An ecdysone response element (EcRE) has been located in the Sciara II/9A origin of replication (ORI) directly adjacent to the binding site for the origin recognition complex (ORC). Preliminary chromatin immunoprecipitation (CHIP) experiments demonstrate that EcR binds at this EcRE during DNA amplification, but not prior to amplification. It is hypothesized that direct or indirect interactions between EcR and the replication machinery modulate origin firing, perhaps by stabilizing proteins of the pre-replicative complex, thus serving as a """"""""re-replication factor"""""""". This could be a mechanism by which certain gene loci become amplified in some cancers. Sciara EcR isoforms and the homologue of its hetero-dimerization partner, ultraspiracle (USP) will be cloned. These cDNAs will be used for protein expression and generation of antibodies. With these antibodies, ChIP data on the in vivo occupancy of EcR and its heterodimerization partner USP at the II/9A locus will be extended. Next, it will be determined if EcR interacts with known components of the replication machinery through co-immunoprecipitation. Finally, it will be determined if the presence of EcR is required for DNA amplification at the Sciara II/9-A locus by constructing P-element tranformants of Drosophila that contain mutations in the origin EeRE. ? ?
