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

通过实时单分子荧光研究复制起点许可机制

基本信息

批准号：
10707170
负责人：
Stephen P. Bell
金额：
$ 38.43万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-20 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10707170
关键词：
Address Affect Binding Binding Sites Biochemical Biochemistry Biological Assay Bypass Cell Cycle Cell division Cells Chromosomes Collaborations Complement Complex Cryoelectron Microscopy DNA DNA Binding DNA biosynthesis DNA replication fork DNA replication origin Defect Deposition Development Elements Ensure Eukaryotic Cell Event Experimental Genetics Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer G1 Phase Genome Genomic Instability Head Human In Vitro Individual Kinetics Lead Licensing Maintenance Malignant Neoplasms Mediating Molecular Molecular Biology Molecular Genetics Molecular Machines Monitor Mutation N-terminal Nature Nucleosomes Organism Pathway interactions Process Productivity Proteins Reaction Regulation Replication Initiation Replication Origin Research Role S phase Saccharomycetales Shapes Site Specificity Structure Techniques Testing Time Yeasts experimental study genome integrity helicase in vivo insight mutant novel origin recognition complex prevent protein protein interaction reconstitution recruit single molecule targeted treatment tool tumorigenesis yeast protein

项目摘要

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.

项目摘要 DNA复制对于维持所有生物的基因组至关重要。在每一轮细胞分裂期间，真核细胞必须建立数百至数千的复制叉，以协调每个复制染色体。这些事件始于G1，当时有两个复制性解旋酶，即MCM2-7 复合物在DNA复制的所有潜在起源上加载。一旦加载，两个环形，杂己聚集MCM2-7复合物包围DNA并通过其N末端结构域紧密相互作用。尽管不活动，但由此产生的头对头MCM2-7双重六聚体许可证各自来源进入S阶段时的双向启动。与它们的重要性，突变或不正义相一致介导解旋酶负荷的蛋白质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治疗剂的潜在靶标。使用发芽酵母蛋白的解旋酶负载的生化重构是一种强大的但是，了解这些事件的工具，大量的生化测定不适合研究该复合物由于它们经常不完整和异步性，导致解旋酶负荷涉及的动力学。单身的- 分子荧光显微镜实验绕过了这些问题，通过监视个人实时DNA分子，定义生化事件的序列，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。我们提出了单一在体外使用重构的酵母蛋白进行解旋酶负荷的分子实验，并补充在活细胞上进行分子遗传学实验。这些研究将共同提供对解旋酶负荷的动态机制，并将补充和有助于解释静态在最近的低温电子显微镜研究中揭示的结构。拟议的研究主要集中于在所有人中保守的解旋酶负荷事件真核生物。酵母和后生兽人在结合后会引起DNA的强弯。在特定目的一，我们研究了该活动在原点选择中的作用，并确定哪些步骤解旋酶加载需要此功能。 MO建筑群是一个关键的旋转酶加载中间体，可确保第二次招募的MCM2-7与第一个形成了正面的互动。在目标两个中，我们将确定角色在DNA周围MCM2-7环关闭中的这种复合物中，并定义了兽人和MCM2-的途径 7形成这个复合物。在最终目标中，我们将确定核小体和序列非特异性兽人如何 DNA结合变化解旋酶负载，这是后生种类中原点选择的两个关键要素。