Structural Basis of Eukaryotic Replication Initiation by Cryo-EM

 描述（由申请人提供）：真核细胞染色体复制的启动是一个严格调控的多步骤过程。未能确保每个细胞分裂周期一次且仅一次起始事件可导致不受控制的细胞增殖和肿瘤发生。的核心 复制起始是识别染色体复制起点的起点识别复合物（ORC）。在复制启动子Cdc 6和复制许可因子Cdt 1的帮助下，ORC招募并将Mcm 2 -7解旋酶加载到DNA上，形成高度稳定的Mcm 2 -7双六聚体。这完成了第一个里程碑-复制前复合物（pre-RC）的形成。一系列的后续事件最终形成前起始复合物（pre-IC）并导致细胞进入S期。原核生物复制起始的关键蛋白质的X射线晶体结构已有报道，但对相应的真核生物复合物的结构知之甚少。这是因为真核生物的复制起始物组装成大的和短暂的复合物，这对于晶体学来说是非常困难的。在硬件和软件的最新进展，使冷冻EM的理想方法来研究这些复杂的分辨率大大提高。因此，我们建议研究如何通过冷冻EM建立复制前复合物。这项工作将推进我们的知识在真核细胞染色体复制的启动。

 DESCRIPTION (provided by applicant): Initiation of the eukaryotic chromosome replication is a tightly regulated multi-step process. Failure to ensure once and once only initiation event per cell division cycle can result in uncontrolled cell proliferation and tumorigenesis. Central to the replication initiation is the origin recognition complex (ORC) that recognizes the chromosomal origins of replication. With the help of replication initiator Cdc6 and replication licensing facto Cdt1, ORC recruits and loads the Mcm2-7 helicase onto DNA, forming a highly stable Mcm2-7 double hexamer. This completes the first milestone - formation of pre-replication complex (pre-RC). A series of subsequent events culminate to formation of pre-initiation complex (pre-IC) and leading cell to enter into the S phase. X-ray crystal structures have been reported for key proteins involved in prokaryotic replication initiation, but little is known about the structures o the corresponding eukaryotic complexes. This is so because the eukaryotic replication initiators assemble into large and transient complexes, which have been exceedingly difficult for crystallography. Recent advance in hardware and software has made cryo-EM an ideal approach to studying these complexes at much improved resolution. We therefore propose to study how the pre- Replication Complex is established by cryo-EM. This work will advance our knowledge in the initiation of eukaryotic chromosome replication.