The mechanisms controlling duplication of metazoan chromosomes are only beginning to be understood. In multicellular eukaryotes, it is not known what organization of DNA sequences constitutes an origin of DNA replication, nor is it fully understood how cell cycle controls result in the firing of origins only once in a cell cycle. Based on work in yeast, the prediction is that assembly of a pre-replication complex (pre-RC) onto origins in Gl is the central control point for once per cell cycle replication. However, specific association of the pre-RC with a metazoan origin of replication has not been reported. It is also predicted that cyclin dependent kinases (CDKs), in addition to activating the pre-RC in S, also inhibit pre- RC assembly in G2 and M; however, the mechanisms underlying these dual functions are unclear. Among the best understood origins of replication in higher eukaryotes are those used for developmental amplification of eggshell (chorion) genes during Drosophila oogenesis. The regions that are critical for origin function have been mapped to less than 1Kb. Recent results indicate that chorion amplification requires CDKs, pre-RC subunits, and other molecules required for Gl/S progression. We propose to use the chorion system to study origin structure and the cell cycle regulation of preRC assembly in metazoa. To begin, we will map DNA sequences to which the pre-RC binds in relation to replication start sites, and ask how this binding is modulated in response to cell cycle regulation. Next, we will investigate the structure of the pre-RC in vivo through genetic and molecular manipulation of a gene implicated in pre-RC function. Finally, we will use modern tools of Drosophila genetics to conduct Fl genetic screens for mutations which disrupt amplification. These screens should identify the cadre of proteins associated with origins, and the regulatory pathways that control their activity. Moreover, by examining what differs between amplification and normal replication, we should gain insight into the mechanisms by which origin firing is restricted to once per cell cycle. Combined with the advantages inherent in Drosophila as a model organism, this system represents an opportunity for the genetic and molecular dissection of the cell cycle control of metazoan replication.
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