CUL-4 is a key regulator of DNA replication in C. elegans. Inactivation of
cul-4 by RNAi produces an L2 stage arrest with enlarged postembryonic somatic blast cells. The DNA content of these cells continues to increase in subsequent days post-hatch, with over 100C DNA content observed in seam cells. The increased ploidy results from DNA re-replication caused by origin refiring (Feng et al., 2003, Nature 423: 885). The re-replication is linked to a failure during S phase to degrade the DNA replication licensing factor CDT-1 and to export the licensing factor CDC-6 from the nucleus. Based on structural similarity with its cullin paralogs, CUL-4 is expected to form a ubiquitin ligase complex with multiple components. In both humans and yeast, CUL-4 orthologs interact with the DDB1 protein. We found that
ddb-1 RNAi produced L2 larval arrest and re-replication phenotypes in C. elegans similar to that of
cul-4 RNAi. In addition to increased DNA ploidy in seam cells, we have confirmed that CDT-1, which is normally degraded in S phase, is not degraded in
ddb-1 RNAi larvae, similar to what is seen in
cul-4 RNAi larvae. We are seeking additional CUL-4 complex components through genetic and biochemical approaches. In humans, many CUL4A-associated proteins that are proposed to function as substrate-binding components (SBCs) have WD repeats, e.g., CSA, DDB2, and hCOP1. We would like to determine if any C. elegans WD proteins interact with CUL-4. There are 153 WD repeat genes in the C. elegans genome. We initially screened for an RNAi re-replication phenotype, e.g., greatly enlarged seam cells with high DNA content. So far, of the 39 tested WD repeat genes, none have produced a re-replication phenotype upon RNAi. It is possible that distinct SBCs target CDT-1 and CDC-6 regulation and that inactivation of one SBC independently of the other will not induce re-replication. Therefore, we are also directly screening for defects of CDT-1 degradation and CDC-6 export during S-phase. In addition we are in the process of affinity purifying CUL-4 complexes to identify associated proteins. We have tagged CUL-4 with the epitopes MYC, FLAG and GFP and are using strains expressing these genes to isolate the CUL-4 complex with antibodies specific for the tag. Associated proteins will be identified by mass spectrometry. We have already identified the cullin-associated protein CAND1, which is known to bind the inactive, deneddylated form of cullins (Liu et al, 2002, Mol. Cell 10:1511).