In eukaryotic cells the mechanism and regulation of DNA replication, a process of fundamental importance for normal cell proliferation, is not understood. The long term objective is to gain a better understanding of this process on the biochemical and genetic level. The investigators believe that progress in this area hinges upon their ability to elucidate the mechanism of initiation of replication at a single replicon. Thus understanding of the structural organization of an origin of replication is necessary and the identification and characterization of the proteins responsible for DNA replication are crucial. The investigators have recently completed a systematic structural analysis of the yeast nuclear origin, the ARS121. The investigators have also purified and characterized the OBF1 protein and cloned its gene. Based on these studies the investigators have suggested that OBF1, a multiple phosphorylated protein in vivo, acts as an enhancer of DNA replication. The investigators objective in the proposal is to elucidate the mechanism of OBF1 function as it pertains to its role in DNA replication. To attain this objective the short term specific aims are: (I) to develop an in vivo functional assay for OBF1 action in replication by (i) constructing and expressing lexA-OBF1 protein fusions and (ii) testing the action of these fusions on chimeric origins containing the bacterial lexA-binding site instead of the OBF1 recognition sequences; (II) To use such an assay to dissect the OBF1 protein in order to identify the origin activation domain. This will be performed by generating truncated OBF1 fused to lexA; (III) to isolate, by affinity chromatography procedures, and clone the genes of the remaining components of the ARS initiation replication apparatus. These will entail fractionation of yeast extracts using specific DNA-cellulose and OBF-1 sepharose affinity procedures; (IV) to identify OBF1 phosphorylated domains and correlate it with the origin activation domain; (V) to identify the OBF1 protein kinase and analyses the phosphorylation process in the context of the cell cycle. By achieving these short term goals, the investigators will be able to resolve and reconstitute in vitro the initiation replication apparatus, which will provide the means for a biochemical analysis of initiation of replication of a eukaryotic replicon. In addition, the investigators will gain a deeper understanding of the regulatory mechanisms that govern the activation of origins of replication in eukaryotic cells.
