The central objective of cell cycle regulation is the faithful duplication of genomic DNA and the segregation of that DNA to two daughter cells. Genomic DNA must be replicated completely each cell cycle, yet this replication must be limited to a single round of per cell cycle. Protein degradation through the ubiquitin-mediated proteolytic pathway is employed extensively by eukaryotic cells to regulate cell cycle transitions. Ubiquitin ligases facilitate the polyubiquitination of substrate proteins, which marks them for degradation. One of the major classes of ubiquitin-ligases that regulate the cell cycle are cullin/RING finger complexes. We have found that the C. elegans cullin CUL-4 is required to limit DNA replication to once per cell cycle. In the absence of CUL-4, cells arrest in S phase and continuously re-replicate their genomic DNA. The molecular pathway through which CUL-4 functions to limit genomic DNA replication is not known. The related cullin proteins CUL-1 and CUL-2 function in multi-protein cullin/RING finger complexes with dedicated substrate-binding components. Currently the protein complex(es) in which CUL-4 functions have not been described in any organism. C. elegans is being used as a model system in which to study CUL-4 function, to allow the use of powerful genetic and molecular approaches.The experiments in this proposal will clarify the molecular functions of C. elegans CUL-4 in three ways. First, immunoaffinity and two-hybrid methods will be used to identify proteins that function in CUL-4-complexes. Second, immunoaffinity, two-hybrid, and suppressor screens will be used to identify substrates of CUL-4 complexes. The molecular and genetic characterization of these CUL-4 complex components and substrates is expected to provide significant insights into the fundamental processes that regulate the initiation of DNA replication. Finally, the interaction between CUL-4 and homologs of the eukaryotic DNA replication licensing factors Cdc6 and Cdt1 will be determined. In addition to providing insights into the fundamental aspects of DNA replication, this work has implications for understanding cancer etiology as human CUL4A has been found to be overexpressed in breast cancer, and gene amplification of oncogenes, which involves DNA re-replication, is a contributing factor in multiple cancers.
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