9505907 Gilbert Successful development of the first cell-free system in which DNA replication is preferentially initiated at a physiologically utilized origin of replication has been achieved by this laboratory. These initial observations will be fully investigated by using a combination of cell and molecular biological tools to identify the components of nuclear structure that designate an origin of replication. Specific aims are: 1. To determine if origins of replication are designate at the time of nuclear assembly, 2. To determine if pre-designated origins in Gl nuclei are disrupted by a Xenopus early cleavage mitotic cycle, 3. To determine if the mammalian nuclear membrane is necessary for site-specific initiation of replication in Xenopus egg extracts. %%% DNA replication is central to the life cycle of every living organism and, although much is known about the process in simple organisms, the regulatory mechanisms in higher eukaryotes are largely unknown. DNA replication initiates within defined chromosomal loci in eukaryotic cells but naked DNA in systems derived from metazoan organisms do not have any such requirements. This proposal describes a novel approach based upon the hypothesis that DNA replication in metazoan chromosomes is regulated by aspects of nuclear structure that cannot be easily reconstructed when starting with cloned DNA templates. Cell-free extracts from Xenopus eggs provide an excellent model system in which to test the hypothesis that the structural and functional organization of the nucleus is crucial in regulating DNA replication. *** ??
