how nucleosomes and sequence-nonspecific ORC DNA binding change helicase loading, both key elements of origin selection in metazoan species.