The functions of C. elegans CUL-4/DDB-1 complexes in DNA replication and cell cycle regulation
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The replication of genomic DNA is strictly regulated to occur only once per cell cycle. This regulation centers on the temporal-restriction of replication licensing factor activity. In all eukaryotic species examined, the regulation of Cdt1 is critical to prevent re-replication. In humans, two distinct ubiquitin ligase (E3) complexes, CUL4/DDB1 and SCF(Skp2), have been shown to target the replication licensing factor Cdt1 for ubiquitin-mediated proteolysis. However, it is unclear to what extent these two distinct Cdt1 degradation pathways are conserved. The work described here shows that C. elegans CUL-4/DDB-1 functions as the major E3 ligase to target CDT-1 for degradation. A ddb-1 null mutant exhibits extensive DNA re-replication in post-embryonic blast cells, similar to what is observed in cul-4(RNAi) larvae. In contrast, SKPT-1 has no appreciable role in CDT-1 degradation during S phase. This dissertation also describes additional CUL-4/DDB-1 functions. The CUL-4/DDB-1 complex is required to target the degradation of the CIP/KIP family CKI-1 and cyclin E homolog CYE-1. It is shown that CKI-1 is required for DNA re-replication associated with loss of CUL-4 or DDB-1. Evidence is provided that the ÔWDXRÕ motif protein CDT-2 functions as the SRS (substrate recognition subunit) for a C. elegans CUL-4/DDB-1 complex that targets the degradation of CKI-1. CDT-2 is required to target both CDT-1 and CKI-1 for degradation. CDT-2 can directly bind to both CDT-1 and CKI-1, as expected for an SRS. Germ cells in ddb-1 mutants and cul-4(RNAi) larvae exhibit corrupted cell and nucleolar morphology. The ddb-1 mutant germ cells do not exhibit re-replication phenotypes, suggesting that the CUL-4/DDB-1 ubiquitin ligase is required for germ cell integrity independently of its known function in regulating DNA replication. It is shown that a ÔWDXRÕ motif protein VprBP phenocopies ddb-1 in the germ cells, suggesting that it functions as an SRS to regulate germ cell integrity. In total, this work demonstrates that the C. elegans CUL-4/DDB-1 E3 ligase is a central regulator that controls the extent of DNA replication, is required for germ cell viability, and regulates the critical cell cycle regulators CKI-1 and cyclin E.