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• W2115314080 abstract "Article23 November 2006free access A Cdt1–geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus Malik Lutzmann Malik Lutzmann Institute of Human Genetics, CNRS, Montpellier, France Search for more papers by this author Domenico Maiorano Domenico Maiorano Institute of Human Genetics, CNRS, Montpellier, France Search for more papers by this author Marcel Méchali Corresponding Author Marcel Méchali Institute of Human Genetics, CNRS, Montpellier, France Search for more papers by this author Malik Lutzmann Malik Lutzmann Institute of Human Genetics, CNRS, Montpellier, France Search for more papers by this author Domenico Maiorano Domenico Maiorano Institute of Human Genetics, CNRS, Montpellier, France Search for more papers by this author Marcel Méchali Corresponding Author Marcel Méchali Institute of Human Genetics, CNRS, Montpellier, France Search for more papers by this author Author Information Malik Lutzmann1, Domenico Maiorano1 and Marcel Méchali 1 1Institute of Human Genetics, CNRS, Montpellier, France *Corresponding author. Institute of Human Genetics, CNRS, Genome Dynamics and Development, 141, rue de la Cardonille, Montpellier 34396, France. Tel.: +33 499 619 917; Fax: +33 499 619 920; E-mail: [email protected] The EMBO Journal (2006)25:5764-5774https://doi.org/10.1038/sj.emboj.7601436 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Initiation of DNA synthesis involves the loading of the MCM2–7 helicase onto chromatin by Cdt1 (origin licensing). Geminin is thought to prevent relicensing by binding and inhibiting Cdt1. Here we show, using Xenopus egg extracts, that geminin binding to Cdt1 is not sufficient to block its activity and that a Cdt1–geminin complex licenses chromatin, but prevents rereplication, working as a molecular switch at replication origins. We demonstrate that geminin is recruited to chromatin already during licensing, while bulk geminin is recruited at the onset of S phase. A recombinant Cdt1–geminin complex binds chromatin, interacts with the MCM2–7 complex and licenses chromatin once per cell cycle. Accordingly, while recombinant Cdt1 induces rereplication in G1 or G2 and activates an ATM/ATR-dependent checkpoint, the Cdt1–geminin complex does not. We further demonstrate that the stoichiometry of the Cdt1–geminin complex regulates its activity. Our results suggest a model in which the MCM2–7 helicase is loaded onto chromatin by a Cdt1–geminin complex, which is inactivated upon origin firing by binding additional geminin. This origin inactivation reaction does not occur if only free Cdt1 is present on chromatin. Introduction Within each cell cycle, chromatin is faithfully replicated exactly once. To ensure that no part of the genome is rereplicated, chromatin must acquire replication competence before S phase in the so-called licensing reaction (Blow and Laskey, 1988). Entry into S phase then blocks further licensing for the remaining cell cycle (reviewed in Machida et al, 2005; DePamphilis et al, 2006). Chromatin licensing takes place at the end of mitosis, beginning with the binding of the ORC complex to chromatin at multiple potential origins of replication, and is followed by the assembly of two other factors, Cdc6 and Cdt1 (reviewed in Bell and Dutta, 2002). The latter interacts with the MCM2–7 DNA helicase complex (Tanaka and Diffley, 2002; Yanagi et al, 2002; Cook et al, 2004; Ferenbach et al, 2005) and loads it onto chromatin to form prereplication complexes (pre-RCs). The resulting licensed replication origins are activated at the start of S phase in a reaction mediated by two main kinase/cyclin systems, CDC7/Dbf4 and cyclinE/CDK2. Cdt1 is ubiquitinylated at the onset of S phase, and its consequent destruction is one mechanism which may prevent the relicensing of replication origins (Liu et al, 2004; Nishitani et al, 2004, 2006; Sugimoto et al, 2004; Arias and Walter, 2005; Li and Blow, 2005; Hu and Xiong, 2006; Senga et al, 2006). In metazoans, relicensing is further inhibited by an additional mechanism involving geminin, a protein that binds to Cdt1 and blocks the loading of the MCM2–7 helicase onto chromatin (McGarry and Kirschner, 1998; Wohlschlegel et al, 2000; Nishitani et al, 2001; Quinn et al, 2001; Tada et al, 2001). Geminin is degraded by the anaphase-promoting complex (APC), suggesting that its destruction at the end of mitosis liberates Cdt1 and enables a new round of origin licensing. However, the mechanism of licensing is probably more complex, as recent reports have shown that polyubiquitination of geminin in anaphase does not necessarily destroy the protein. Instead, it becomes incapable of interacting with Cdt1, whereas subsequent nuclear import of the protein in G1 restores its ability to bind to and block Cdt1 (Hodgson et al, 2002; Li and Blow, 2005). The molecular event that reactivates geminin upon nuclear import remains unknown. It is also unclear why a part of the geminin pool in egg extracts is already complexed with Cdt1 in the absence of chromatin and what the function of this complex might be during the licensing reaction (Hodgson et al, 2002; Maiorano et al, 2004; Yoshida et al, 2005). In addition to its negative role in the control of DNA replication, geminin was recently shown to be required for cell cycle progression, as it complexes Cdt1 during mitosis and protects it from degradation, thereby maintaining sufficient amounts of Cdt1 for the licensing of replication origins in the following cell cycle (Ballabeni et al, 2004). This result is consistent with the unexpected finding that geminin is present at high levels in fast-dividing cells and also in cancer cells, contrary to what one might expect from its established role in blocking licensing and DNA replication (Wohlschlegel et al, 2002; Gonzalez et al, 2004; reviewed in Pitulescu et al, 2005). In Xenopus, depletion of geminin results in relicensing and limited rereplication (Yoshida et al, 2005). This result was explained by assuming that geminin must be present before the degradation of Cdt1 in order to prevent rereplication. Adding recombinant Cdt1 during G2 is also sufficient to give rise to relicensing and massive rereplication (Arias and Walter, 2005; Li and Blow, 2005; Maiorano et al, 2005; Yoshida et al, 2005). In this study, we show that geminin does not necessarily block the activity of Cdt1, but rather controls it through formation of a Cdt1–geminin complex, which acts as a molecular switch, turning DNA replication origins on or off. During licensing, geminin is recruited to chromatin together with Cdt1 and does not block licensing activity. Indeed, a complex of recombinant Cdt1 and geminin is capable of loading the MCM2–7 helicase onto chromatin to license DNA for replication. Furthermore, whereas the addition of free Cdt1 either in G1 or G2 leads to rereplication and an ATM/ATR-dependent checkpoint response, adding the active Cdt1–geminin complex, even in large excess, induces neither rereplication nor a checkpoint response. The stoichiometry of the Cdt1–geminin complex is critical for its licensing activity, as the addition of more geminin to the complex blocks its ability to load the MCM2–7 helicase onto chromatin. Taken altogether, these data suggest that a Cdt1–geminin complex, and not free Cdt1, is the natural MCM2–7 loader whose activity can be precisely regulated throughout the cell cycle by changing its stoichiometry, explaining geminin's essential function in cell cycle progression. Results A fraction of geminin binds to chromatin already during the licensing reaction in a Cdt1-dependent manner Western blot analysis has shown that the bulk of geminin in Xenopus egg extracts binds to chromatin at the onset of S phase (Maiorano et al, 2004; Arias and Walter, 2005; Li and Blow, 2005; Yoshida et al, 2005). Accordingly with these findings, using immunofluorescence, we also detected significant loading of geminin onto chromatin at S-phase onset (here at 40 min), followed by a decrease at the end of S phase/G2 (Figure 1A). However, we reproducibly detected earlier binding of geminin to chromatin, within 5 min after adding sperm chromatin to egg extract (Figure 1A). The early chromatin binding of geminin was not detected in a control reaction using a nonspecific antibody (Figure 1B, left panel, pre-immune), nor when geminin was depleted from the extract (Figure 1B, right panel and Supplementary Figure S1), demonstrating that the observed signal was geminin-specific. We next confirmed geminin binding during the first 5 min after sperm chromatin addition by Western blotting. Figure 1C shows that geminin is recruited to chromatin with the same kinetics as Cdt1 and before the loading of the MCM2–7 complex. Interestingly, further recruitment of bulk geminin occurs at onset of S phase, which appears to be synchronized with the previously reported polyubiquitination of Cdt1 (Figure 1C, Cdt1 long exposure, Arias and Walter, 2005). Finally, we tried to estimate the amount of Cdt1 and geminin bound to chromatin by a quantification experiment shown in Figure 1D. Chromatin was purified after different incubation times and the signal intensities of Cdt1 and geminin were compared to dilutions of the corresponding recombinant proteins (see Figure 2A) loaded onto the same gel. Whereas the Cdt1 signal remained constant during the time course of the experiment, geminin levels increased considerably at a time corresponding to the onset of DNA synthesis. From these and other independent experiments, the Cdt1–geminin ratio was estimated to be about 1:2.4 during licensing and to exceed 1:4 after initiation. We have previously shown that the mean inter-origin spacing is around 20 kbp in such replication experiments (Lemaitre et al, 2005). We estimate that five Cdt1 molecules and 12 geminin molecules are bound per origin during licensing. After initiation, about 20 geminin molecules are bound per origin while the Cdt1 level remains unchanged. We conclude that Cdt1 and geminin are recruited synchronously to chromatin during the licensing reaction, and that the stoichiometric ratio of geminin to Cdt1 is increased after initiation of DNA replication. Figure 1.A fraction of geminin assembles in a Cdt1-dependent manner during licensing onto chromatin. (A) Upper panel: kinetics of the replication reaction performed as described in Materials and methods and expressed as the percentage of replicated sperm DNA compared to the total input DNA. Lower panel: sperm chromatin incubated in egg extract for indicated times and further detergent extracted and processed for immunofluorescence. Geminin was visualized by FITC (green) and MCM3 by Texas red (red). DNA was stained with Hoechst and is shown in blue. (B) Left panel: immunofluorescence of chromatin purified as in (A) after 5 min incubation in egg extract and stained either with geminin antibody or with preimmune serum. Right panel: immunofluorescence of chromatin after 5 min incubation in egg extract that was either Mock- or geminin-depleted. (C) Western blot analysis of Cdt1, MCM2–7, geminin and ORC2 (as a loading control) on chromatin purified after the indicated time. Also shown is a Mock purification (first lane), without added sperm chromatin, to determine background staining by non-chromatin bound proteins. A longer exposure of the Cdt1 blot is also presented to show polyubiquitination of Cdt1 at the onset of S phase. (D) Western blot analysis of purified chromatin following the indicated times of incubation in egg extract (S-phase entry at 40 min). A dilution series of recombinant Cdt1 and geminin was loaded on the same gel. The amount of loaded chromatin is indicated, as well as the corresponding number of origins (Lemaitre et al, 2005). (E) Geminin signal on chromatin incubated 5 min in egg extract, either Mock-depleted, Cdt1-depleted, or Cdt1-depleted and complemented with geminin before addition of sperm chromatin. Download figure Download PowerPoint Figure 2.The recombinant Cdt1–geminin complex is active in licensing DNA for replication. (A) Coomassie-stained SDS–PAGE of purified Cdt1, Cdt1–geminin complex and geminin. Also shown is a molecular weight marker. Protein concentrations were determined by comparison with BSA dilution series. (B) Kinetics of DNA replication in either Mock-depleted extract (squares) or in Cdt1-depleted extract supplemented by buffer (diamonds), 40 nM Cdt1 (triangles) or 40 nM Cdt1–geminin complex (circles). The insert shows a Western blot for Cdt1 and ORC2 (as loading control) of Mock- and Cdt1-depleted extract. (C) Percentage of replicated sperm chromatin after 100 min in egg extract supplemented by buffer, 40 nM geminin, 40 nM geminin plus 40 nM Cdt1, or 40 nM geminin plus 40 nM Cdt1–geminin complex. (D) Percentage of replicated sperm chromatin after 100 min in egg extract supplemented by buffer or by increasing amounts of Cdt1–geminin complex (upper panel) or by geminin (lower panel). (E) Incubation of Cdt1–geminin complex immobilized by GST-geminin to GSH-Sepharose (left panels) or GST-geminin alone immobilized in the same way (right panels) either in buffer or in interphasic egg extract for 40 min at room temperature (Coomassie stain). Additional bands visible after incubation in egg extract are proteins that bound nonspecifically to the GSH-Sepharose (marked by asterisks). Download figure Download PowerPoint As geminin is present in egg extracts both in a free form as well as in a complex with Cdt1 (Hodgson et al, 2002; Maiorano et al, 2004; Yoshida et al, 2005), depleting Cdt1 only removes the Cdt1-bound geminin pool. If the endogenous Cdt1–geminin complex is recruited to chromatin, then depletion of Cdt1 should leave only free geminin in the extract, unable to bind chromatin. Figure 1E shows that this is indeed the case: Cdt1 depletion abolished geminin binding to chromatin during licensing, strongly suggesting that geminin can only be recruited to chromatin when it is complexed with Cdt1. In agreement with this result, addition of an excess of free geminin to the Cdt1-depleted extract failed to restore geminin binding to chromatin (Figure 1E, right panel and Supplementary Figure S2). Further evidence for an endogenous Cdt1–geminin complex bound to chromatin during licensing was obtained from co-immunoprecipitation experiments. Sperm chromatin was assembled in egg extract for 10 min, purified and salt-washed to remove Cdt1 as described (Maiorano et al, 2000a, 2000b). Co-immunoprecipitations from the soluble fraction, performed with Cdt1 and geminin antibodies, showed that both proteins co-precipitate in both cases (immunoprecipitation of geminin co-precipitates Cdt1 and immunoprecipitation of Cdt1 co-precipitates geminin, Supplementary Figure S3). From these experiments, we conclude that geminin is recruited in a complex with Cdt1 to chromatin during the licensing reaction. A Cdt1–geminin complex is active in origin licensing To investigate more in detail the recruitment of the Cdt1–geminin complex to chromatin, we expressed and purified recombinant free Cdt1, free geminin, as well as the Cdt1–geminin complex. To purify the complex, we coexpressed both proteins and used a double-affinity tag strategy (His6-tagged Cdt1 and GST-TEV-tagged geminin) to ensure that no free Cdt1- or geminin subunits were present in the final purified fraction (Figure 2A, see also Materials and methods. Although the complex can also be assembled from individual purified proteins in vitro (Supplementary Figure S4), we found coexpression more efficient to obtain a stable, homogeneous complex). Figure 2A shows the purified recombinant proteins used in this study. The Cdt1 protein in the Cdt1–geminin complex lane migrates slightly slower on SDS–PAGE than free Cdt1, as expected due to its His6-tag. Further characterization of the purified complex by dynamic light scattering demonstrated that the complex is a homogeneous and monodispersed particle with a diameter of about 13.5 nm (Supplementary Figure S5 and Discussion). We next addressed whether the Cdt1–geminin complex would be active in licensing. To test this, egg extracts were depleted of Cdt1 and then complemented by either free recombinant Cdt1 or by the Cdt1–geminin complex. As shown in Figure 2B, a Cdt1-depleted extract did not replicate sperm chromatin, but DNA replication could be restored by addition of 40 nM free Cdt1, as reported before (Maiorano et al, 2000b). Importantly, addition of 40 nM of Cdt1–geminin complex to the Cdt1-depleted extracts restored DNA replication with the same efficiency as addition of free Cdt1. Moreover, like free Cdt1, the Cdt1–geminin complex also restored block of DNA replication in egg extracts treated with an excess of free geminin (Figure 2C). Finally, Figure 2D shows that the Cdt1–geminin complex did not block DNA replication even when added at high concentrations to egg extracts (up to 200 nM), whereas a 30 nM addition of free geminin led to complete inhibition of DNA replication. We conclude that the Cdt1–geminin complex is active in licensing chromatin for DNA replication. These observations also confirm that the Cdt1–geminin complex is stable, as its disassembly would produce, apart from free Cdt1, free geminin, which could block licensing. To further address the stability of the Cdt1–geminin complex, it was bound to GSH-Sepharose via the GST-tag on geminin and washed with more than 1000 volumes of buffer. No loss of Cdt1 was detected (see below and data not shown), and no dissociation was observed upon addition of up to 2 M MgCl2, confirming previous results showing that the complex is stable up to 4 M urea (Tada et al, 2001; Saxena et al, 2004 and data not shown). The tight binding of Cdt1 to geminin was also not affected upon incubation of the immobilized complex in egg extract (Figure 2E). The possibility that, during this incubation, recombinant Cdt1 was exchanged for endogenous Cdt1 is unlikely for two reasons. First, we did not detect the presence of the faster-migrating endogenous Cdt1 after recovery of the Sepharose-bound complex. Second, recombinant geminin that was similarly immobilized to GSH-Sepharose did not bind detectable amounts of endogenous Cdt1 under the same conditions (Figure 2E, right panel). These results confirm that Cdt1 and geminin form a highly stable complex, which is as active as free Cdt1 in replication licensing. The Cdt1–geminin complex is recruited to chromatin and interacts with the MCM2–7 complex We further examined the licensing activity of the Cdt1–geminin complex by analyzing the binding of geminin and the MCM2–7 helicase to chromatin in the presence of identical amounts of Cdt1, geminin, or the Cdt1–geminin complex. Adding free Cdt1 did not change the level of geminin bound to chromatin, whereas adding free geminin slightly increased it (Figure 3, compare E, F and G). In contrast, adding the same amount of the Cdt1–geminin complex caused a dramatic increase of geminin on chromatin (Figure 3H). Importantly, while a small amount of additional free geminin on chromatin blocked MCM2–7 loading, a much higher level recruited to chromatin as part of the Cdt1–geminin complex did not interfere with MCM2–7 loading (Figure 3, compare K and L). These data confirm that geminin is efficiently recruited to chromatin during licensing only when it is added in a complex with Cdt1. They also confirm the high stability of the Cdt1–geminin complex in egg extract, as free geminin is poorly recruited to chromatin (Figure 3, compare G and H). Similar results were obtained using a high-speed extract that is devoid of membranes and unable to support DNA replication, in agreement with the conclusions that the observed reactions occurred during licensing, before initiation of DNA synthesis (data not shown). Figure 3.Geminin is massively recruited to chromatin without blocking MCM2–7 loading only if added in a complex with Cdt1. Immunofluorescence of geminin (FITC, green) and MCM3 (Texas red, red) on sperm chromatin (Hoechst, blue) after 5 min incubation in egg extract. As indicated, equal amounts of Cdt1, geminin or the Cdt1–geminin complex were added to the egg extract 10 min before the addition of sperm chromatin. Download figure Download PowerPoint Together, these data show that the Cdt1–geminin complex can be recruited to chromatin during licensing to load the MCM2–7 complex, and that the amount of geminin that is bound to chromatin before S phase depends on the Cdt1–geminin complex present in the extract. Whereas in yeast a preformed, non-chromatin bound complex of Cdt1 and MCM2–7 has been reported (Tanaka and Diffley, 2002), there has not been any evidence for such a complex in vertebrates. However, in vitro reconstitution experiments using recombinant and purified metazoan proteins were successfully employed to detect such a direct interaction, and it was further suggested that geminin binding would sterically prevent Cdt1 from interacting with the MCM2–7 complex (Yanagi et al, 2002; Cook et al, 2004; Lee et al, 2004; Ferenbach et al, 2005). We therefore tested the ability of the Cdt1–geminin complex to interact with the MCM2–7 complex under in vitro conditions. The MCM2–7 complex was purified from egg extracts as previously described (Maiorano et al, 2000a) and incubated with GSH-Sepharose bound Cdt1, geminin or Cdt1–geminin complex. As shown in Supplementary Figure S6, the MCM2–7 complex could interact equally well with Cdt1 and with the Cdt1–geminin complex, but not with geminin alone. These data confirm that, as for Cdt1, the Cdt1–geminin complex can bind directly to the MCM2–7 proteins in vitro. Cdt1 recruitment to chromatin without geminin induces a checkpoint response It has been reported that the addition of recombinant Cdt1 to egg extract in G1 induces limited rereplication, while its addition in G2 induces massive rereplication and checkpoint activation (Arias and Walter, 2005; Li and Blow, 2005; Maiorano et al, 2005; Yoshida et al, 2005). We reproducibly observed that adding free Cdt1 to egg extract before the addition of sperm chromatin significantly slowed down DNA replication. Interestingly, such an effect was not observed when the Cdt1–geminin complex was added (Figure 4A, left panel). The addition of caffeine counteracted this Cdt1-specific slow down of replication, suggesting that a surplus of free Cdt1, but not of the Cdt1–geminin complex, may induce a checkpoint response during the first round of DNA replication (Figure 4A, right panel). Figure 4.Free Cdt1, but not the Cdt1–geminin complex, leads to rereplication and checkpoint activation. (A) Left panel: kinetics of sperm chromatin replication supplemented with buffer (circles), 40 nM free Cdt1 (rectangles) or 40 nM Cdt1–geminin complex (triangles). Right panel: the same reactions were performed in the presence of 5 mM caffeine. (B) Western blot analysis of purified nuclei with a phospho-Chk1 antibody. Nuclei were purified after 100 min incubation of sperm chromatin in egg extract supplemented with either buffer, free Cdt1, Cdt1–geminin complex (all 40 nM), or Aphidicolin (50 μg/ml). A Western blot with ORC2 antibody as loading control is also shown. (C) Analysis of replicated DNA half-substituted with BrdUTP (‘heavy–light’, HL) or double-substituted after rereplication (‘heavy–heavy’, HH) on a CsCl gradient. Before sperm chromatin addition, reactions were supplemented either with buffer (circles), free Cdt1 (rectangles) or Cdt1–geminin complex (diamonds). (D) Immunofluorescence of G2 chromatin (120 min after sperm chromatin addition) and 20 min after addition of either buffer, free Cdt1, Cdt1–geminin complex, or free geminin. DNA is shown in blue stained with Hoechst, geminin in green (FITC) and MCM3 in red (Texas red). (E) Analysis of replicated DNA half-substituted with BrdUTP after one round of replication (‘heavy–light’, HL) or double-substituted after rereplication (‘heavy–heavy’, HH) on a CsCl gradient in reactions supplemented in G2 (120 min after sperm DNA was added) either with buffer (circles), free Cdt1 (rectangles) or Cdt1–geminin complex (diamonds). Download figure Download PowerPoint To confirm our observations, we supplemented egg extracts with equivalent amounts of free Cdt1 or Cdt1–geminin complex, added sperm chromatin, isolated nuclei at the end of S phase and tested for checkpoint activation by detection of phosphorylated Chk1 (Guo et al, 2000; Liu et al, 2000; Capasso et al, 2002). As controls, parallel reactions were supplemented with either buffer or 50 μg/ml aphidicolin. Only those reactions supplemented with aphidicolin or free Cdt1 showed phosphorylation of Chk1, indicating checkpoint activation (Figure 4B). We next tested for rereplication by using a CsCl gradient to detect double BrdUTP-substituted DNA. As shown in Figure 4C, the addition of free Cdt1 before the initiation of DNA synthesis gave rise to partial rereplication. In contrast, no rereplication (and no checkpoint activation, see above) was detected when the Cdt1–geminin complex was added. These data strongly suggest that disturbing the Cdt1:geminin ratio by adding free Cdt1 before the onset of S phase results in rereplication, activates an ATM/ATR-dependent checkpoint response during S phase, and thus delays DNA replication. Therefore, the recruitment of the Cdt1–geminin complex during licensing to chromatin appears essential to prevent rereplication during S phase. The activity of the Cdt1–geminin complex is faithfully regulated by the cell cycle We and others recently reported that Cdt1 added during G2 can cross the nuclear membrane and induce massive rereplication (Arias and Walter, 2005; Li and Blow, 2005; Maiorano et al, 2005; Yoshida et al, 2005). Given that the Cdt1–geminin complex is as efficient as free Cdt1 in licensing, we tested whether its addition in G2 would similarly induce rereplication. We purified G2 chromatin 20 min after adding geminin, Cdt1 or the Cdt1–geminin complex and found that addition of free Cdt1 or the Cdt1–geminin complex, but not free geminin, induced the rerecruitment of geminin to chromatin (Figure 4D, compare e–h). Rerecruitment of Cdt1 to chromatin in G2 also led to the rerecruitment of the MCM2–7complex and to rereplication (Figure 4D and E). In contrast, the addition of the Cdt1–geminin complex in G2 induced neither the rerecruitment of the MCM2–7 complex to chromatin (Figure 4D, compare j and k) nor rereplication, as seen by the absence of double BrdUTP-substituted DNA (Figure 4E). Interestingly, the rerecruitment of geminin to chromatin induced by the addition of free Cdt1 during G2 could neither prevent relicensing nor rereplication (Figure 4D, compare f, j and g, k). We conclude that free Cdt1 on chromatin causes uncontrolled licensing in both G1 and G2, resulting in rereplication. In contrast, the Cdt1–geminin complex is faithfully controlled in both G1 and G2. In G1, it licenses chromatin only once for replication, and in G2 it does not allow (re-) licensing of chromatin, thereby preventing a second round of replication within the same cell cycle. The stoichiometry of the Cdt1–geminin complex determines its activity The above findings prompted us to ask how the Cdt1–geminin complex could be active in licensing without promoting relicensing, and how geminin can block the loading of the MCM2–7 complex onto chromatin when appropriate despite the Cdt1–geminin complex being active. One possibility could be that the nuclear environment inactivates the Cdt1–geminin complex. We have previously shown that MCM2–7 loading is blocked following accumulation of geminin on chromatin (Maiorano et al, 2004). Two different oligomerization states of geminin have been reported. The first, a dimer of either a free geminin domain or a geminin domain complexed with a Cdt1 geminin-binding domain, was identified by crystallization studies (Lee et al, 2004; Saxena et al, 2004; Thépaut et al, 2004). Whereas the second (using small angle scattering and electron microscopy to visualize full-length geminin) was shown to be a tetramer (Okorokov et al, 2004; Thépaut et al, 2004). We explored the possibility that such changes of the Cdt1–geminin complex stoichiometry could account for its inactivation. We have shown that the Cdt1–geminin complex is stable in solution. However, we also showed that further recruitment of geminin to chromatin occurs after initiation of DNA synthesis. Thus, we investigated whether the complex might change its stoichiometry when incubated with an excess of free geminin. Figure 5A shows that the active Cdt1–geminin complex can incorporate additional geminin, which was detectable through the use of a higher molecular weight myc-tagged form of free geminin in the binding assay outlined in Figure 5A. Binding of the additional geminin produced an ‘I-complex’ (for Inactive complex, see below). In parallel, immobilized Cdt1–geminin complex was incubated with buffer only and processed similarly, and named the ‘A-complex’ (for Active complex, see below). An immobilized, unrelated control GST-protein (the yeast Seh1 nucleoporin), as well as GST-geminin itself were unable to bind additional free geminin (Supplementary Figure S7), showing that the association of free geminin with the complex was specific. Quantification of Cdt1 and geminin (untagged and myc-tagged) on the Coomassie-stained SDS–PAGE shown in Figure 5A indicated that the Cdt1–geminin A-complex has a Cdt1:geminin stoichiometry of 1:3, and that this stoichiometry was changed upon incubation with myc-tagged geminin by the addition of one geminin molecule per complex (I-complex, 1:4 stoichiometry). It is es" @default.
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• W2115314080 date "2006-11-23" @default.
• W2115314080 modified "2023-10-14" @default.
• W2115314080 title "A Cdt1–geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus" @default.
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• W2115314080 doi "https://doi.org/10.1038/sj.emboj.7601436" @default.
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