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The nucleotide exchange activity of RCC1, the only known nucleotide exchange factor for Ran, a Ras-like small guanosine triphosphatase, was required for microtubule aster formation with or without demembranated sperm in Xenopus egg extracts arrested in meiosis II. Consistently, in the RCC1-depleted egg extracts, Ran guanosine triphosphate (RanGTP), but not Ran guanosine diphosphate (RanGDP), induced self-organization of microtubule asters, and the process required the activity of dynein. Thus, Ran was shown to regulate formation of the microtubule network.

Previous reports showed that chromatin-associated PCNA couples DNA replication with Cul4-DDB1^Cdt2-dependent proteolysis of the licensing factor Cdt1. The CDK inhibitor p21, another PCNA-binding protein, is also degraded both in S phase and after UV irradiation. Here we show that p21 is degraded by the same ubiquitin-proteasome pathway as Cdt1 in HeLa cells. When PCNA or components of Cul4-DDB1^Cdt2 were silenced or when the PCNA binding site on p21 was mutated, degradation of p21 was prevented both in S phase and after UV irradiation. p21 was co-immunoprecipitated with Cul4A and DDB1 proteins when expressed in cells. The purified Cul4A-DDB1^Cdt2 complex ubiquitinated p21 in vitro. Consistently, p21 protein levels are low during S phase and increase around G₂ phase. Mutational analysis suggested that in addition to the PCNA binding domain, its flanking regions are also important for recognition by Cul4-DDB1^Cdt2. Our findings provide a new aspect of proteolytic control of p21 during the cell cycle.

S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in G₁, and passage through S-phase removes the license to replicate. In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring that replication occurs once per cell cycle. We show that the human Cdt1 homologue (hCdt1), a nuclear protein, is present only during G₁. After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G₂. hCdt1 can associate with the DNA replication inhibitor Geminin, however these two proteins are mostly expressed at different cell cycle stages. hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase-arrested cells, and its levels do not change dramatically during a cell cycle, suggesting that proteolytic rather than transcriptional controls ensure the timely accumulation of hCdt1. Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase. In contrast, hCdc6/18 levels are constant through most of the cell cycle and are only low for a brief period at the end of mitosis. These results suggest that the presence of active hCdt1 may be crucial for determining when licensing is legitimate in human cells.