To maintain genome stability, it is essential that genomic DNA is duplicated precisely during each cell cycle. The regulation of DNA replication is largely focused on controlling the formation of the pre-replicative complex (pre-RC) on replication origins during G1 phase. The pre-RC includes Cdt1 and Cdc6, which load the replicative helicase, the MCM2-7 complex. Cdt1 and Cdc6 activities are restricted either by degradation or by nuclear export at the beginning of S phase to prevent re-replication. CUL-4, which is a cullin-based ubiquitin ligase, plays a major role in controlling DNA replication in C. elegans (Zhong et al., Nature 2003). CUL-4 targets CDT-1 for ubiquitin-medicated proteolysis during S phase. However, a role of CUL-4 in regulating CDC-6 has not been described.
In higher eukaryotes, Cdc6 is exported from the nucleus in S phase. The phosphorylation of Cdc6 triggers its nuclear export. We found that in C. elegans, CDC-6 is also exported from the nucleus in S phase. In
cul-4(RNAi) larvae, CDC-6 fails to be exported during S phase, indicating that CUL-4 is required for this process. We hypothesized that CDC-6 nuclear export was induced by phosphorylation, as is the case in humans. The status of CDC-6 phosphorylation during S phase was tested by immunofluorescence with a phospho-specific anti-CDC-6 antibody, which recognizes phosphorylation of the T131 residue. We observed that T131 is phosphorylated during S phase in wild-type larvae, but is not phosphorylated in
cul-4(RNAi) larvae. To verify the functional relevance of CDC-6 phosphorylation in promoting nuclear export, we generated mutant versions of CDC-6 that are unable to be phosphorylated on specific CDK consensus sites. We observed that CDC-6 nuclear export is regulated by the phosphorylation of multiple sites. CDT-1 is degraded by CUL-4 during S phase, and CDT-1 accumulates in
cul-4 mutant cells. Therefore, We tested the hypothesis that the accumulation of CDT-1 indirectly causes the nuclear retention of CDC-6 in
cul-4 mutants by examining localization of CDC-6 in the presence of a stable form of CDT-1 in wild type. Stable CDT-1 does not block CDC-6 nuclear export in S phase, indicating that CDC-6 nuclear export occurs independently of CDT-1 degradation. Our data indicates that CUL-4 independently regulates CDT-1 by degradation and CDC-6 by nuclear export.