Genes whose protein products modulate the cell cycle and ensure that cells enter S-phase only under appropriate conditions are frequently mutated in cancer. Cells with such mutations have an increased frequency of chromosome breakage, and spawn the genetic variants that fuel cancer progression. Whereas there has been an explosion of knowledge about cell cycle, our understanding of how the cell cycle machinery communicates with the DNA duplex to initiate replication in mammalian cells is rudimentary. This proposal has three interrelated goals. The first is to develop structure-function maps of regions of the mammalian genome previously shown to initiate DNA replication. This goal will be expedited by a functional initiation assay developed in the last grant cycle. The strategy uses site-specific recombination to transfer wild-type and mutant replicators into fixed chromosomal sites in which initiation can be quantified. Since the chromatin environment is fixed in this analysis, Dr. Wahl is able to evaluate the specific contribution of short sequences to replicator function. The second goal is to analyze these replicators in their native loci using the same site-specific recombination approach. This will allow Dr. Wahl to evaluate the role of chromosome environment on the initiation of DNA replication. The third goal is to determine whether specific regions of replicators undergo changes in chromatin structure during the cell cycle. Such changes may be prominent at the anaphase-G1 transition when replicators are licensed to initiate in the subsequent S-phase. The genetic strategy described above provided the first evidence for a replicator in the human b-globin and Chinese hamster DHFR loci, and established the validity of the replicon model in mammalian cells. The recent discovery from the principal investigator's laboratory that a 200 bp fragment of the DHFR replicator is necessary for initiation has significantly advanced the knowledge of metazoan replicator structure. Dr. Wahl will test the hypothesis that the deleted DNA contains a structural motif important for initiation as he has found similar motifs in the b-globin replicator. Genetic and biochemical studies are proposed to uncover the mechanism by which these regions contribute to initiation. The integrated genetic and biochemical approach outlined in this proposal should elucidate the molecular structures of mammalian replicators, and will advance our understanding of how replicators, replication control proteins, and the cell cycle control machinery are interconnected.
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