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

描述（申请人提供）：真核生物的染色体复制是一个复杂的过程，需要许多分子机器的协调和严格调节。如果不能确保每个细胞周期只有一次复制启动，就会导致不受控制的增殖和基因组不稳定，这是肿瘤发生的两个标志。起源识别复合体（ORC）是一种在酵母中首次发现的六亚基蛋白机器，在所有真核生物中都是保守的。酵母ORC在整个细胞周期中组成性地结合并标记复制起点。当关键细胞分裂周期蛋白Cdc6p与ORC结合时，DNA复制起源的许可就开始了。最近纯化组分的生化研究表明，Cdc6p和特定的起源DNA序列激活ORC中的atp酶开关。这在原始DNA上诱导了一个扩展的复制前复合体（pre-RC）样核酸酶保护足迹，这在以前只在体内观察到。我们的初步EM工作揭示了ORC-Cdc6p复合体的环状结构特征，其大小与假定的复制六聚体MCM解旋酶相似。这一结果支持了一个新兴的概念，即解旋酶是由复制启动子加载的，其机制类似于DNA聚合酶夹片PCNA由RF-C夹片加载复合物加载。ORC和Cdc6p形成扩展的pre- rc样足迹是复制起始许可的关键事件，依赖于atp结合和-水解。我们有兴趣通过研究ORC- cdc6p - dna在ATP结合形式（pre-RC足迹形成的地方）和在非水解ATP （atpmas）结合形式（扩展足迹不形成的地方）的结构来揭示ORC中这种ATP酶开关的结构基础。我们假设扩展的pre-RC足迹是cdc6p诱导的起源DNA在ORC周围弯曲的结果。我们计划通过确定酵母ARS1起源DNA的三个关键元素（A， B1和B2）的近似结合位点来验证这一假设。在与ORC和Cdc6p结合进行EM分析之前，将原始DNA片段进行生物素化和链亲和素标记。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。

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.