The Structural Basis of Eukaryotic Replication Origin Licensing by Cryo-EM

冷冻电镜真核复制起源许可的结构基础

基本信息

批准号：
7916806
负责人：
Huilin Li
金额：
$ 29.64万
依托单位：
BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-01 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7916806
关键词：
ATP phosphohydrolase Address Back Binding Binding Sites Biochemical Cell Cycle Cell Cycle Proteins Complex DNA DNA Binding DNA Sequence DNA Structure DNA biosynthesis DNA replication origin DNA-Directed DNA Polymerase Electron Microscopy Elements Ensure Eukaryota Event Failure Genomic Instability Hydrolysis Image Analysis Label Length Licensing Methods Molecular Molecular Machines Nucleotides Pattern Pattern Formation Play Pre-Replication Complex Preparation Process Protein Subunits Proteins Regulation Replication Initiation Replication Origin Research Personnel Role Saccharomyces cerevisiae Side Site Staging Streptavidin Structure Testing Width Winged Helix Work Yeasts activator 1 protein adenosine 5&apos -O-(3-thiotriphosphate)base dalton helicase in vivo insight interest melting molecular mass nuclease origin recognition complex particle programs tumor

项目摘要

DESCRIPTION (provided by applicant): Eukaryotic chromosomal replication is an intricate process that requires the coordinated and tightly regulated action of numerous molecular machines. Failure to ensure once only replication initiation per cell cycle can result in uncontrolled proliferation and genomic instability, two hallmarks of tumor genesis. The origin recognition complex (ORC), first discovered in yeast, is a six-subunit protein machine conserved in all eukaryotes. Yeast ORC constitutively binds to and marks the replication origin throughout the cell cycle. Licensing of the DNA replication origin starts when the critical cell division cycle protein Cdc6p binds to ORC. Recent biochemical studies with purified components indicate that Cdc6p and specific origin DNA sequence activate an ATPase switch in ORC. This induces an extended pre-replication complex (pre-RC)-like nuclease protection footprint on origin DNA that was previously observed only in vivo. Our preliminary EM work reveals a ring-like structural feature in the ORC-Cdc6p complex that is similar in size to the presumptive replicative hexameric MCM helicase. This result supports the emerging concept that the helicase is loaded by replication initiators in a mechanism similar to the loading of the DNA polymerase clamp PCNA by the RF-C clamp loader complex. The formation of the extended pre-RC-like footprint by ORC and Cdc6p, a crucial event in replication origin licensing, is ATP-binding and -hydrolysis dependent. We are interested in revealing the structural basis of this ATPase switch in ORC by studying the structures of ORC-Cdc6p-DNA in the ATP-bound form where the pre-RC footprint is formed, and in a non-hydrolyzable ATP (ATPgammaS)-bound form where the extended footprint is not formed. We hypothesize that the extended pre-RC footprint is a result of Cdc6p-induced origin DNA bending around ORC. We plan to test this hypothesis by determining the approximate binding sites of the three critical elements (A, B1 and B2) of the yeast ARS1 origin DNA. The origin DNA fragments will be biotinylated and labeled with streptavidin before binding with ORC and Cdc6p for EM analysis. These detailed structural studies will provide a long-awaited molecular mechanism for the origin licensing stage of the intricate replication initiation process.

描述（由申请人提供）：真核染色体复制是一个复杂的过程，需要众多分子机的协调和严格调节的作用。无法确保每一个细胞周期仅复制起始，就会导致不受控制的增殖和基因组不稳定性，这是肿瘤起源的两个标志。原点识别复合物（ORC）最初是在酵母中发现的，它是所有真核生物中保守的六亚基蛋白机。酵母菌在整个细胞周期内结合并标记复制起源并标记。当关键细胞分裂周期蛋白CDC6P与ORC结合时，DNA复制起源的许可开始。最近使用纯化成分的生化研究表明，CDC6P和特定的原点DNA序列激活ORC中的ATPase开关。这会诱导扩展的预复合物（PRE-RC）类似核酸酶的核酸酶保护足迹，以前仅在体内观察到。我们的初步EM工作揭示了ORC-CDC6P复合物中的环状结构特征，其大小与推定复制性六聚体MCM解旋酶相似。该结果支持了新兴概念，即在与RF-C夹具装载机复合物相似的机制中，通过复制启动器加载了解旋酶。 ORC和CDC6P（在复制起源许可中的关键事件）形成了扩展的RC样足迹，这是ATP结合和 - 氢解。我们有兴趣通过以ATP结合形式研究ORC-CDC6P-DNA的结构，在ATP结合形式中揭示了ORC中此ATPase开关的结构基础，在该形式中形成了前RC足迹，并以不可用的ATP（ATPGAMMAS）形式形成 - 未形成伸展足迹。我们假设扩展的前RC足迹是CDC6P诱导的原点DNA周围弯曲的结果。我们计划通过确定酵母ARS1原点DNA的三个关键元素（A，B1和B2）的近似结合位点来检验这一假设。原点DNA片段将是生物素化的，并在与ORC和CDC6P结合之前，用链霉亲和素标记进行EM分析。这些详细的结构研究将为复杂的复制起始过程的起源许可阶段提供期待已久的分子机制。