Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication Andrea Ballabenia, Raffaella Zamponib, Jodene K. Moorea, Kristian Helinc,d, and Marc W. Kirschnera,1 aDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115; bNovartis Institutes for Biomedical Research, Cambridge, MA 02139; cBiotech Research and Innovation Centre and dCentre for Epigenetics, University of Copenhagen, 2200 Copenhagen, Denmark Contributed by Marc W. Kirschner, June 7, 2013 (sent for review March 4, 2013) Cdc10-dependent transcript 1 (Cdt1) is an essential DNA replication the initiation of S phase and its duration. We show that although protein whose accumulation at the end of the cell cycle promotes Cdt1 protein accumulates in G2 phase, it still turns over very the formation of pre-replicative complexes and replication in the quickly and that to produce high Cdt1 levels when cells exit next cell cycle. Geminin is thought to be involved in licensing rep- mitosis into G1, the accumulation in G2 must overcome degra- lication by promoting the accumulation of Cdt1 in mitosis, because dation. This regulation is a product of Geminin’s positive regu- decreasing the Geminin levels prevents Cdt1 accumulation and lation of Cdt1 protein and RNA in the preceding G2 phase. impairs DNA replication. Geminin is known to inhibit Cdt1 func- Degradation of Cdt1 is not a consequence of DNA damage, be- tion; its depletion during G2 leads to DNA rereplication and check- cause Cdt1 levels decrease upon Geminin depletion even in point activation. Here we show that, despite rapid Cdt1 protein presence of inhibitors of DNA synthesis. Metaphase unleashes turnover in G2 phase, Geminin promotes Cdt1 accumulation by a precipitous degradation of Geminin via APC, leading to the increasing its RNA and protein levels in the unperturbed cell cycle. activation of Cdt1 in early G1 for pre-RC formation. Overall, Therefore, Geminin is a master regulator of cell-cycle progression these results show that Geminin is a master regulator of DNA that ensures the timely onset of DNA replication and prevents replication in the cell cycle of metazoans, ensuring that each DNA its rereplication. segment of the chromosome is replicated on time and only once before each cell division. n eukaryotic cells DNA replication occurs at a specific point of Ithe cell cycle known as S phase, which is flanked by two peri- Results ods, G1 and G2, during which there is no replication or cell Cdt1 in G2 Phase Is Both Abundant and Unstable. It has been shown division. The timing of S phase follows the formation of the pre- previously that Cdt1 levels increase after S phase and that Cdt1 replicative complexes (pre-RCs) on chromatin during the pre- protein is stabilized during G2 phase (4, 9). However, the levels ceding G1 phase and the activation of the cyclin-dependent kinase of a protein are not necessarily related to its turnover rate. (CDK) and dumbbell forming 4 (Dbf4)-dependent kinase (DDK) Therefore, we measured the turnover of Cdt1 protein in G2 in S phase (1). Cdc10-dependent transcript 1 (Cdt1) protein is phase by treatment with the protein synthesis inhibitor cyclo- indispensable for pre-RCs formation (2, 3); its levels fluctuate heximide. We first synchronized U2OS cells in S phase by during the cell cycle, being high in G1 phase, allowing pre-RC a double thymidine block. We then released the cells and started formation, low in S phase, preventing pre-RC formation and im- the cycloheximide treatment 8 h after the second thymidine re- mediate reinitiation, and high again in G2 and mitosis, presumably lease, when cells were still in S phase. We then collected cells for to prepare for G1 (3–5). Cdt1 activity is limited to G1 through the the next 4 h with time points at every hour (Fig. 1A). As shown by control of its synthesis, degradation, and activity. The low level in Western blot analysis, U2OS cells accumulate Cdt1 in G2, as S phase is thought to result from targeted degradation (6–8), they exit S phase, 10 h after release from thymidine. As expected, whereas its higher level in G2 is thought to result from its stabi- Cdt1 protein does not accumulate in G2 phase if the cells first lization (9). However, the increase of Cdt1 in G2 poses a potential are treated with cycloheximide during S phase. When cells were risk in allowing rereplication, which could occur if there were treated in parallel with a proteasome inhibitor, Cdt1 levels residual activity of the DNA-replicating enzymes in G2. The control of Cdt1 levels also is a response to Geminin (4, 10), Significance an unstable protein present only in metazoans, which is targeted for degradation by the anaphase-promoting complex (APC) (11). The master cell-cycle processes governing DNA replication and Geminin has two putative roles in the cell cycle: inhibiting Cdt1 mitosis in eukaryotic cells are regulated by cyclin/cyclin de- and promoting the accumulation of Cdt1 during mitosis. Both pendent kinase 1 and the anaphase-promoting complex, with Geminin and Cdt1 are expressed at high levels in G2, where checkpoint activity on these regulators. It is not these regu- Geminin binds Cdt1 and prevents DNA rereplication (12–14). A lators but rather intermediaries that communicate to the pro- critical role of Geminin in regulating the accumulation of Cdt1 cesses. Here we show that the protein Geminin acts centrally in levels has been inferred by the observation that the depletion of controlling DNA replication by ensuring that DNA is replicated Geminin leads to decreased Cdt1 protein levels in mitosis (4) during S phase and only once. This paper describes the Geminin and meiosis (10). However, it also has been suggested that “sub-master” regulatory circuit and the central role of Geminin Geminin actively inhibits Cdt1, because depletion of Geminin in in controlling events of the cell cycle. G2 phase activates Cdt1 and causes DNA rereplication and, consequentially, DNA damage (12). Because Cdt1 and cell Author contributions: A.B. and M.W.K. designed research; A.B., R.Z., and J.K.M. per- division cycle 6 (Cdc6) replication factors have been shown to be formed research; A.B., R.Z., K.H., and M.W.K. contributed new reagents/analytic tools; degraded after DNA damage (15–19), the Cdt1 decrease upon A.B., R.Z., K.H., and M.W.K. analyzed data; and A.B. and M.W.K. wrote the paper. Geminin depletion simply may be an indirect consequence of The authors declare no conflict of interest. DNA rereplication. 1To whom correspondence should be addressed. E-mail: [email protected]. In this paper we clarify the role of Geminin in regulating Cdt1 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and show more clearly how APC contributes to the regulation of 1073/pnas.1310677110/-/DCSupplemental. E2848–E2853 | PNAS | Published online July 8, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1310677110 Downloaded by guest on October 1, 2021 A B PNAS PLUS CD Fig. 1. Cdt1 is abundant in G2 phase but is highly unstable. (A) Schematic of the experiment. (B) U2OS cells were synchronized by double thymidine treatment and then were released for the indicated times. At 8 h after release (i.e., at the end of S phase) cells were left untreated or were treated with either cycloheximide (CHX) or carbobenzoxy-Leu-Leu-leucinal (MG132). Western blotting analysis for the indicated proteins is shown. APC2 protein was used as loading control. As, nonsynchronized cells; Thy, cells arrested in S phase. (C) Schematic of the experiment. (D) U2OS cells were synchronized by double thymidine treatment and then were released for the indicated times. At 11 h after release (i.e., in early G2 phase) cells were left untreated or were treated with either cycloheximide or MG132. Western blotting analysis for the indicated proteins is shown. APC2 protein was used as loading control. increased progressively during S and G2 phases (Fig. 1B), in- By Western blot the decrease of Cdt1 protein levels upon siRNA dicating that Cdt1 protein is both synthesized and degraded. for Geminin was unchanged when DNA replication was inhibi- Turnover continues in G2. Repeating the experiment with cy- ted (Fig. 2C). Furthermore the levels of Cdc6, which previously cloheximide and proteasome inhibitor treatments in G2 (Fig. had been shown to decrease upon DNA damage (15, 16), and of 1C), we found that Cdt1 protein disappeared completely 1 h Cdc7 remained unchanged upon Geminin depletion (Fig. 2C). after the addition of cycloheximide (Fig. 1D) but accumulated These results suggest that depletion of Geminin leads to a de- when the proteasome is inhibited (Fig. 1D). In both experiments, crease of Cdt1 independently of DNA rereplication. Moreover, Geminin and cyclin A remain stable. These experiments show that the stability of Cdc6 and cell division cycle 7 (Cdc7) proteins Cdt1 accumulates in the G2 phase of the cell cycle, but even in G2 suggests that any DNA rereplication in these experimental it remains extremely unstable with a half-life of less than 30 min. conditions is unlikely to have caused significant levels of DNA damage. These experiments therefore suggest that Geminin Geminin Acts Upstream of Cdt1, but Its Regulation Is Not Mediated by regulates Cdt1 in the normal cell cycle. DNA Damage and Replication. It is known that depletion of SYSTEMS BIOLOGY Geminin blocks the accumulation of Cdt1 protein in mitosis (4) Geminin Is a Positive Regulator of Cdt1 mRNA. The most obvious and meiosis (10); at the same time Geminin depletion leads to way Geminin would regulate the increase in Cdt1 levels in G2 unscheduled DNA replication.