The central hypothesis in this proposal is that the factors that determine the temporal and spatial activities of DNA replication initiation sites are intimately connected to those regulating gene expression. The extent to which on selection and activity influences promoter function requires further exploration. Our work focusing upon the Drosophila ORC and the regulation of DNA replication mediated gene amplification in the follicle cells of the egg has uncovered a direct role for the Drosophila homologue of the proto-oncogene Myb in replication control. DmMyb is found in complex with four other proteins and we have direct biochemical and molecular biological evidence that suggests that the Myb complex interacts with E2.F:DP complexes. One ultimate long-term goal is to recreate in vitro an integrated DNA replication system that mimics both repression and activation of specific origins mediated by these proteins. To achieve this goal we must continue a broad based program focusing upon the biochemical steps that lead to DNA replication initiation and to understand the general mechanistic roles affected by the DmMyb complex and E2F proteins.
Aims i nclude: 1) Understanding how ORC specifically binds to the regulatory sequences controlling amplification and what DNA regions replicate when repression of other potential origin sites is lost 2). Define the chromosomal positions for the DmMyb complex throughout the Drosophila genome and ask which sites regulate transcription and if some of these sites control DNA replication zones that are coordinately or separately controlled with nearby promoters. 3). We will determine the identity of the proteins and the specific interfaces that bring the E2F2: DP complex together with the DmMyb complex. We propose to ask if humans Myb-B is found associated in a complex that also interacts with an E2F family member. 4). We propose to study the structure and function of the Drosophila ORC protein to investigate its role in wrapping DNA and in the early steps of replication initiation. These steps must include DNA melting and the loading a processive-helicase, likely provided by the MCM complex and ancillary factors.
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