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.
|Pacek, Marcin; Tutter, Antonin V; Kubota, Yumiko et al. (2006) Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Mol Cell 21:581-7|