Role of MCM10 in DNA Replication Initiation In Vitro

DNA复制是一个高度受监管的过程。在细胞分裂过程中，每个细胞都必须以极高的保真度快速复制其基因组。在真核生物中，染色体DNA复制的启动是通过复制起始处大量蛋白质的逐步组装而发生的。最早的步骤发生在细胞周期的G1期，是六亚单位起源识别复合体(ORC)与复制起点的结合，其次是CDC6、CDT1和MCM2-7复合体。总的来说，这些蛋白质来自复制前复合体(Pre-RC)。在G1期向S期的转变过程中，CDC7和CDK2激活了Pre-RC。在细胞周期从G1期向S期转变的过程中，CDC7和CDK2激活了Pre-RC，刺激了Pre-RC的募集，将Pre-RC转化为一个活跃的复制分叉。在酿酒酵母中的实验表明，在G1期参与前RC组装的一种蛋白质是MCM10。沃尔特实验室最近克隆了MCM10的非洲爪哇同源物，并使用一种重组真核DNA复制步骤的可溶性非洲爪哇提取物来表征其功能。出乎意料的是，MCM对于Pre-RC组装并不是必需的，但对于CDC45加载步骤是必不可少的。这项建议的目的是阐明MCM10刺激CDC45负载的分子机制。此外，我还将探讨MCM10是否可能是细胞周期检查点的目标。

DNA replication is a highly regulated process. During cell division each cell must replicate its genome rapidly, yet with extremely high fidelity. In eukaryotes, the initiation of chromosomal DNA replication occurs through the stepwise assembly of numerous proteins at origins of replication. The earliest step, which occurs in the G1 phase of the cell cycle, is the binding of the six-subunit origin, recognition complex (ORC) to replication origins, followed by Cdc6, Cdt1 and the MCM2-7 complex. Collectively, these proteins from the pre-replicative complex (pre-RC). During the transition from G1 to S phase, Cdc7 and Cdk2 kinases activate the pre-RC. During the transition from G1 to S-phase, Cdc7 and Cdk2 kinases activate the pre-RC stimulating the recruitment of Cdc45, converting the pre-RC into an active replication fork. Experiments in S. cerevisiae suggests that one protein involved in pre- RC assembly during G1 phase is MCM10. The Walter laboratory has recently cloned the Xenopus homolog of MCM10, and has characterized its function using a soluble Xenopus extract that reconstitutes the steps involved in eukaryotic DNA replication. Unexpectedly, MCM is not required for pre-RC assembly, but is essential for the Cdc45 loading step. The goal of this proposal is to elucidate the molecular mechanism by which MCM10 stimulates CDC45 loading. In addition, I will explore whether MCM10 may be the target of cell cycle checkpoints.