MCM9 Binds Cdt1 and Is Required for the Assembly of Prereplication Complexes

Molecular Cell

Article MCM9 Binds Cdt1 and Is Required for the Assembly of Prereplication Complexes Malik Lutzmann1 and Marcel Me´chali1,* 1Institute of Human Genetics, CNRS, 141 rue de la Cardonille, 34396 Montpellier, France *Correspondence: [email protected] DOI 10.1016/j.molcel.2008.07.001

SUMMARY

Prereplication complexes (pre-RCs) define potential origins of DNA replication and allow the recruitment of the replicative DNA helicase MCM2-7. Here, we characterize MCM9, a member of the MCM2-8 family. We demonstrate that MCM9 binds to chromatin in an ORC-dependent manner and is required for the recruitment of the MCM2-7 helicase onto chromatin. Its depletion leads to a block in pre-RC assembly, as well as DNA replication inhibition. We show that MCM9 forms a stable complex with the licensing factor Cdt1, preventing an excess of geminin on chromatin during the licensing reaction. Our data suggest that MCM9 is an essential activating linker between Cdt1 and the MCM2-7 complex, required for loading the MCM2-7 helicase onto DNA replication origins. Thus, Cdt1, with its two opposing regulatory binding factors MCM9 and geminin, appears to be a major platform on the pre-RC to integrate cell-cycle signals. INTRODUCTION DNA replication is tightly controlled to ensure that the whole genome is replicated only once during each cell cycle. A major step in this process is the licensing reaction, which assembles prereplication complexes (pre-RCs) onto origins of replication and is restricted to late mitosis and G1 phase of the cell cycle. First, ORC-complex binds to potential DNA replication origins, recruiting CDC6 and Cdt1. Once Cdt1 is chromatin bound, the MCM2-7 helicase complex is loaded by an unknown mechanism (Takahashi et al., 2005). This step completes the assembly of the pre-RC and the licensing reaction. ORC and CDC6 are ATPases and their ATP hydrolysis activity is essential for the iterative loading of MCM2-7 complexes onto DNA (Bowers et al., 2004; Speck et al., 2005; Randell et al., 2006). In contrast, Cdt1 lacks known enzymatic activity, although it is also essential for MCM2-7 loading (Maiorano et al., 2000). Cdt1 regulation is crucial for ordered DNA replication origin firing and, with its negative regulator geminin, also appears to participate in cellular decisions involving proliferation and differentiation (Del Bene et al., 2004; Luo et al., 2004; Seo et al., 2005). It is the only pre-RC member that causes illegitimate rereplication in metazoans during the same cell cycle when present in excess

190 Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc.

during S phase or G2 (Arias and Walter, 2005; Li and Blow, 2005; Maiorano et al., 2005; Yoshida et al., 2005, Lutzmann et al., 2006). Cells have developed several ways to negatively control Cdt1 activity through degradation, nuclear export, or inhibition by geminin (for review see Blow and Dutta, 2005). A direct interaction between Cdt1 and the MCM2-7 helicase cannot be detected in vivo in higher eukaryotes, although both proteins can be forced to interact in vitro using purified proteins (Yanagi et al., 2002; Cook et al., 2004; Lee et al. 2004; Fehrenbach et al. 2005; Lutzmann et al., 2006). Thus, it is not understood how, in complex eukaryotes, Cdt1 recruits MCM2-7 at the assembling pre-RC. Here, we show that a member of the MCM2-8 gene family, MCM9, is required for this reaction. We show that the in silico-predicted MCM9 protein (Lutzmann et al., 2005) is expressed and required for loading the MCM2-7 helicase onto chromatin during pre-RC formation. Furthermore, we demonstrate that MCM9 directly forms a complex with Cdt1 and prevents an elevated and inhibitory ratio of geminin to Cdt1 on chromatin. Our data suggest that MCM9, in contrast to geminin, is a positive regulator of Cdt1 and plays the role of a colicenser of DNA replication. RESULTS MCM9, an MCM Family Protein Expressed in Vertebrates The predicted Xenopus and human MCM9 proteins consist of 1143 amino acids with a molecular weight of approximately 130 kDa. They have an N-terminal half, which shares high homology with MCM2-8 proteins and an additional C terminus of 650 amino acids, which does not show clear homology with any other protein in the databanks (Figure 1A). We raised antibodies against the C terminus of the Xenopus protein and against two peptides from the C terminus of the human protein in order to avoid crossreactions with the known MCM2-8 proteins. The antibody raised against the Xenopus protein recognized a band of the predicted size of 130 kDa in egg extracts (Figure 1B) and also recognized recombinant Xenopus MCM9 (Figure 1C). The antibody further recognized Xenopus MCM9 proteins, which were expressed in SF9 cells via the baculovirus system (Figure S1 available online). In human cells, MCM9 was also expressed as shown by the specific antibody raised against the peptides from the human protein (Figure 1D). MCM9 Depletion Abolishes DNA Replication To investigate the function of MCM9, we took advantage of the Xenopus egg extract system. In this cell-free system, added

Molecular Cell MCM9 Is Required for Pre-RC Formation

Figure 1. MCM9 Is Expressed as a 130 kDa Protein (A) Domain organization of the MCM9 protein. (B) Western blot analysis of egg extracts with preimmune antibody (left) and XLMCM9 antibody (right). (C) Comparison of western blot MCM9 signals in egg extracts and in reticulocyte-lysates translating XLMCM9 (or not) with the 35S-autoradiography of labeled XLMCM9 translated in the same recticulocyte lysate. (D) Western blot analysis of HeLa cell extracts with preimmune antibody (right) and HSMCM9 antibody (right).

sperm DNA is converted into chromatin, prereplication complexes are then assembled and, after nuclear membrane formation, DNA is replicated exactly once. We depleted the protein from extracts using the antibody against the Xenopus protein and then compared the ability of MCM9-depleted extracts to replicate chromosomal DNA with that of extracts mock depleted with an unspecific antibody. As shown in Figure 2A, MCM9 could be removed from extracts to undetectable levels, whereas ORC levels remained unchanged. Chromosomal DNA replication was abolished in the MCM9depleted extracts, whereas mock-depleted extracts replicated normally (Figure 2B). Figure 2C shows that replication of ssDNA, which does not require the formation of a replication fork, was not affected by the absence of MCM9. Also not affected was the assembly of the replicated ssDNA into chromatin (Figure S2). These results show that MCM9 is not involved in complementary DNA strand synthesis, which requires the RNA primase-DNA polymerase machinery (Me´chali and Harland, 1982), or in nucleosome assembly coupled to DNA synthesis (Almouzni and Me´chali, 1988). We also analyzed nuclear membrane formation in MCM9-depleted extracts, as its assembly is required for double-stranded DNA replication (Blow and Laskey, 1988). Nuclear membrane formation appeared to be normal in both mock- and MCM9depleted extracts (Figure S3).

MCM9 Is Required for Loading the MCM2-7 Complex onto Chromatin The replication defect observed in MCM9-depleted extracts suggested a defect at or before replication initiation. We investigated the recruitment of pre-RC proteins to chromatin in the presence or absence of MCM9, using histone H3 as an independent loading control (Figure 2D). ORC levels on chromatin did not

change upon depletion of MCM9. In contrast, the amount of chromatin-bound CDC6 in MCM9-depleted extracts was higher, while Cdt1 levels were reduced, which suggests either a destabilization of Cdt1 in the absence of MCM9 or an interaction with MCM9 itself. Most strikingly, we observed a complete absence of the MCM2-7 complex on chromatin formed in MCM9-depleted extracts (Figure 2D). We tried to rescue this phenotype by adding recombinant MCM9, but did not succeed in expressing and purifying, from baculovirus-infected SF9 cells or from reticulocyte lysates, an active, recombinant protein, which could significantly restore the phenotype. We then produced recombinant MCM9 directly in depleted egg extracts via in vitro transcribed mRNA (Matthews and Colman, 1991) to facilitate proper folding and possible modifications of the protein. We validated this method of in extracto translation from in vitro transcribed mRNA by the rescue of Cdt1-depleted extracts by Cdt1 mRNA (Figure S4). The in extracto translated MCM9 protein was able to restore MCM2-7 recruitment to chromatin (Figure 2E) and was also able to significantly rescue chromosomal DNA replication (Figure 2F; Figures S5 and S6). Since we observed an incomplete rescue of DNA replication by recombinant MCM9, we cannot rule out that other factors might be (partially) codepleted upon depletion of MCM9, which, together with MCM9, are necessary for a full restoration of DNA replication. However, our data clearly show that MCM9 is required for the recruitment of the MCM2-7 helicase on chromatin during the assembly of pre-RCs. MCM9 Binds Rapidly to Chromatin in an ORC-Dependent Manner We next investigated whether MCM9 itself associates with chromatin. The analysis of the pre-RC assembly reaction shown in Figure 3A demonstrates that MCM9 rapidly binds to chromatin, much earlier as the MCM2-7 helicase and with kinetics similar to that of ORC and Cdt1. Addition of geminin completely blocked MCM2-7 recruitment (last lane of Figure 3A) and increased CDC6 on chromatin, as expected (Tada et al., 2001; Maiorano et al., 2004), but did not influence MCM9 binding to chromatin. These results show that MCM9 is recruited to chromatin upstream and independently of the MCM2-7 loading step and is in agreement

Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc. 191

Molecular Cell MCM9 Is Required for Pre-RC Formation

Figure 2. MCM9 Depletion Abolishes Replication of Sperm Chromatin, but Not of Single-Stranded DNA, and Is Essential for Pre-RC Assembly (A) Western blot analysis of MCM9 depletion from egg extracts. As a loading control, extracts were also blotted for ORC2. (B) Kinetics of sperm chromatin replication in either mock- (circles) or in MCM9-depleted egg extracts (squares). (C) Replication kinetics of either ssDNA (squares) or sperm chromatin (circles) in MCM9-depleted egg extracts. Also shown is ssDNA replication in a mock-depleted egg extract (triangles). (D) Western blot analysis of Histone H3 (as loading control), ORC2, ORC1, CDC6, Cdt1, and MCM2-7 proteins on chromatin assembled in mock- and MCM9-depleted egg extracts. (E) Left panel: in extracto translation of MCM9 mRNA in MCM9-depleted extracts. Shown is a western blot analysis of mock- and MCM9depleted egg extracts, supplemented or not with MCM9 mRNA at 0, 30, and 60 min after mRNA addition. Right panel: western blot analysis of ORC2 (as loading control) and MCM2-7 proteins on chromatin assembled in the extracts shown in the left panel. (F) Left panel: western blot analysis of egg extracts that were mock depleted, MCM9 depleted, and MCM9 depleted supplemented with translated MCM9 protein. Right panel: replication kinetics of sperm chromatin in these extracts.

dently of CDC6, and that MCM9 is required to load the MCM2-7 helicase to complete pre-RC assembly.

with the phenotype observed in MCM9-depleted extracts (Figure 2D). Figure 3B shows that MCM9 also remained chromatin bound throughout S phase, and this association gradually decreased at the end of S phase. As shown in Figure 3C, ORC2-depletion from egg extracts abolished MCM9 binding to chromatin. In contrast, depletion of CDC6 neither affected binding of MCM9 to chromatin (Figure 3D) nor the binding of Cdt1, as reported previously. These results show that chromatin recruitment of MCM9 requires ORC, but not CDC6 association to chromatin. Figure 3E shows that depletion of Cdt1 reduced both MCM9 levels in the extract and its recruitment to chromatin, interestingly, like the MCM9 depletion did with Cdt1 (Figure 2D, see also below). From these data, we concluded that MCM9 is recruited to an assembling pre-RC in an ORC-dependent manner, but indepen-

192 Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc.

Licensed Chromatin Does Not Require Non-Chromatin-Bound MCM9 to Replicate Since MCM9 is essential for the recruitment of the MCM2-7 complex during licensing, it should be possible to rescue replication in a MCM9-depleted extract by licensed sperm chromatin as a template, which has already loaded MCM2-7 complexes. To test this, we assembled sperm chromatin in (nondepleted) extracts for 12 min, the time required for licensing to happen (Rowles et al., 1999; Maiorano et al., 2004; Lutzmann et al., 2006), but not enough to build a nuclear membrane around chromatin and to recruit proteins downstream of pre-RC formation. Figure 3F shows that this chromatin template could replicate both in mock- and MCM9-depleted extracts. Figure 3F also demonstrates that the MCM9-depleted extract could not replicate unlicensed sperm chromatin and that the licensed chromatin could not be replicated in a high speed extract, which does not contain the membranes necessary to form a nuclear envelope. These experiments indicate that once MCM9 is loaded onto chromatin, no additional loading of MCM9 is required during the initiation or elongation phases of DNA synthesis.

Molecular Cell MCM9 Is Required for Pre-RC Formation

Figure 3. Characterization of the Chromatin Recruitment of MCM9 (A) Western blot analysis of MCM9, MCM2-7, Cdt1, CDC6, ORC2, and Histone H3 (as a loading control) during a pre-RC assembly time course. Chromatin was assembled in egg extracts and purified at the indicated times. Also shown is a mock purification (first lane) without addition of sperm chromatin to determine background staining and a reaction of chromatin assembled in egg extract blocked by 40 nM recombinant geminin (last lane). (B) Left panel: Western blot analysis for MCM9, MCM2-7, Cdt1, and ORC2 during a time course of chromatin assembled in egg extracts such as in (A) that covers pre-RC assembly and S phase. Also shown is a mock purification (last lane) without added sperm chromatin to determine background staining. Right panel: replication kinetics of the time course. (C) Left panel: western blot analysis of egg extract either mock depleted or ORC2 depleted for MCM9 and ORC2. Right panel: western blot analysis of chromatin assembled in these extracts for MCM9, Cdt1, ORC2, and histone H3 as a loading control. (D) Left panel: western blot analysis of egg extract either mock depleted or CDC6 depleted for MCM9 and CDC6. Right panel: western blot analysis of chromatin assembled in these extracts for MCM9, MCM2-7, Cdt1, CDC6, and ORC2 as a loading control. (E) Left panel: western blot analysis of egg extract either mock depleted or Cdt1 depleted for MCM9 and Cdt1. Right panel: western blot analysis of chromatin assembled in these extracts for MCM9, MCM2-7, Cdt1, and ORC2 as loading control. (F) Kinetics of replication of sperm chromatin and licensed chromatin (containing loaded MCM2-7 complexes) in mock-depleted (open circles for sperm chromatin, closed circles for licensed chromatin) and MCM9-depleted (open squares for sperm chromatin, closed squares for licensed chromatin) egg extracts. Also shown is the inability of high-speed egg extract devoid of membranes to replicate the licensed chromatin (triangles).

Rowles et al. (1999) showed that a salt extraction (150–250 mM KCl) of licensed chromatin dissociates ORC complex from chromatin, and Maiorano et al. (2000) showed similar results for Cdt1. Importantly, this salt extraction does not remove chromatin-bound MCM2-7 complexes and does not affect the ability of the salt extracted chromatin to replicate (Rowles et al., 1999; Maiorano et al., 2000) We tried to remove MCM9 from chromatin by a similar salt wash, but the majority of MCM9 resisted a salt extraction even at 250 mM KCl (Figure S7). We conclude that MCM9, like the MCM2-7 complex, is tightly bound to chromatin and that after MCM2-7 loading, soluble MCM9 is not further required for DNA replication. MCM9 and Cdt1 Interact Directly and Form a Stable Complex Since MCM9 depletion reduces chromatin-bound Cdt1 levels (Figure 2D) and Cdt1 depletion affects MCM9 levels on chromatin (Figure 3E), we asked whether MCM9 might interact with Cdt1. Figure 4A shows that the soluble amounts of ORC1, ORC2, CDC6, and MCM2-7 proteins in egg extract (as well as MCM8) were not affected by depletion of MCM9. In contrast,

we detected a reduction of Cdt1 levels, such as that observed on chromatin assembled in the absence of MCM9 (Figure 2D). We next depleted, in parallel, equivalent aliquots of the same egg extract of either Cdt1 or MCM9. As shown in Figure 4B, depletion of MCM9 reduced Cdt1 levels in that extract, and depletion of Cdt1 reduced MCM9 levels, respectively. In contrast, MCM2-7 levels remained unchanged in both reactions, compared to the mock depletion. The reduced levels of Cdt1 in MCM9-depleted extracts and of MCM9 in Cdt1-depleted extracts, respectively, were not due to an accelerated degradation of the proteins as remaining proteins were as stable as in mockdepleted control extracts (Figure S8). These results suggested a specific interaction between MCM9 and Cdt1. We then asked whether a stable interaction between MCM9 and Cdt1 could be detected through coimmunoprecipitation. Figure 4C, lane 2, shows that MCM9 antibodies indeed coprecipitated Cdt1 from the extract. Reciprocally, we found that MCM9 was present in immunoprecipitates of Cdt1, although to a lesser extent (Figure 4C, lane 3). We did not detect other pre-RC proteins like ORC, CDC6, or MCM3 in MCM9 immunoprecipitations, and immunoprecipitations of these proteins did not contain

Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc. 193

Molecular Cell MCM9 Is Required for Pre-RC Formation

Figure 4. MCM9 Interacts Stably and Directly with Cdt1 (A) Western blot analysis of MCM9, MCM8, MCM2-7, Cdt1, CDC6, ORC1, and ORC2 in mock- and MCM9-depleted egg extracts. (B) Western blot analysis of MCM9, MCM2-7, and Cdt1 in egg extracts mock depleted (lane 1), MCM9 depleted (lane 2), or Cdt1 depleted (lane 3). (C) Western blot analysis of immunoprecipitations of MCM9 (lane 2) and Cdt1 (lane 3). Also shown is a mock immunoprecipitation to determine background signals (lane 1). Further shown is the signal obtained for the IgGs to control the quantity of antibody used in each reaction. (D) Western blot analysis of fractionated egg extracts on a linear 7% to 23% sucrose gradient. Fractions were analyzed for the presence of MCM9 (two different exposures of the western blot are shown), MCM2-7, Cdt1, and geminin. The size and positions of globular marker proteins used for calibration are also indicated. (E) Western blot analysis of MCM9 and Cdt1 in pooled fractions 13–15 (high molecular weight pool) and pooled fractions 17–19 (low molecular weight pool). These pools were subjected to an immunoprecipitation of MCM9 and a mock immunoprecipitation, which were analyzed by western blot for MCM9 and Cdt1. Also shown is the signal obtained for the IgG to control the amount of antibody used in each immunoprecipitation. (F) Interaction of GST-Cdt1 and (untagged) MCM9 coexpressed in SF9 cells. The left lane shows a mock purification with GSH-Sepharose of untagged MCM9 expressed alone in SF9 cells to determine unspecific background binding of the (untagged) MCM9 to GSH-Sepharose. The right lane shows the purification of GST-Cdt1 coexpressed with untagged MCM9 by GSH-Sepharose.

MCM9 (Figure S9). We concluded that MCM9 interacts specifically, directly or indirectly, with Cdt1, but not with the MCM2-7 helicase or other pre-RC proteins. We further investigated whether the sedimentation properties of MCM9 were in agreement with formation of a complex with Cdt1. Sucrose gradient analysis (Figure 4D) shows that most of the MCM9 in egg extract sediments at an S value corresponding to 150 kDa, suggesting a mainly monomeric pool of the protein. However, a fraction of the protein sediments at higher molecular weight. As previously shown (Maiorano et al., 2004), Cdt1 (70 kDa) sediments as a broad peak between 100 and 300 kDa, which overlaps with the MCM9 sedimentation profile (Figure 4D). This would be in agreement with the presence of a (lower molecular weight) fraction of Cdt1, mainly associated

194 Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc.

with geminin (fractions 17–19), and a fraction mainly associated with MCM9 (fractions 13–15). To test this, we pooled the corresponding MCM9-containing high and low molecular weight fractions (fractions 13–15 and 17–19) and performed immunoprecipitation of MCM9 from these pools. As shown in Figure 4E, Cdt1 coimmunoprecipitated with MCM9 from the high molecular weight pool much more efficiently than from nonfractionated egg extracts. In addition, very little Cdt1 coprecipitated with MCM9 from the low molecular weight pool (despite the higher absolute amount of MCM9 in this pool), in agreement with an enrichment of a stable MCM9-Cdt1 complex by the sucrose gradient in fractions 13–15. We conclude that MCM9 and Cdt1 form a stable complex in egg extract. Since depletion of MCM9 reduces Cdt1 levels in egg extract (Figure 4A), we also investigated whether the addition of recombinant Cdt1 could rescue the replication defect of MCM9depleted extracts. However, neither restoration of physiological, nor addition of excessive amounts of, Cdt1 resulted in an important rescue of DNA replication (Figures S10A and S10B). We

Molecular Cell MCM9 Is Required for Pre-RC Formation

Figure 5. MCM9 Affects the Geminin to Cdt1 Ratio on Chromatin during Licensing (A) Left panel: western blot analysis of egg extract either mock depleted or MCM9 depleted for MCM9, Cdt1, and ORC2. Right panel: western blot analysis of chromatin assembled in these extracts for MCM2-7, Cdt1, geminin, and ORC2 (as a loading control). (B) Left panel: western blot analysis of egg extract either double depleted mock/mock, MCM9/mock, or MCM9/geminin for MCM9, Cdt1, and geminin. Right panel: western blot analysis of chromatin assembled in these extracts for MCM9, MCM2-7, Cdt1, ORC2 (as a loading control), and geminin. (C) Kinetics of sperm chromatin replication in these double-depleted extracts.

concluded that MCM9 interacts stably with Cdt1 and is required for the MCM2-7 helicase loading onto chromatin, in a reaction, which cannot be substituted by Cdt1 alone. Next, we asked whether the interaction between Cdt1 and MCM9 was direct. We coexpressed both proteins via the baculovirus system in SF9 cells, Cdt1 was expressed as a GST-fusion protein, and MCM9 was coexpressed without affinity tag. GST-affinity purification of Cdt1 clearly revealed an interaction between MCM9 and Cdt1, showing that both proteins can directly interact. (Figure 4F; Figure S11). We conclude that MCM9 can form a stable and direct complex with Cdt1. We also tested the recombinant MCM9-Cdt1 complex for its ability to rescue an MCM9-depleted extract, and the recombinant complex restored replication activity to 30% (Figure S12). This relatively low effect might be due to the substoichiometric assembly of MCM9 in this recombinant complex (Figures S11 and S12). MCM9 Affects the Amount of Geminin Bound to Cdt1 on Chromatin during Licensing Cdt1 binds its negative regulatory partner geminin tightly, and both proteins can be found in immunoprecipitations of the partner protein (Wohlschlegel et al., 2000; Nishitani et al., 2001). Therefore, we tested if geminin would also coimmunoprecipitate with MCM9 through its interaction with Cdt1, and we could detect small amounts of geminin in immunoprecipitations of MCM9 (Figure S13, which shows the geminin analysis of the immunoprecipitations shown in Figure 4C). This suggests that MCM9, Cdt1, and geminin may interact in egg extract. Since an excess of geminin blocks MCM2-7 recruitment through its association with Cdt1, we next analyzed geminin

levels on chromatin that was assembled either in mock-or MCM9-depleted egg extracts (Figure 5A). In the absence of MCM9, Cdt1 levels on chromatin were reduced as shown before (Figure 2D), but not geminin levels. This caused a significant increase in the ratio of geminin to Cdt1 on chromatin (see also the corresponding geminin level of the independent experiment presented in Figure 2D, presented in Figure S14). These data suggested that one function of MCM9 might be to control the geminin to Cdt1 stoichiometry on chromatin during licensing. To test this further, we double-depleted egg extracts for MCM9 and geminin to test if the absence of geminin could restore pre-RC formation in MCM9-depleted extracts. Double depletion of MCM9 and geminin removed entirely Cdt1 from extracts (Figure 5B), suggesting that most of the Cdt1 in egg extract is either bound to geminin or MCM9 (or both). Consequently, MCM9 and geminin double-depleted extracts cannot recruit MCM2-7 complexes (Figure 5B) and do not initiate DNA replication (Figure 5C). These data confirm our previous results (Figure 4) and show in a different way that MCM9 is an important stoichiometric regulator of the Cdt1-geminin ratio. This is confirmed in the chromatin analysis shown in Figure 5B, which shows that the geminin-to-Cdt1 ratio is altered both in the extract and on chromatin upon depletion of MCM9. In addition, we further tested if replication could be rescued through the addition of recombinant Cdt1 in an extract double-depleted for MCM9 and geminin. As shown in Figure S15 and in accordance with Figure S10, the addition of recombinant Cdt1 to double-depleted extracts could neither rescue replication, nor restore MCM2-7 levels on chromatin to significant amounts. From these data altogether, we first concluded that a primary function of MCM9 is to regulate the Cdt1-to-geminin ratio on chromatin during licensing, preventing an excess of geminin to be loaded before the recruitment of the MCM2-7 helicase. Second, the observation that depletion of geminin cannot rescue replication, even if free Cdt1 is present in excess, suggests that

Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc. 195

Molecular Cell MCM9 Is Required for Pre-RC Formation

Figure 6. Chromatin Transfer Studies Reveal that MCM9 Limits Geminin Binding to Cdt1 during Pre-RC Formation (A) Upper panel: replication kinetics of sperm chromatin assembled first in mock-depleted extract and subsequently transferred into either mock- or Cdt1-depleted extract. Lower panel: western blot analysis of chromatin assembled first in mock-depleted extract after being subsequently transferred into mock- or Cdt1-depleted extract. (B) Upper panel: replication kinetics of sperm chromatin assembled first in Cdt1-depleted extract and subsequently transferred into either mock- or Cdt1-depleted extract. Lower panel: western blot analysis of chromatin assembled first in Cdt1-depleted extract after being subsequently transferred into mock- or Cdt1-depleted extract. (C) Upper panel: replication kinetics of sperm chromatin assembled first in MCM9-depleted extract and subsequently transferred into either mock- or Cdt1-depleted extract. Lower panel: western blot analysis of chromatin assembled first in MCM9-depleted extract after being subsequently transferred into mock- or Cdt1-depleted extract. (D) Upper panel: replication kinetics of sperm chromatin assembled first in mock-depleted extract and subsequently transferred into either mock- or MCM9-depleted extract. Lower panel: western blot analysis of chromatin assembled first in mock-depleted extract after being subsequently transferred into mock- or MCM9-depleted extract. (E) Upper panel: replication kinetics of sperm chromatin assembled first in Cdt1-depleted extract and subsequently transferred into either mockor MCM9-depleted extract. Lower panel: western blot analysis of chromatin assembled first in Cdt1depleted extract after being subsequently transferred into mock- or MCM9-depleted extract. (F) Upper panel: replication kinetics of sperm chromatin assembled first in MCM9-depleted extract and subsequently transferred into either mockor MCM9-depleted extract. Lower panel: western blot analysis of chromatin assembled first in MCM9-depleted extract after being subsequently transferred into mock- or MCM9-depleted extract.

MCM9 has also a direct role in pre-RC formation and DNA replication. Chromatin-Transfer Experiments Confirm the Regulatory Interplay between Geminin, Cdt1, and MCM9 during Pre-RC Formation To gain further evidence that MCM9 controls the geminin-toCdt1 stoichiometry during licensing, we performed chromatintransfer experiments in which chromatin was first assembled in either mock-, Cdt1-, or MCM9-depleted extracts to allow pre-RC formation (or not, depending on the depleted protein). This chromatin was then purified and added to (new) mock-, MCM9-, or Cdt1-depleted extracts. By this approach, we could perform a detailed analysis for each depleted

196 Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc.

chromatin/depleted extract combination not only for pre-RC formation, but also for geminin to Cdt1 ratio on chromatin and, finally, DNA replication. Figure 6 shows the data obtained from this series of experiments, and Table S1 summarize these results. Mock-depleted chromatin still contained MCM2-7 complexes and replicated after transfer into all depleted extracts (mock-, Cdt1-, and MCM9-depleted, Figures 6A and 6D) as expected. Cdt1-depleted chromatin did not recruit MCM2-7 complexes and did not replicate after transfer to Cdt1-depleted extracts (Figure 6B). MCM9-depleted chromatin similarly did not recruit MCM2-7 complexes or replicate in MCM9-depleted extract (Figure 6F). Importantly, both Cdt1- and MCM9-depleted chromatin recruited MCM2-7 complexes and replicated when they

Molecular Cell MCM9 Is Required for Pre-RC Formation

were transferred to mock-depleted extracts, as also expected (Figures 6B, 6C, 6E, and 6F). A new result was that mock-depleted chromatin lost most of its bound Cdt1 (and geminin) when it was incubated in Cdt1-depleted extract (Figure 6A), indicating a dynamic chromatin association of Cdt1. However, chromatin-bound MCM2-7 complexes were less affected, in agreement with their salt-resistant association to chromatin (Rowles et al., 1999; Maiorano et al., 2004), and replication was not significantly affected (Figure 6A). We previously showed that chromatin assembled in MCM9depleted extracts bound more geminin for less Cdt1 recruited. Cdt1 has a dual function during pre-RC assembly. It first recruits MCM2-7 complexes, essential for the initiation of DNA replication, and recruits geminin to prevent reinitiation of replication. One function of MCM9 might therefore be to prevent Cdt1 from recruiting an excess of geminin during the licensing reaction. If this was the case, chromatin assembled in MCM9-depleted extracts would be inactivated by an excess of geminin and should not be able to recruit MCM2-7 complexes when transferred into a Cdt1-depleted extract. As shown in Figure 6C, this is exactly what we observed. This experiment further revealed that chromatin assembled in MCM9-depleted extracts and subsequently transferred into Cdt1-depleted extracts contained less MCM9 as when transferred into mock-depleted extract. We believe this is because in the former case, the Cdt1 depletion removed the Cdt1-associated fraction of MCM9 and, thus, only a non-Cdt1 associated fraction of MCM9 could be recruited (Figure 6C). Altogether, these results confirm our data presented in Figure 3E, showing that in the absence of Cdt1, less MCM9 is bound to chromatin. Next, we found that Cdt1-depleted chromatin transferred into MCM9-depleted extract did not recruit MCM2-7 complexes and could not replicate (Figure 6E), in contrast to its ability to do so in mock-depleted extracts (Figures 6B and 6E). Our interpretation of this result is that Cdt1-depleted chromatin, which is transferred into MCM9-depleted extract could only recruit geminin-inactivated Cdt1, since MCM9-associated Cdt1 had been codepleted before, together with MCM9. As shown in Figure 6E, this situation indeed led to a reduced recruitment of Cdt1 compared to an incubation in mock-depleted extract (as also shown in Figures 2D, 5A, and 7B and Figure S14, see below), but concordantly did not reduce geminin recruitment compared to the mock control. This observation is in total agreement with the inability to recruit MCM2-7 complexes and the absence of DNA replication. These data confirm our previous evidence (Figures 2D, 3E, 5, and 7 and Figure S14, see below) that the absence of MCM9 leads to an elevated ratio of geminin to Cdt1 on chromatin, blocking recruitment of the MCM2-7 helicase and DNA replication. When purified chromatin assembled in the absence of MCM9 was incubated in MCM9-depleted extract, we observed, as expected, the same characteristics as already presented, which is an increased geminin-to-Cdt1 ratio on chromatin, the absence of MCM2-7 complexes, and the inability to replicate. Finally, we show that it is possible to restore a licensing-active geminin to Cdt1 ratio on chromatin through addition of recombinant MCM9 to MCM9-depleted extract (Figure 7). Once again, chromatin assembled in MCM9-depleted extract contains an excess of geminin relative to Cdt1 (Figure 7B, compare lanes 1 and

Figure 7. Addition of MCM9 to MCM9-Depleted Extract Restores a Licensing Active Geminin-to-Cdt1 Ratio (A) Western blot analysis of mock- or MCM9-depleted egg extract and MCM9depleted extract supplemented by translated MCM9 or translated MCM9 and Cdt1. (B) Western blot analysis of chromatin assembled in these extracts for MCM27, Cdt1, geminin, and ORC2 (as a loading control).

2). Importantly, addition of MCM9 can restore the ‘‘active’’ geminin to Cdt1 ratio on chromatin (lane 3). This restoration subsequently permits the recruitment of MCM2-7 complexes. The restoration of MCM2-7 recruitment might be partial, possibly due to an inefficient or slow formation of an active MCM9-Cdt1 complex in the depleted egg extract. Addition of Cdt1 and MCM9 together increased the amount of recruited MCM2-7 complex. Altogether, these data establish an essential function of MCM9 in preventing an excess of geminin that blocks Cdt1 activity on chromatin during the licensing reaction. DISCUSSION Our data show that MCM9 is a protein involved in the regulation of the licensing factor Cdt1. MCM9 is recruited to chromatin very early, and its absence blocks pre-RC assembly between the recruitment of Cdt1 and that of the MCM2-7 helicase. This phenotype is strikingly similar to the phenotype observed when pre-RC assembly is blocked by geminin. We also demonstrate that MCM9 binds Cdt1 and, in contrast to geminin, which negatively regulates Cdt1, is an activating binding partner of Cdt1. Altogether, our results show that MCM9 is acting as a colicenser to recruit MCM2-7 complexes onto chromatin, preventing geminin to be recruited in excess to Cdt1 during pre-RC formation. MCM9 Is an Essential Pre-RC Protein MCM9 is the biggest MCM protein and divided into a well-conserved, MCM2-8 family-like N terminus and a unique C-terminal part. MCM9 binds to chromatin in an ORC-dependent manner during pre-RC assembly, independently of CDC6 recruitment to chromatin and well before the recruitment of the MCM2-7 helicase. Importantly, MCM9 recruitment is not blocked by exogenous geminin, also arguing against an interaction with the MCM2-7 helicase independently from chromatin. However, MCM9 seems to be less abundant than the MCM2-7 complex

Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc. 197

Molecular Cell MCM9 Is Required for Pre-RC Formation

on chromatin. MCM9 recruitment to chromatin appears to be dispensable for DNA synthesis after licensing has occurred, since licensed sperm chromatin loaded with MCM2-7 complexes replicates in MCM9-depleted extracts (Figure 3F). Gillespie et al. (2001) reported the in vitro reconstitution of pre-RC formation using purified ORC, CDC6, Cdt1, and MCM27 complexes. Interestingly, the Cdt1-containing fraction that was purified from egg extract (RLF-B) clearly contained a band at 130 kDa that we believe was MCM9. We also previously noticed a band in silver-stained Cdt1 immunoprecipitates at the size of MCM9 (in Maiorano et al., 2004 as data not shown, here presented in Figure S16). In addition, when Gillespie et al. used recombinant Cdt1 instead of extract-purified RLF-B in their licensing assays, 400-fold more Cdt1 protein was required to restore licensing in 6-DMAP treated extracts (to around 20%). We tried to rescue DNA replication in an MCM9-depleted extract with very high amounts of Cdt1, and we could also observe some incorporation corresponding to up to 20% of added sperm chromatin (Figure S10B). However, even when added in great excess, Cdt1 does not efficiently rescue the role of MCM9 in DNA replication. MCM9 and Cdt1 Form a Stable, Direct Complex Cdt1 plays a particularly important regulatory role in licensing, since in contrast to all other replication proteins, its aberrant presence at the wrong stage of the cell cycle is sufficient to relicense and rereplicate the genome (Arias and Walter, 2005; Li and Blow, 2005; Maiorano et al., 2005; Yoshida et al., 2005; Lutzmann et al., 2006). Cdt1 is an oncogene (Arentson et al., 2002), and a recent report also suggests that in human cells, Cdt1 possesses chromosome-damaging activities when overexpressed as it is seen in many cancers (Tatsumi et al., 2006). Thus, cells may have developed a variety of different ways to tightly control the activity or the protein level of Cdt1 (reviewed in Maiorano and Me´chali, 2002; Arias and Walter, 2007). Here, we identified MCM9 as a binding partner of Cdt1. Both proteins are partially codepleted from egg extracts by depletion of the partner protein, and we show a stable interaction between MCM9 and Cdt1 by immunoprecipitations either from egg extracts or from fractionated extracts. Thus, MCM9 might participate in the regulation of Cdt1 beyond pre-RC formation. Geminin and MCM9: Two Opposing Factors Controlling Cdt1 The phenotype after MCM9 depletion mimics the situation on chromatin after a geminin block, which is more recruited CDC6, a higher ratio of geminin to Cdt1, and the absence of the MCM2-7 helicase (Figures 2D, 5, 6E, 6F, and 7B and Figure S14). Geminin can bind Cdt1 with different stoichiometries (Lutzmann et al., 2006), and our data support that the binding of MCM9 to Cdt1 keeps the Cdt1:geminin stoichiometry in its active state on chromatin during formation of the pre-RC. Thus, MCM9 might provide an explanation to why active Cdt1, which is not saturated with (and blocked by) geminin, is present and able to license DNA, despite of free geminin in egg extracts. Besides controlling the Cdt1-to-geminin ratio on chromatin, MCM9 might be directly necessary to load MCM2-7 complexes onto chromatin, by providing an essential ATPase and helicase activity to open the replication origin and to load the MCM2-7

198 Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc.

complex. ORC and CDC6 build an ATP-driven ‘‘molecular motor’’ whose ability to hydrolyze ATP is indispensable for reiterative loading of MCM2-7 complexes (Bowers et al., 2004; Speck et al., 2005; Randell et al., 2006). ORC and CDC6 might use ATP for a chromatin remodeling activity, providing the topological features, which enable MCM9 to unwind DNA locally and subsequently load MCM2-7 complexes together with Cdt1. MCM9, Geminin, and Cdt1 Build a Platform of Licensing Regulation in Complex Organisms Along with geminin, MCM9 is another factor involved in pre-RC assembly that is present in multicellular organisms but absent in unicellular eukaryotes. Furthermore, Cdt1 from yeast has a rather weak homology (about 12%) with the protein from multicellular organisms. In yeast, Cdt1 binds directly and in solution to the MCM2-7 complex. In contrast, in multicellular organisms, a direct interaction of Cdt1 with the MCM2-7 complex independently from chromatin is not found in vivo. Why multicellular organisms avoid a preformed complex of Cdt1 and the MCM2-7 helicase is not known. However, in multicellular organisms, cells have to divide in very different time scales: some cells exit the cell cycle completely, whereas others might be somewhere between proliferation and differentiation, and yet others have to keep on dividing on a daily basis. Cdt1 might be a platform for this regulation, as it is negatively regulated by geminin, a protein also involved in differentiation control (Del Bene et al., 2004; Luo et al., 2004; Seo et al., 2005; Spella et al., 2007). While geminin binding to Cdt1 blocks licensing, MCM9 binding to Cdt1 enables licensing. It will thus be important to investigate whether, like geminin, MCM9 may also have additional roles in proliferationdifferentiation control through the regulation of licensing activity. EXPERIMENTAL PROCEDURES Xenopus Egg Extracts and Replication Reactions Egg extracts were prepared as described before (Menut et al., 1999; Lemaitre et al., 2005; Lutzmann et al., 2006). After thawing, extracts were supplemented with an energy regeneration system (10 mg/ml creatine kinase, 10 mM creatine phosphate, 1mM ATP, and 1mM MgCl2). DNA replication was followed by incorporation of a-(32P)dNTP into replicated DNA. For a standard reaction, 1 ml of a-(32P)dNTP (3000 Ci/mmol) was added to 50 ml egg extract. Percentages were calculated relative to the amount of replication in control reactions, which reproducibly replicate 85% to 100% of the chromatin template. Depletions were performed as described for Cdt1 (Maiorano et al., 2000). For complementation assays, protein translated in depleted egg extract (see below) was added 10–50 min before sperm chromatin addition. Alternatively, extracts were first depleted of MCM9 and then directly supplemented with 1% reticulocyte lysate, and MCM9 mRNA (or not in controls) and sperm chromatin was finally added 1 hr later. Antibodies Antibodies against the Xenopus and Human MCM9 were raised in rabbits by immunization with a His6-tagged, recombinant peptide (aa 606–1143) of Xenopus MCM9 and with synthetic peptides of aa 809–928 and aa 989–008 of human MCM9. The anti-Cdt1 and ORC2 antibodies were raised in rabbits using the full-length recombinant proteins for immunization. The anti-geminin and MCM8 antibodies were described before (Maiorano et al., 2004, 2005). Immunoprecipitations Immunoprecipitations from egg extracts were performed by first incubating the antibody with ProtA-Sepharose for 1 hr at room temperature. After washing with buffer XB (10 mM HEPES-KOH pH 7.7, 100 mM KCl, 0.1 mM CaCl2, 1 mM

Molecular Cell MCM9 Is Required for Pre-RC Formation

MgCl2, and 5% sucrose), beads were incubated with egg extracts for 1 hr at 4 C in the presence of protease inhibitors (leupeptin, aprotinin, and pepstatine, 10 mg/ml each). Beads were then washed at least three times with 100 volumes of XB, transferred to a new tube, and again washed at least three times with the same volume. Proteins were finally eluted with SDS sample buffer and analyzed by SDS-PAGE. Chromatin Purifications Chromatin was obtained by diluting reactions with five volumes of XB + 0.3% Triton X-100, supplemented with protease inhibitors. Diluted reactions were purified through a sucrose cushion as described before (Coue et al., 1996; Maiorano et al., 2000). Licensed sperm chromatin was prepared as follows. After incubation of sperm DNA in 50 ml egg extract for 12 min (in depleted extracts for 20 min), the reaction was diluted 5-fold with buffer CPB (50 mM KCl, 5mM MgCl2, 20 mM HEPES/KOH pH 7.7, and 2% sucrose) and purified as described (Coue et al., 1996; Maiorano et al., 2000). The pelleted chromatin was either directly used for further studies or resuspended in CPB complemented by 10% glycerol, aliquoted, and frozen in liquid nitrogen. Aliquots were then thawed and used as templates in subsequent reactions. Salt extractions were performed with indicated amounts of KCl added to CPB. Sucrose Gradients Egg extracts were diluted 5-fold in XB and centrifuged for 10 min at 12,000 g at 4 C. Diluted and centrifuged egg extract (200 ml) was then layered onto a 7% to 23% percent linear sucrose gradient prepared in XB supplemented with protease inhibitors. The gradient was then centrifuged at 26,000 g for 22 hr at 4 C in a SW 55Ti rotor. Fractions were collected, and aliquots were analyzed by SDS-PAGE and western blotting. Recombinant Protein Expression Translations in reticulocyte lysates were performed according to the manufacturer’s instructions (TNT Reticulocyte Lysate System, Promega). For expression of MCM9 in SF9 cells, recombinant viruses were produced using the Bac-to-Bac system (Invitrogen). MCM9 was cloned using EcoRI/SalI into the pFastBac vector to be expressed as a His-tagged protein. For coexpression of GST-Cdt1 and MCM9 via baculovirus infection of SF9 cells, GST-Cdt1 was cloned as an XmaI/SphI, and MCM9 was cloned as a SalI/NotI fragment into the pFastBacDual vector. Recombinant GST-Cdt1-MCM9 complex was purified as GST purifications described in Lutzmann et al. (2002). For in vitro translation of recombinant MCM9, mRNA coding for MCM9 was produced using the mRNAmMachine system (Ambion) according to the manual’s instructions. As template vector, a pCMV-Sport6 vector containing the Xenopus MCM9 ORF linearized by ClaI after the SV40 poly(A) signal was used. Egg extracts were first depleted of the endogenous MCM9 and then complemented with 1% volume of reticulocyte lysate. Two micrograms of mRNA were added to a reaction of 50 ml egg extract. After 90 min, the extract was aliquoted and frozen in liquid nitrogen.

SUPPLEMENTAL DATA The Supplemental Data include 16 figures and one table and can be found with this article online at http://www.molecule.org/cgi/content/full/31/2/190/ DC1/. ACKNOWLEDGMENTS This work was supported by the ‘‘Association pour la Recherche contre le Cancer,’’ the ‘‘Ligue Nationale contre le Cancer,’’ and the ANR. M.L. is supported by a Liebig-Stipendium des Fond der chemischen Industrie (VCI) Deutschland. We would like to thank Sabine Figaro for help in some experiments described in this study and S. Bocquet for antibody production. We also thank D. Maiorano for helpful discussions and D. Maiorano and P. Pasero for critical reading of the manuscript.

Received: August 7, 2007 Revised: March 4, 2008 Accepted: July 1, 2008 Published: July 24, 2008 REFERENCES Almouzni, G., and Me´chali, M. (1988). Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity. EMBO J. 7, 4355–4365. Arentson, E., Faloon, P., Seo, J., Moon, E., Studts, J.M., Fremont, D.H., and Choi, K. (2002). Oncogenic Potential of the DNA replication licensing protein CDT1. Oncogene 21, 1150–1158. Arias, E.E., and Walter, J.C. (2005). Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts. Genes Dev. 19, 114–126. Arias, E.E., and Walter, J.C. (2007). Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Dev. 21, 497–518. Blow, J.J., and Laskey, R.A. (1988). A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature 332, 546–548. Blow, J.J., and Dutta, A. (2005). Preventing re-replication of chromosomal DNA. Nat. Rev. Mol. Cell Biol. 6, 476–486. Bowers, J.L., Randell, J.C., Chen, S., and Bell, S.P. (2004). ATP hydrolysis by ORC catalyzes reiterative Mcm2–7 assembly at a defined origin of replication. Mol. Cell 16, 967–978. Cook, J.G., Chasse, D.A., and Nevins, J.R. (2004). The regulated association of Cdt1 with minichromosome maintenance proteins and Cdc6 in mammalian cells. J. Biol. Chem. 279, 9625–9633. Coue, M., Kearsey, S.E., and Me´chali, M. (1996). Chromotin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication. EMBO J. 15, 1085–1097. Del Bene, F., Tessmar-Raible, K., and Wittbrodt, J. (2004). Direct interaction of geminin and Six3 in eye development. Nature 427, 745–749. Fehrenbach, A., Li, A., Brito-Martins, M., and Blow, J.J. (2005). Functional domains of the Xenopus replication licensing factor Cdt1. Nucleic Acids Res. 33, 316–324. Gillespie, P.J., Li, A., and Blow, J.J. (2001). Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins. BMC Biochem. 2, 15. Lee, C., Hong, B., Choi, J.M., Kim, Y., Watanabe, S., Ishimi, Y., Enomoto, T., Tada, S., Kim, Y., and Cho, Y. (2004). Structural basis for inhibition of the replication licensing factor Cdt1 by geminin. Nature 430, 913–917. Lemaitre, J.M., Danis, E., Pasero, P., Vassetzky, Y., and Me´chali, M. (2005). Mitotic remodeling of the replicon and chromosome structure. Cell 123, 787–801. Li, A., and Blow, J.J. (2005). Cdt1 downregulation by proteolysis and geminin inhibition prevents DNA rereplication in Xenopus. EMBO J. 24, 395–404. Luo, L., Yang, X., Takihara, Y., Knoetgen, H., and Kessel, M. (2004). The cell cycle regulator geminin inhibits Hox function through direct and polycombmediated interactions. Nature 427, 749–753. Lutzmann, M., Kunze, R., Buerer, A., Aebi, U., and Hurt, E. (2002). Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins. EMBO J. 21, 387–397. Lutzmann, M., Maiorano, D., and Mechali, M. (2005). Identification of full genes and proteins of MCM9, a novel, vertebrate-specific member of the MCM2–8 protein family. Gene 5, 51–56. Lutzmann, M., Maiorano, D., and Me´chali, M. (2006). A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus. EMBO J. 25, 5764–5774. Maiorano, D., and Me´chali, M. (2002). Many roads lead to the origin. Nat. Cell Biol. 4, E58–E59. Maiorano, D., Moreau, J., and Me´chali, M. (2000). XCdt1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature 404, 622–625.

Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc. 199

Molecular Cell MCM9 Is Required for Pre-RC Formation

Maiorano, D., Rul, W., and Me´chali, M. (2004). Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis. Exp. Cell Res. 295, 138–149. Maiorano, D., Krasinska, L., Lutzmann, M., and Me´chali, M. (2005). Recombinant Cdt1 induces rereplication of G2 nuclei in Xenopus egg extracts. Curr. Biol. 15, 146–153. Matthews, G., and Colman, A. (1991). A highly efficient, cell-free translation/ translocation system prepared from Xenopus eggs. Nucleic Acids Res. 19, 6405–6412. Me´chali, M., and Harland, R.M. (1982). DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro. Cell 30, 93–101. Menut, S., Lemaitre, J.M., Hair, A., and Me´chali, M. (1999). DNA Replication and Chromatin Assembly using Xenopus Egg Extracts (Oxford, UK: Oxford University Press).

Speck, C., Chen, Z., Li, H., and Stillman, B. (2005). ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. Nat. Struct. Mol. Biol. 12, 965–971. Spella, M., Britz, O., Kotantaki, P., Lygerou, Z., Nishitani, H., Ramsay, R.G., Flordellis, C., Guillemot, F., Mantamadiotis, T., and Taraviras, S. (2007). Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle. Neuroscience 147, 373–387. Tada, S., Li, A., Maiorano, D., Me´chali, M., and Blow, J.J. (2001). Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat. Cell Biol. 3, 107–113. Takahashi, T.S., Wigley, D.B., and Walter, J.C. (2005). Pumps, paradoxes and ploughshares: mechanism of the MCM2–7 DNA helicase. Trends Biochem. Sci. 30, 437–444.

Nishitani, H., Taraviras, S., Lygerou, Z., and Nishimoto, T. (2001). The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase. J. Biol. Chem. 276, 44905–44911.

Tatsumi, Y., Sugimoto, N., Yugawa, T., Narisawa-Saito, M., Kiyono, T., and Fujita, M. (2006). Deregulation of Cdt1 induces chromosomeal damage without rereplication and leads to chromosomal instability. J. Cell Sci. 119, 3128–3140.

Randell, J.C., Bowers, J.L., Rodriguez, H.K., and Bell, S.P. (2006). Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2–7 helicase. Mol. Cell 21, 29–39.

Wohlschlegel, J.A., Dwyer, B.T., Dhar, S.K., Cvetic, C., Walter, J.C., and Dutta, A. (2000). Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science 290, 2309–2312.

Rowles, A., Tada, S., and Blow, J.J. (1999). Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins. J. Cell Sci. 112, 2011–2018.

Yanagi, K., Mizuno, T., You, Z., and Hanaoka, F. (2002). Mouse geminin inhibits not only Cdt1–MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity. J. Biol. Chem. 277, 40871–40880.

Seo, S., Herr, A., Lim, J.W., Richardson, G.A., Richardson, H., and Kroll, K.L. (2005). Geminin regulates neuronal differentiation by antagonizing Brg1 activity. Genes Dev. 19, 1723–1734.

Yoshida, K., Takisawa, H., and Kubota, Y. (2005). Intrinsic nuclear import activity of geminin is essential to prevent re-initiation of DNA replication in Xenopus eggs. Genes Cells 10, 63–73.

200 Molecular Cell 31, 190–200, July 25, 2008 ª2008 Elsevier Inc.