The Caenorhabditis elegans Replication Licensing Factor CDT-1 Is Targeted for Degradation by the CUL-4/DDB-1 Complex

Abstract

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. Two distinct ubiquitin ligase (E3) complexes, CUL4/DDB1 and SCF^Skp2, have been reported 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. Here, we show that Caenorhabditis elegans DDB-1 is required for the degradation of CDT-1 during S phase. DDB-1 interacts specifically with CUL-4 but not with other C. elegans cullins. A ddb-1 null mutant exhibits extensive DNA rereplication in postembryonic BLAST cells, similar to what is observed in cul-4(RNAi) larvae. DDB-1 physically associates with CDT-1, suggesting that CDT-1 is a direct substrate of the CUL-4/DDB-1 E3 complex. In contrast, a deletion mutant of the C. elegans Skp2 ortholog, skpt-1, appears overtly wild type with the exception of an impenetrant gonad migration defect. There is no appreciable role for SKPT-1 in the degradation of CDT-1 during S phase, even in a sensitized ddb-1 mutant background. We propose that the CUL-4/DDB-1 ubiquitin ligase is the principal E3 for regulating the extent of DNA replication in C. elegans.

We thank S. Mitani and the National Bioresource Project for the Experimental Animal Nematode C. elegans for providing the ddb-1(tm1769) and gmn-1(tm2212) alleles, the C. elegans Gene Knockout Consortium for providing the cul-4(gk434) allele, A. Fire for providing plasmid vectors, Y. Kohara for providing cDNA clones, the Caenorhabditis Genetics Center for providing strains, and members of the Kipreos laboratory for critical reading of the manuscript.

This work was supported by grant R01 GM055297 from the National Institute of General Medical Sciences (NIH).