DNA replication in somatic cells is an asynchronous process. Many regions of DNA replicate early while others replicate late. The molecular mechanism which generates these differences in timing of replication are not well understood. Recently, using a completely soluble system which we have developed for investigating DNA replication we have found that initiation of replication at one DNA region activates a regulatory pathway which actively inhibits initiation of DNA replication at other potential sites of initiation of replication. Specifically, we find that in this replication system initiation of replication on one plasmid DNA template inhibits replication of a second plasmid template. When the first template completes replication, the inhibitory signal attenuates and the second plasmid initiates replication. Our results suggest that this signaling pathway may normally regulate S-phase progression during the somatic cell cycle. In this project, experiments designed to identify the components of this pathway are described. Specifically, we will look for a replication- dependent inhibition of factors such as cdk2 and cdc7 kinase and cdc45 protein, which are essential for initiation of DNA replication. We will also examine whether a kinase ATM/R which has been implicated in regulating other DNA-dependent checkpoints is a component of this new pathway. We will also determine whether this pathway is responsible for regulating both the rate of replication during S-phase and the timing of replication in somatic cells. We have also been investigating how ongoing DNA replication inhibits premature initiation of mitosis. Results from these studies show that weel, a negative regulator of the mitotic kinase, cdc2, is stabilized while DNA replication is ongoing and then rapidly degraded in a cdc34- dependent manner following completion of replication. Our results suggest that stabilization of weel by replication prevents initiation of mitosis during S-phase. Experiments to identify the essential components (cdk2kinase, cds1 kinase and an F box receptor) which cause weel degradation are described in the current proposal.
