The nature of DNA replication origins in eukaryotic chromosomes needs to be defined before we can understand how origin activation is specified and controlled in living cells. A commitment to activate origins in cells is, at the same time, a commitment to undergo cell division and proliferation. An understanding of the mechanisms that control origin activation is essential for a full understanding of the regulation of cell proliferation and the regulatory defects that lead to uncontrolled proliferation, as in cancer. At present, chromosomally-derived origins are best defined in the yeast, S. cerevisiae, where they have been characterized as autonomously replicating sequences (ARS) in plasmids. However, little is known about the determinants of origin activity within cellular chromosomes. In this proposal, two ARS elements have been selected for further study based on their unique properties in terms of origin function within yeast chromosomes: (l) the rDNA ARS, present in each of the 100-200 ribosomal DNA repeats, only a minority of which are actually used as replication origins in the chromosome, and (2) ARS3O3, a functional ARS element in a plasmid which is silent as an origin in its native chromosomal location. We will address two major questions of general importance to the understanding of the nature of chromosomal origins and the factors that regulate their usage: (1) what is the molecular basis for the low frequency of rDNA origin usage in a chromosome? (2) what is the molecular basis for origin silencing at ARS303 in the chromosome? To address these questions, we will (a) identify the cis-acting components that are sufficient to account for the full activity of the rDNA ADS and ARS3O3 within a plasmid, (b) identify the cis-acting elements, both positive and negative, as well as other factors that govern the level of replication origin usage in a chromosome (rDNA) and the silencing of an origin within a chromosome (ARS303) and, (c) identify DNA-protein interactions and DNA structural alterations that occur in active and silent origins, and identify the cis-components required for those interactions and structural alterations. Identification of the genetic requirements for chromosomal origin function will be achieved by taking advantage of the facility with which yeast undergo homologous DNA recombination, permitting the precise replacement of a wild-type chromosomal locus with a site-specific mutant locus carried on a plasmid vector. The proposed studies are made feasible as a result of novel approaches we have devised to assay a single-copy rDNA ARS in the chromosome and to detect origin activity associated with the normally silent ARS3O3.
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