Cdt1 and Cdc6 Are Destabilized by Rereplication-induced DNA Damage

Abstract

The replication factors Cdt1 and Cdc6 are essential for origin licensing, a prerequisite for DNA replication initiation. Mechanisms to ensure that metazoan origins initiate once per cell cycle include degradation of Cdt1 during S phase and inhibition of Cdt1 by the geminin protein. Geminin depletion or overexpression of Cdt1 or Cdc6 in human cells causes rereplication, a form of endogenous DNA damage. Rereplication induced by these manipulations is however uneven and incomplete, suggesting that one or more mechanisms restrain rereplication once it begins. We find that both Cdt1 and Cdc6 are degraded in geminin-depleted cells. We further show that Cdt1 degradation in cells that have rereplicated requires the PCNA binding site of Cdt1 and the Cul4 DDB1 ubiquitin ligase, and Cdt1 can induce its own degradation when overproduced. Cdc6 degradation in geminin-depleted cells requires Huwe1, the ubiquitin ligase that regulates Cdc6 after DNA damage. Moreover, perturbations that specifically disrupt Cdt1 and Cdc6 degradation in response to DNA damage exacerbate rereplication when combined with geminin depletion, and this enhanced rereplication occurs in both human cells and in Drosophila melanogaster cells. We conclude that rereplication-associated DNA damage triggers Cdt1 and Cdc6 ubiquitination and destruction, and propose that this pathway represents an evolutionarily conserved mechanism that minimizes the extent of rereplication.One of the critical events in the cell division cycle is complete and precise duplication of the genome. In eukaryotic cells, origins of DNA replication acquire replication competence through the assembly of a prereplication complex (preRC) 3 in the G 1 phase of the cell cycle. PreRCs are assembled by the sequential origin binding of the origin recognition complex (ORC), Cdc6, Cdt1, and the minichromosome maintenance complex (MCM). Origins harboring preRCs are licensed for replication but do not initiate DNA synthesis until S phase begins and the Cdc7 and Cdk2 kinases are activated (1-3). The large genomes of metazoan cells necessitate the utilization of thousands of origins, but each origin that initiates DNA synthesis must do so only once. Failure to maintain this control has been linked to genome instability and oncogenesis (4, 5), but the cellular consequences of rereplication are not fully understood. Multiple regulatory mechanisms operate to ensure that any origins that have "fired" do not fire again by blocking preRC assembly after the G 1 to S phase transition. Among the most important of these mechanisms are degradation of Cdt1 during S phase and inhibition of Cdt1 by the geminin protein. Geminin depletion, overexpression of Cdt1, or overexpression of Cdc6 causes rereplication, which ultimately triggers a DNA damage response (6 -11) and can promote tumorigenesis (4, 5). Rereplication induced by these manipulations is however incomplete, with the extent of rereplication varying widely among different cell lines. These observations suggest that in addition to the mechanisms that bloc...

A spontaneous Cdt1 mutation in 129 mouse strains reveals a regulatory domain restraining replication licensing

“…S5F) and strongly induced re-replication ( Supplementary Fig. S5G), as previously reported 39 31.7% ΔPEST ( Supplementary Fig. S5G, see also Fig.…”

Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication

“…2C). Furthermore the levels of Cdc6, which previously had been shown to decrease upon DNA damage (15,16), and of Cdc7 remained unchanged upon Geminin depletion (Fig. 2C).…”

“…However, it also has been suggested that Geminin actively inhibits Cdt1, because depletion of Geminin in G2 phase activates Cdt1 and causes DNA rereplication and, consequentially, DNA damage (12). Because Cdt1 and cell division cycle 6 (Cdc6) replication factors have been shown to be degraded after DNA damage (15)(16)(17)(18)(19), the Cdt1 decrease upon Geminin depletion simply may be an indirect consequence of DNA rereplication.…”

CDK1-dependent Inhibition of the E3 Ubiquitin Ligase CRL4CDT2 Ensures Robust Transition from S Phase to Mitosis

“…Flow cytometry analysis was performed using a Cyan FACScan (DakoCytomation) and Summit version 4.3 software (DakoCytomation) as described previously (36).…”