The events of the cell cycle occur in a temporally conserved fashion. In particular, DNA replication or S-phase always precedes mitosis. For a somatic cell to enter mitosis before completion of DNA replication would be a lethal event. In order to insure that this does not occur the cell must contain a feedback or fidelity mechanism which actively suppresses initiation of mitosis until DNA replication is complete. Several recent observations have provided definitive evidence that such a feedback mechanism exists. Genetic screens have identified specific genes (RCC1, cdc25, NimA) which appear to be proteins which play an integral role in maintaining the feedback mechanism. For example, mutations in these genes or overexpression allow cells to enter mitosis prior to completion of mitosis. The long term objective of this proposal is to identify the molecular components involved in this feedback system and to determine precisely how these components couple completion of DNA replication to the initiation of mitosis. The primary experimental system which will be used for these investigations will be a cell-free system derived from Xenopus eggs. In the absence of feedback controls this in vitro system spontaneously oscillates between S- phase and mitosis with a regular periodicity. We have shown that inhibition of DNA replication blocks this spontaneous oscillation and causes the cycle to arrest in S-phase. In this proposal we intend to use this regulated in vitro system to investigate how unreplicated DNA suppresses the initiation of mitosis. Specifically, we intend to: 1) determine the DNA substrate which serves to generate this suppressing signal; 2) investigate whether the phosphatases and kinases which regulate cdc2 activity are themselves subject to regulation by the feedback pathway activated by unreplicated DNA; 3) isolate these phosphatases and kinases; 4) determine whether the positive regulator of mitosis, the cdc25 protein, controls the sensitivity of the feedback pathway; 5) determine whether the feedback pathway regulates MPF activity by modulating the amount of cdc25 protein; 6) determine whether cdc25 interacts with cdc2 specific kinases and phosphatases; 7) isolate and characterize the DNA-binding protein RCC1; and 8) determine if this DNA-binding protein prevents activation of MPF when the DNA is incompletely replicated. These investigations should provide valuable information about how cell division is normally regulated as well as how a cell ensures that it neither over nor under replicates its DNA prior to initiating mitosis.
