Strict regulation of DNA replication is essential to ensure proper duplication and segregation of chromosomes during the cell cycle, as its deregulation can lead to genomic instability and cancer. Thus, eukaryotic organisms have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. Here, we show that inactivation of Geminin, an inhibitor of origin licensing, leads to rereplication in human normal and tumor cells within the same cell cycle. We found a CHK1-dependent checkpoint to be activated in rereplicating cells accompanied by formation of γH2AX and RAD51 nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show that Geminin is required for maintaining genomic stability in human cells.

Geminin is an unstable inhibitor of DNA replication that negatively regulates the licensing factor CDT1 and inhibits pre-replicative complex (pre-RC) formation in Xenopus egg extracts. Here we describe a novel function of Geminin. We demonstrate that human Geminin protects CDT1 from proteasome-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle. Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate that Geminin is both a negative and positive regulator of pre-RC formation in human cells, playing a positive role in allowing CDT1 accumulation in G2-M, and preventing relicensing of origins in S-G2.

ES cells proliferate with very short gap phases yet maintain their capacity to differentiate. It had been thought that the levels of cyclins and other substrates of ubiquitin ligase APC/C remain nearly constant and Cdk activity remains constitutively high in mouse ES cells. Here we demonstrate that APC/C (anaphase-promoting complex/cyclosome) enzyme is active in ES cells but attenuated by high levels of the Emi1 (early mitotic inhibitor-1) protein. Despite the presence of high Cdk activity during the G1 phase, chromatin can be effectively licensed for DNA replication and fast entry into the S phase can still occur. High Cdk activity during S-G2-M phases produces high levels of the DNA replication factor Cdt1, and this leads to efficient Mcm proteins loading on chromatin after mitotic exit. Although disturbing the usual balance between Cdk activity and APC/C activity found in somatic cells, a few key adaptations allow normal progression of a very rapid cell cycle.E mbryonic stem cells show unusual cell-cycle features: the duration of the S phase is comparable to somatic cells but they have remarkably short G1 and G2 phases (1-3). In somatic cells, the duration of G1 and G2 is determined by relative levels of Cdk kinase activity and other cell cycle-related proteins (4). Many of these proteins, including Cyclin A, Cyclin B, Cdt1, Cdc6, and Geminin fluctuate along the cell cycle because of degradation mediated by E3 ubiquitin ligase APC/C (anaphase-promoting complex/ cyclosome) together with E2 enzymes, such as UbcH10 and UBE2S (5-8). APC/C is activated at the end of mitosis by interaction with Cdc20 and Cdh1 proteins and inactivated just before the S phase by the pseudosubstrate inhibitor Emi1 (early mitotic inhibitor-1) and by the phosphorylation and degradation of Cdh1 (6, 9, 10). Cdk kinases are activated by Cyclins and phosphorylate a number of cell-cycle proteins important for mitotic and S phase progression. Cdk activity is inhibited during G1 in somatic cells because of degradation of Cyclins and presence of inhibitor proteins, like p21 (11). Inhibition of Cdk activity in the G1 phase allows the replication factors Cdt1 and Cdc6 to recruit Mcm proteins on chromatin, form prereplicative complexes (pre-RCs), and license DNA for replication (12)(13)(14). Geminin protein inhibits Cdt1 during the S phase and promotes its stabilization during mitosis (3,13,(15)(16)(17)(18)(19)(20). A puzzling feature of ES cells is that APC/C substrates were shown to be constant and Cdk activity to be high throughout the ES cell cycle (1,3,21), raising the question of whether the APC/C complex is functional and how ES cells regulate pre-RC assembly at G1. Remarkably, APC/C substrates and other positive cell-cycle regulators decrease after differentiation (1,3,22). We carefully reinvestigated cell-cycle dynamics in ES cells. Contrary to previous conclusions, APC/C substrate levels and Cdk activity both oscillate, although in a more muted manner compared with most studied somatic models. A few key adaptations promote an abbreviated...

In somatic cells, the length of the G1 phase of the cell cycle is tightly linked to differentiation, and its elongation can drive differentiation in many cases. Although it has been suggested that the situation is very similar in embryonic stem cells (ESCs), where a rapid cell cycle and a short G1 phase maintain the pluripotent state, evidence has been contradictory. Here we show that, in murine ESCs, elongation of the cell cycle and elongation of G1 are compatible with their pluripotent state. Multiple methods that lengthen the cell cycle and that target cyclin-dependent kinase, retinoblastoma protein, and E2F activity all fail to induce differentiation on their own or even to facilitate differentiation. The resistance of murine ESCs to differentiation induced by lengthening G1 and/or the cell cycle could allow for separate control of these events and provide new opportunities for investigation and application.stemness | proliferation | decoupling