The functions of CUL-2 and CUL-4 in C. elegans cell cycle regulation

Abstract

CUL-2 and CUL-4 are members of the cullin family of ubiquitin-ligases that facilitate ubiquitin-mediated proteolysis. Here we demonstrate that in Caenorhabditis elegans, CUL-2 and CUL-4 are both essential cell cycle regulators. CUL-2 is required at two distinct points in the cell cycle, the G1-to-S phase transition and mitosis. cul-2 mutant germ cells undergo a G1 arrest that correlates with accumulation of CKI-1, a member of the CIP/KIP family of cyclin-dependent kinase inhibitors. In cul-2 mutant embryos, mitotic chromosomes are unable to condense, leading to unequal DNA segregation, chromosome bridging, and the formation of multiple nuclei in cells. C. elegans CUL-4 functions as a central regulator of DNA replication licensing to prevent DNA re-replication. Inactivation of CUL-4 causes massive re-replication of genomic DNA. To maintain genetic stability, eukaryotic cells must precisely replicate the entire genome only once per cell cycle. The presence of licensing proteins, such as Cdt1 and Cdc6, is required to allow the formation of pre-replicative complexes at replication origins. The removal of licensing proteins from chromatin in S phase is crucial to ensure that origins fire only once per cell cycle. Here we show that the C. elegans orthologs of Cdt1 and Cdc6 are both required for DNA replication. Both proteins accumulate in cul-4 re-replicating cells. In wild-type, CDT-1 protein levels peak in G1 phase and drop rapidly in S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease in S phase. Removal of one genomic copy of the cdt-1 gene suppresses the cul- 4 re-replication phenotype, indicating that CUL-4 prevents DNA re-replication at least partially by negatively regulating CDT-1 protein levels.