Cell division is one of the most fundamental processes in living organisms. A critical part of cell division is the duplication of the genome during S phase, an event not well characterized in higher eukaryotes. Recent work has shown that the ubiquitin conjugating enzyme, Cdc34, in a large molecular size complex is required for the onset of DNA replication in egg extracts of the frog, Xenopus laevis. How Cdc34 and its as yet unidentified associated components function to regulate DNA replication in a vertebrate is not known. This research will test the hypothesis that Cdc34 and other associated components target key cell cycle regulators for ubiquitin-mediated degradation, triggering the initiation of DNA replication in vertebrates. Xenopus interphase egg extracts will be used as a cell-free vertebrate model system to study the regulatory events surrounding the initiation of DNA replication. The specific goal of this research is to accomplish the following aims: (1) To identify and functionally characterize the Cdc34-associated protein complex that regulates the onset of DNA replication. This will be achieved by the identification and cloning of the components associated with Cdc34, which are required for the onset of DNA replication. These components will then be studied to determine for the functional and biophysical characteristics of the complex in association with Cdc34. (2) To define the role of Cdc34 in regulating the initiation of DNA replication by determining how Cdc34 may influence the assembly of the replication complex. This will be accomplished by performing cell localization and chromatin centrifugation studies to determine whether Cdc34 activity influences the formation of the replication complex during the onset of S phase.
