Role of MCM10 in DNA Replication Initiation In Vitro

DNA复制是一个高度调控的过程。在细胞分裂过程中，每个细胞都必须快速复制其基因组，但要具有极高的保真度。在真核生物中，染色体DNA复制的起始通过在复制起点处的许多蛋白质的逐步组装而发生。最早的步骤发生在细胞周期的G1期，是六亚基起点识别复合物（ORC）与复制起点的结合，随后是Cdc 6、Cdt 1和MCM 2 -7复合物。总的来说，这些蛋白质来自复制前复合物（pre-RC）。在从G1期到S期的转变过程中，Cdc 7和Cdk 2激酶激活前RC。在从G1期到S期的过渡期间，Cdc 7和Cdk 2激酶激活前RC，刺激Cdc 45的募集，将前RC转化为活跃的复制叉。在S.酿酒酵母表明，在G1期期间参与前RC组装的一种蛋白是MCM 10。 Walter实验室最近克隆了MCM 10的爪蟾同源物，并使用可溶性爪蟾提取物表征了其功能，该提取物重建了真核DNA复制中涉及的步骤。 出乎意料的是，预RC组装不需要MCM，但对于Cdc 45加载步骤是必需的。该提案的目标是阐明MCM 10刺激CDC 45负载的分子机制。此外，我将探讨MCM 10是否可能是细胞周期检查点的靶点。

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.