The function of CUL-4 ubiquitin ligase complexes in DNA replication and enhancers of cullin-inhibitor CAND-1 in C. elegans
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Ubiquitin-mediated proteolysis plays a central role in specific and selective degradation of cell cycle regulators. E3 ubiquitin ligases determine the specificity of targets in the pathway. CUL-4, a member of the cullin-RING ubiquitin ligase (CRL) family, has a major function in restricting DNA replication in C. elegans. Genome stability is a fundamental issue for survival and is achieved by precise duplication of DNA. The temporal separation of the assembly of pre-replicative complexes from initiation of DNA replication prevents DNA re-replication. The replication licensing factors Cdc6 and Cdt1 participate in the formation of pre-replicative complexes during G1 phase, but they are regulated in different ways. Metazoan CUL4 mediates Cdt1 degradation and vertebrate Cdc6 is exported from the nucleus during S phase. However, because residual Cdc6 remains in the nucleus during S phase, it has been unclear whether Cdc6 translocation prevents re-replication. We show that C. elegans CDC-6 translocates from the nucleus during S phase in a multiple-phosphorylation-dependent manner. CUL-4 promotes CDC-6 phosphorylation and its subsequent nuclear export via negatively regulating the CDK-inhibitor CKI-1. Co-expression of non-exportable CDC-6 with non-degradable CDT-1 induces re-replication, but re-replication is not observed upon expression of deregulated CDT-1 or CDC-6, indicating that CDC-6 translocation and CDT-1 degradation redundantly prevent re-replication. CUL-4 independently controls both replication-licensing factors. CRL activity is regulated by Nedd8 conjugation that enhances CRL activity and also by CAND-1, a cullin-inhibitor. Although CAND-1 blocks assembly of CRL complexes, the absence of cand-1 leads to inactivation of CRLs, suggesting that CAND1 is required for proper CRL activity. However, the mechanism by which CAND1 promotes CRL activity is ambiguous. To understand the roles of CAND-1, the cand-1 enhancers dynein and hpk-1 (Homeodomain interacting Protein Kinase) are analyzed. Inactivation of dynein causes a reduction of CUL-2 deneddylation. However, CUL-2 is still functional and dynein(RNAi) does not lead to cul-2 mutant phenotypes. Combining the cand-1(tm1683) mutant with hpk-1(RNAi) causes embryonic lethality but is not obviously defective in CRL function or cell fate. Further investigation is necessary to understand the causes of embryonic lethality when both cand-1 and hpk-1 are co-inactivated.