The Human Licensing Factor for DNA Replication Cdt1 Accumulates in G1 and Is Destabilized after Initiation of S-phase

Abstract

S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in G 1 , 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 G 1 . After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G 2 . 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.

Involvement of CUL4 Ubiquitin E3 Ligases in Regulating CDK Inhibitors Dacapo/p27Kip1 and Cyclin E Degradation

“…21 CDT1 is also degraded in S phase in mammalian cells and such an event can be reproduced in Xenopus egg extracts in which CDT1 was found to undergo ubiquitin-dependent proteolysis once DNA replication starts. 37,38 In C. elegans, loss of CUL4 stabilizes CDT1 in S phase and causes the accumulation of polyploid nuclei containing 100C DNA content. 39 It is possible that CUL4 may also regulate the proteolysis of Dacapo or p27 in similar processes in Drosophila or human cells.…”

DNA replication licensing and cell cycle kinetics of oligodendroglial tumours

“…Moreover, cell synchronisation by membrane elution has revealed a similar cell cycle-dependent periodicity in Mcm5 and Mcm7 expression (Eward et al, manuscript submitted), findings that may have been previously obscured by metabolic perturbation due to the use of chemical synchronisation agents. Previous work has suggested that Geminin is present during S, G2 and early mitosis of the proliferative cell cycle before ubiquitination by the anaphasepromoting complex (McGarry and Kirschner, 1998;Wohlschlegel et al, 2000;Nishitani et al, 2001;Tada et al, 2001). Using our newly generated affinity-purified polyclonal anti-Geminin antibody G95 ( Figure 1A), we found little to no expression of this repressor of origin licensing during G1 phase, with detectable levels at the G1/S transition and increasing linearly during the remainder of the cycle before mitotic degradation ( Figure 1D).…”

Idas, a Novel Phylogenetically Conserved Geminin-related Protein, Binds to Geminin and Is Required for Cell Cycle Progression

“…Geminin, initially identified as a substrate of the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase during mitosis (2), was shown to regulate DNA replication by directly binding to and inhibiting the DNA replication licensing factor Cdt1 (3)(4)(5). Geminin binding to Cdt1 during the S and G 2 phases of the cell cycle ensures that licensing of a further round of DNA replication is inhibited until the end of mitosis (6,7).…”