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

项目摘要 DNA复制对维持所有生物体的基因组至关重要。在每一轮细胞分裂过程中， 真核细胞必须建立数百到数千个复制分叉，以协调复制每个分叉 染色体。这些事件在G1期间开始，当时复制的解旋酶的两个拷贝，McM2-7 复合体，在DNA复制的所有潜在起始处都加载了。一旦加载，两个环形， 异六聚体Mcm2-7复合体包围DNA，并通过其N-末端结构域紧密相互作用。 尽管处于非活动状态，但最终得到的头对头McM2-7双六角体许可每个原点用于后续 进入S阶段时的双向启动。与它们的重要性、突变或调控不当相一致 介导解旋酶负荷的蛋白质会导致癌症和发育异常。因此， 了解这些过程的机制将提供有关维护的关键信息 基因组完整性和潜在的治疗靶点。 利用发芽酵母蛋白进行解旋酶的生化重组是一种强有力的方法。 了解这些事件的工具，然而，批量生化分析不太适合研究复杂的 由于解旋酶经常不完整和不同步的性质，因此涉及解旋酶负载的动力学。单人- 分子荧光显微镜实验通过监测个体事件绕过了这些问题 实时DNA分子，定义生化事件序列，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。我们建议单人- 重组酵母蛋白体外负载解旋酶的分子实验 活细胞的分子遗传学实验。总而言之，这些研究将为 解旋酶负载的动态机制，将补充和帮助解释静态 在最近的低温电子显微镜研究中发现的结构。 拟议中的研究主要集中在解旋酶加载事件上，这些事件在所有 真核生物。酵母和后生动物ORC在结合时都会在DNA中引起强烈的弯曲。在……里面 具体目标一，我们调查这一活动在起源选择中的作用，并确定在 解旋酶的装载需要这个功能。MO复合体是关键的解旋酶负载中间体，确保 第二个招募的McM2-7与第一个形成了面对面的互动。在第二个目标中，我们将确定 关闭DNA周围的Mcm2-7环，并定义ORC和McM2- 7形成这个复合体。在最终目标中，我们将确定核小体和序列非特异性ORC是如何 DNA结合改变了解旋酶的负荷，这两个因素都是后生动物物种起源选择的关键因素。

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.