Recombinant Cdt1 Induces Rereplication of G2 Nuclei in Xenopus Egg Extracts

Cdt1 Triggers Rereplication in G2Maiorano et al.

Current Biology Immediate Early Publication Copyright 2004 by Cell Press

Published Online December 2, 2004DOI: 10/1016/S0960982204009418

Recombinant Cdt1 Induces Rereplication of G2 Nuclei in Xenopus Egg Extracts Domenico Maiorano, Liliana Krasinska,1 Malik Lutzmann, and Marcel Mechali* Institute of Human Genetics Centre National de la Recherche Scientifique 141 rue de la Cardonille 34396 Montpellier France *Correspondence: [email protected] 1Present address: Centre de Recherches de Biochimie Macromoleculaire, CNRS, 1919 Route de Mende, 34293 Montpellier, France.

Summary

A crucial regulation for maintaining genome integrity in eukaryotes is to limit DNA replication in S phase to only one round. Several models have been proposed; one of which, the licensing model, predicted that formation of the nuclear membrane restricts access to chromatin to a positive replication factor [1]. Cdt1, a factor binding to origins and recruiting the MCM2-7 helicase, has been identified as a component of the licensing system in Xenopus and other eukaryotes [2]. Nevertheless, evidence is missing demonstrating a direct role for unscheduled Cdt1 expression in promoting illegitimate reinitiation of DNA synthesis. We show here that Xenopus Cdt1 is absent in G2 nuclei, suggesting that it might be either degraded or exported. Recombinant Cdt1, added to egg extracts in G2, crosses the nuclear membrane, binds to chromatin, and relicenses the chromosome for new rounds of DNA synthesis in combination with chromatin bound Cdc6. The mechanism involves rebinding of MCM3 to chromatin. Reinitiation is blocked by geminin only in G2 and is not stimulated by Cdc6, demonstrating that Cdt1, but not Cdc6, is limiting for reinitiation in egg extracts. These results suggest that removal of Cdt1 from chromatin and its nuclear exclusion in G2 is critical in regulating licensing and that override of this control is sufficient to promote illegitimate firing of origins.

Results and Discussion

Cdt1 Is Removed from the Nucleus upon Initiation of DNA Synthesis Cdt1 is an essential component of the licensing reaction in eukaryotes [3–9]. We have previously shown that in Xenopus Cdt1 is removed from chromatin after initiation of DNA synthesis [3, 10].

However, it is not clear whether Cdt1 is released free in the nucleoplasm or whether it leaves the nucleus. To address this point, nuclei formed in egg extracts at different times during S phase were isolated and both chromatin bound and nucleoplasmic soluble fractions were analyzed. The binding of Cdc45, a DNA polymerase loader that associates with chromatin following nuclear membrane formation [11], was included as a control. Before initiation of DNA synthesis, Cdt1 is chromatin bound in the nucleus (Figure 1A, lane 1) and is absent from the nucleoplasm (Figure 1A, lane 2). However, in late S phase (G2-like state), Cdt1 is absent from both chromatin and nucleosolic fractions (Figure 1A, lanes 3 and 4, respectively), suggesting that it might either be degraded or exported. Most of Cdc45 was bound to chromatin before initiation of DNA synthesis (Figure 1A, lane 1), demonstrating nuclear membrane formation. Interestingly, and unlike Cdt1, Cdc45 was found in the nucleus in late S phase (Figure 1A, lane 4), in both chromatin bound and unbound fractions, which may reflect nuclear import and release from chromatin. These results indicate that G2 nuclei are devoid of Cdt1 but contain the Cdc45 protein.

Recombinant Cdt1 Can Cross the Nuclear Membrane and Induces Reinitiation of DNA Synthesis of G2 Nuclei One postulated feature of licensing factor is that the nuclear membrane blocks its access to chromatin in G2 [1]. To determine whether Cdt1 can cross the nuclear membrane in G2, recombinant Cdt1 was made and purified to homogeneity (Figure 1B). We checked that recombinant Cdt1 could rescue DNA synthesis in egg extract treated with the Cdt1-specific inhibitor geminin (Figure 1C, Geminin + Cdt1) and also rescue replication in Cdt1-depleted extracts (Figure S1, panel C, in the Supplemental Data). We then added an excess of recombinant Cdt1 to G2 nuclei formed in egg extracts (Figure 1D), which have no Cdt1 bound to chromatin (Figure 1E, control). Nuclei were isolated (Figure 1D) and analyzed for the presence of the Cdt1 protein by Western blot (Figure 1E). Recombinant Cdt1 was bound to G2 chromatin, demonstrating that it can cross the nuclear membrane in G2. Only a fraction of recombinant Cdt1 (RecCdt1) was recovered on chromatin (about 20%, + Cdt1). In normal conditions, the import/export and/or degradation equilibrium of Cdt1 may permit its exclusion from the nucleus in G2. An excess of Cdt1 overrides this equilibrium and shows that exogenous Cdt1 can cross the nuclear membrane in G2 and rebind to chromatin. These observations are consistent with a




negative regulation of Cdt1, preventing the MCM2-7 complex from being reloaded at replication origins, and are in agreement with data showing that endogenous Xenopus MCM3 protein can cross the nuclear membrane in G2 [12] but does not bind to chromatin, probably due to lack of a chromatin loader [13]. As Cdt1 loads the MCM2-7 helicase onto DNA replication origins, the absence of Cdt1 in G2 nuclei might prevent reinitiation of DNA replication.

It was previously shown that permeabilized G2 nuclei that have been transferred to fresh egg extract (the licensing assay) reinitiate DNA synthesis, probably by allowing the access to chromatin of licensing factor, which normally cannot cross the nuclear membrane and enter the nucleus during the S and G2 phases [1, 14]. Human G2 nuclei behave in a similar way [14, 15]. We speculated that binding of recombinant Cdt1 to G2 chromatin might be sufficient to induce reinitiation of DNA synthesis in the absence of nuclear membrane permeabilization. G2 nuclei were first exposed to Cdt1 (Figure 1D), then isolated and transferred to fresh extract, as in the licensing assay [1]. Cyclohexymide was added to extracts to suppress protein synthesis and, therefore, block entry into mitosis [16] as nuclear envelope breakdown at mitosis resets the chromatin for a new round of DNA synthesis [1]. Figure 2A shows that upon transfer to fresh extracts, DNA synthesis was observed in G2 nuclei previously exposed to recombinant Cdt1 (+Cdt1) and not in nuclei preincubated with BSA (−Cdt1). To exclude the possibility that relicensing could have been due to nonspecific permeabilization of the nuclear membrane during the nuclear transfer, we have checked the integrity of the nuclear envelope by staining with the membrane-specific dye DIOC. We also monitored the nuclear exclusion of Texas red-conjugated IgGs because IgGs cannot enter the nucleus through nuclear pores [15]. As shown in Figure 2B, G2 nuclei exposed to Cdt1 (+Cdt1) are stained by DIOC and are negative for IgGs, demonstrating that the nuclear envelope was present. When nuclei were permeabilized with detergent (+ NP-40) they became positive for IgGs staining and DIOC negative, demonstrating nuclear membrane permeabilization.

We also analyzed the DNA synthesized upon nuclear transfer by bromodeoxyuridine substitution followed by centrifugation through CsCl gradients. Nuclei were first replicated in egg extracts in presence of BrdUTP and α-[32P] dATP, and G2 nuclei exposed to recombinant Cdt1 were transferred in fresh egg extracts also containing BrdUTP and α-[32P] dATP. One round of DNA replication should produce a hemisubstituted heavy-

light (HL) DNA population in G2 nuclei, whereas reinitiation of DNA synthesis after transfer should result in appearance of heavy-heavy (HH) DNA population, in which the two DNA strands are BrdU substituted. Figure 2C reveals that G2 nuclei relicensed by recombinant Cdt1 (+Cdt1) contain both hemisubstituted DNA (HL) and fully substituted DNA (HH), corresponding to a second round of DNA synthesis. Judging upon the amount of total DNA synthesized in relicensed nuclei (Figure 2A, +Cdt1), replication appeared to correspond to one genome and at later time points (280 min), to two equivalent genomes, suggesting that in the presence of recombinant Cdt1, the entire genome was being rereplicated twice. DNA synthesized in relicensed nuclei was also analyzed by alkaline gel electrophoresis. The resulting single-stranded genomic DNA is expected to migrate as high molecular weight species, while partially replicated DNA should give a smear corresponding to replication intermediates. As shown in Figure 2D, high molecular weight species of DNA are observed in the presence of recombinant Cdt1 (+Cdt1), similar to the control reaction (− Cdt1), although in the presence of Cdt1, the radioactive signal is more intense. Altogether, these results demonstrate that in Xenopus Cdt1 can cross the nuclear membrane in G2 and induce new rounds of DNA synthesis.

Cdt1 Induces Relicensing through Reformation of Pre-RCs on G2 Chromatin The licensing reaction involves loading of the MCM2-7 helicase proteins to replication origins. If Cdt1 induces relicensing of G2 chromatin, it should lead to reloading of MCM2-7 proteins onto chromatin. MCM proteins are removed from chromatin during S phase and remain soluble in the G2 nucleus, so that they can be removed by detergent extraction [17]. To address this issue, G2 nuclei that had been exposed or not to recombinant Cdt1 were isolated, detergent extracted, and double stained with antibodies against the MCM3 protein and Cdc6 as a control. Cdc6, a partner of Cdt1 in licensing [18–20], binds to chromatin after mitotic exit, before nuclear membrane reformation, in agreement with the licensing model, but, unlike Cdt1, Cdc6 is detected on chromatin in G2 [10, 18, 21, 22]. In the presence of recombinant Cdt1 (Figure 3A, +Cdt1), chromatin binding of MCM3, resistant to detergent extraction, was observed in all nuclei, while MCM3 was not chromatin bound in nuclei that had not been exposed to Cdt1 (− Cdt1). Cdc6 was tightly bound to chromatin in the absence of Cdt1, as expected. In the presence of Cdt1, Cdc6 was also chromatin bound, although at




a lower level than in mock-treated extracts (Figure 3A and data not shown). This result shows that recombinant Cdt1 did not remove Cdc6 from chromatin, suggesting that relicensing occurred in combination with chromatin bound Cdc6 and very likely ORC because, in Xenopus, ORC1 is not degraded at the end of S phase but remains chromatin bound in G2 ([21] and our unpublished observations). These results also emphasize the notion that Cdc6 is required to confer the competence to replicate but is not sufficient because Cdt1 is also required to allow firing of DNA replication origins [3, 23–25]. Hence, one major difference between G1 and G2 nuclei is that Cdt1, and not ORC nor Cdc6, is absent from chromatin, thereby causing the reloading of MCM2-7 proteins to be blocked.

Reinitiation of DNA synthesis was not observed by addition of recombinant Cdc6 (Figure 3B), whose overexpression in fission yeast induces rereplication [26]. Analysis of the DNA that had been replicated in the presence of recombinant Cdc6 by density substitution confirms that no reinitiation occurs (Figure S2), although Cdc6 could cross the nuclear membrane and bind to G2 chromatin (Figure 3B, inset; +Cdc6). These results demonstrate that reinitiation of DNA synthesis is specific to recombinant Cdt1 and is not observed with Cdc6. Moreover, rereplication induced by recombinant Cdt1 was efficiently suppressed by geminin (Cdt1 + Gem), a Cdt1-specific inhibitor [9, 27] and was not observed by addition of a nonfunctional Cdt1 mutant lacking the first 372 amino acids (Figure 3B, Cdt1C-ter, and Figure S1). These latter results demonstrate that reinitiation of DNA synthesis in G2 requires a functional Cdt1 protein.

To further demonstrate that relicensing occurs in G2 nuclei, the Cdt1 inhibitor geminin was added either at the time of addition of recombinant Cdt1, in G2 (Figure 3C, protocol I), or to fresh extracts in which G2 nuclei are transferred (protocol II). Geminin was effective in suppressing Cdt1-dependent rereplication when added in G2 at the time of Cdt1 addition (protocol I and panel D), but it was not when added to the fresh extract (protocol II and panel D). These results are consistent with the observation that geminin has no effect on replication once licensing has occurred [28]. They also demonstrate that geminin can function in G2 to prevent illegitimate reinitiation of DNA synthesis by Cdt1.

Cdt1 Induces Reinitiation of DNA Synthesis Only in G2 We asked whether reinitiation of DNA synthesis induced by Cdt1 requires transfer of G2 nuclei to

fresh egg extracts or could be also induced by adding Cdt1 to egg extracts at the end of S phase (G2). As shown in Figure 4A, addition of recombinant Cdt1 (+ Cdt1) resulted in an increased DNA synthesis compared to the control reaction (− Cdt1). A density substitution experiment (Figure 4B) shows that in the presence of Cdt1 (+ Cdt1), hemi- and double-substituted DNA populations (HL and HH) are observed, whereas in the control reaction, only hemisubstituted DNA (HL) is present, confirming results shown in Figures 2A–2C. We conclude that addition of Cdt1 to egg extracts at the end of S phase is sufficient to promote reinitiation of DNA synthesis.

Finally, to determine whether Cdt1 could induce reinitiation at any cell cycle stage, recombinant Cdt1 was introduced at different times during interphase, or at mitosis, and the extent of DNA replication was determined upon prolonged incubation. As shown in Figure 4C, addition of Cdt1 in mitosis (M), in G1, or during ongoing S phase (S) did not result in reinitiation of DNA synthesis, whereas reinitiation was observed by addition of Cdt1 at the end of S phase (G2). These results suggest that during S phase, both endogenous and recombinant Cdt1 might be made incompetent to induce refiring of origins, either by degradation (see below) or by inactivation mediated by the intra-S phase checkpoint, or both. This regulation may be less active or absent in G2. In the latter case, Cdt1 on its own may not be implicated in amplification of segments of the genome but regulates bulk chromosome rereplication in egg extracts. These results also show that recombinant Cdt1 can induce reinitiation of DNA synthesis of G2 nuclei in egg extracts at a time when endogenous Cdt1 is absent from the nucleus.

Previous experiments have shown that permeabilized G2 nuclei can reinitiate DNA synthesis, suggesting access of a positive licensing factor [1]. We have shown that exposing G2 nuclei to recombinant Cdt1, and not Cdc6, is sufficient to relicense chromatin for new rounds of DNA synthesis without nuclear membrane permeabilization. Is Cdt1 the full explanation of the licensing phenomenon? Cdt1 fulfills three out of the four features postulated for the licensing factor [1]. Cdt1 is (1) a chromatin binding protein, (2) an essential replication initiation factor, and (3) removed from the nucleus upon initiation of DNA synthesis. However, our data show that recombinant Cdt1 can cross the nuclear membrane in G2, whereas licensing factor is postulated not to be able to enter the nucleus in G2 [1]. If Cdt1 is the licensing factor, then the nuclear membrane may not impose a physical barrier to Cdt1 in entering




the nucleus but it may instead permit its inactivation. One of its roles could be to permit accumulation in the nucleus of geminin, the Cdt1 inhibitor. Geminin accumulates in the nucleus [28, 29] at the time of initiation of DNA synthesis [10]; however, its removal from egg extracts is not sufficient to induce rereplication [28]. Therefore, an additional mechanism must operate to inactivate Cdt1 during DNA synthesis, which is consistent with the inability of Cdt1 to induce reinitiation in S phase. In Xenopus, nuclear Cdt1 might be degraded by the proteasome (H. Takisawa and J. Walter, personal communication), as in mammalian cells [27, 30–32] and in plants [33]. In contrast, cytoplasmic Cdt1 is stable throughout the cell cycle [10]. Therefore, one role for the nuclear membrane could be to both accumulate the Cdt1 inhibitor geminin, and induce destruction of nuclear Cdt1 upon initiation of DNA synthesis. In this scenario, if cytoplasmic Cdt1 is imported, it will be immediately inactivated by geminin and destroyed by proteolysis. It is conceivable that recombinant Cdt1 added to G2 nuclei permits relicensing by overriding both geminin inhibition and the degradation machinery.

Our findings demonstrate a critical role for Cdt1 in regulating initiation of DNA synthesis in early Xenopus development. In fission yeast, overexpression of Cdt1 in G2 potentiates the rereplication induced by Cdc6 [34]. In C. elegans, a mutation in a proteasome subunit stabilizes Cdt1 and results in extensive rereplication [35]. In plants [33] and human cells, overxpression of Cdt1 in combination with Cdc6 [36], induces rereplication of segments of the genome. Indirectly, in Drosophila [37] and mammalian cells [38, 39], inactivation of geminin, by RNAi, induces rereplication. Finally, formation of tumors have been observed in mice overexpressing Cdt1 [40].

The results presented in this paper demonstrate that deregulation of Cdt1 expression can reset licensing directly through Cdt1 binding to chromatin and formation of prereplication complexes onto postreplicative chromatin. Nuclear exclusion of Cdt1 during S phase is, therefore, a crucial step in regulating the once-per-cell-cycle replication of the eukaryotic genome, and conditions that override this control may lead to loss of genome integrity.

Experimental Procedures

Xenopus Egg Extracts Egg extracts were prepared and frozen as previously described [41]. Upon thawing, egg extracts were supplemented with cycloheximide (250 µg/ml) and an energy regeneration system (10 µg/ml creatine

kinase; 10 mM creatine phosphate; 1 mM ATP; 1 mM MgCl2). For density substitution experiments, standard replication reactions (50 µl) were supplemented with 0.1 mM BrdUTP, 2000 nuclei/µl of egg extracts, and 1 µl α-[32P]dCTP (3000 Ci/mmol). Nuclei isolated from these reactions were transferred to freshly thawed egg extracts supplemented with cyclohexymide and an energy regeneration system as described above, plus 0.1 mM BrdUTP and 1 µl of α-[32P]dCTP. Egg extracts were depleted of the Cdt1 protein as previously described [3].

Expression and Purification of Recombinant Proteins Wild-type, Xenopus Cdt1 protein was expressed in E. coli as 6-His-tagged recombinant protein. For this purpose the Xenopus Cdt1 full-length cDNA was amplified by standard PCR methods and inserted in the BamHI and KpnI sites of the bacterial expression vector pRSETB (Invitrogen) to generate p6HisXCdt1. The N-terminal deletion mutant of XCdt1 (amino acids 141–621) has been previously described [3]. We generated the C-ter XCdt1 mutant (amino acids 372–621) by subcloning the approximately 1.6 kb PvuII-EcoRI DNA fragment of the XCdt1 cDNA into the same sites of the pRSETc expression vector (Invitrogen). Recombinant 6-His-tagged Cdt1 proteins were expressed and purified as previously described [3]. Purified proteins were supplemented with 0.1 mg/ml BSA, concentrated by centrifugation over a Microcon-30 (Millipore) device at 1 mg/ml, and supplemented to egg extracts to a final concentration of 20 µg/ml. Recombinant geminin protein was expressed and purified as previously described [10, 28]. Recombinant Cdc6 expressed in insect cells was purified to homogeneity as previously described [18, 23]. Recombinant Cdt1 proteins were added to egg extracts to 1:20 ratio (protein to egg extracts), to a final concentration of 40 nM. Recombinant cyclin B was prepared as described [42]

Nuclei and Chromatin Isolation Methods Nuclei assembled in egg extracts (1000/µl) were prepared by incubation of Xenopus sperm chromatin in low-speed egg extracts. For nuclear fractionation experiments, nuclei were isolated by 5× dilution of replication reactions in CPB (50 mM KCl; 20 mM Hepes-KOH [pH 7.7]; 5 mM MgCl2; 2% sucrose; leupeptin, aprotinin, and pepstatine, 5 µg/ml each) followed by centrifugation at 6000 g for 5 min at 4°C through a 0.7 M sucrose cushion made in CPB. Detergent extraction was carried out




for 10 min on ice in CPB supplemented with 0.1% NP-40. Unsoluble (chr) and soluble (nuc) fractions were then recovered after centrifugation at 6000 g for 5 min at 4°C. Detergent-extracted chromatin was obtained by 5× dilution of replication reaction with ice-cold CPB buffer supplemented with 0.1% NP-40 and incubation on ice for 1 min. Chromatin was isolated by centrifugation through a sucrose cushion as described above. For experiments involving nuclear transfer (the licensing assay), nuclei assembled in egg extracts were diluted 5× in ice-cold CPB and overlayed onto a 0.7 M sucrose cushion made in CPB. Nuclei were isolated by centrifugation at 6000 g for 5 min at 4°C and resuspended in 10 µl of XB (10 mM Hepes KOH; 100 mM KCl; 0.1 mM CaCl2; 1 mM MgCl2; and 5% sucrose) supplemented with 10% glycerol. An aliquot of these nuclei (4 µl) was then transferred to fresh extracts (40 µl) supplemented with cyclohexymide and an energy regeneration system. To monitor nuclear membrane permeabilization, we isolated nuclei as described above in the absence of detergent and incubated them on ice for 20 min with 1µg of Texas red conjugated IgGs (ICN). To induce permeabilization of the nuclear membrane, one aliquot of the isolated nuclei was diluted 1:2 with XB/10% glycerol containing 0.5% Nonidet P-40 (NP-40) to give a final concentration of NP-40 of 0.25%. Reaction was stopped by addition of BSA to a final concentration of 3%. Nuclei were then processed for immunofluorescence analysis.

Observation of Isolated Nuclei by Indirect Immunofluorescence Replication reactions or isolated nuclei were fixed by 20× dilution in XB + 4% formaldehyde at room temperature for 15 min. Fixed nuclei were recovered on a coverslip by centrifugation through a 0.7 M sucrose cushion at 4°C and postfixed for 5 min with cold EtOH at −20°C. Samples were rehydrated with PBS by incubation at room temperature for 10 min and preblocked with a solution of PBS/1% BSA at room temperature for at least 30 min. Nuclei were stained with primary antibodies by incubation overnight at 4°C in a wet chamber. Samples were then washed thoroughly with PBS/0.2% Tween-20 and incubated with secondary antibodies for 1 hr at room temperature in the dark. After being washed (see above), samples were incubated with a solution of DIOC (0.5 µg/ml, Sigma) and Hoechst (0.1µg/ml) in PBS, for 10 min at room temperature. After three washes of 15 min in PBS, coverslips were mounted on slides with MOVIOL and viewed with a Zeiss inverted microscope.

Analysis of Rereplicated DNA For density substitution experiments, DNA was fractionated by centrifugation through a cesium chloride density gradient as follows. Replication reactions were supplemented with proteinase K (0.5 mg/ml) and incubated at 42°C for 1 hr. DNA was phenol/chloroform extracted twice and ethanol precipitated. DNA was resuspended in 100 µl of TE containing 100 µg/ml RNaseA and incubated at 37°C for 1 hr. DNA was reprecipitated with ethanol and resuspended in 85 µl of TE. DNA was then digested with EcoRI for 1 hr at 37°C.

DNA cut with EcoRI was resuspended in 6 ml of a cesium chloride solution made in TE (0.96 g/ml, giving a density of 1.73). Samples were spun at 36,000 rpm in a Ti50 rotor for 38 hr at 21°C. Thirty-three fractions were collected, and the refractory index of 1 out of 4 fractions was measured to confirm formation of the gradient. The radioactivity of each fraction was measured by scintillation counting. Analysis of DNA by alkaline gel electrophoresis was performed by fractionation through a 1% agarose gel containing 30 mM NaCl and 2.5 mM EDTA run in 30 mM NaOH and 2.5 mM EDTA. After electrophoresis, the gel was soaked in a solution of 7% TCA for 10 min at room temperature and dried. Gel was exposed to a phosphoimager screen (Molecular Dynamics) for autoradiography.

Acknowledgments

We thank: Dr. H. Takisawa (University of Osaka, Japan), Dr. J. Walter (Harvard Medical School) for sharing unpublished results and also for the Cdc45 antibody, and Dr. O. Cuvier for providing recombinant cyclin. B.L.K. was supported by a Training Opportunities for European Ph.D. Students - Marie Curie Training Site - Euro Gen Dis. We thank E. Danis for help in some experiments described here. D.M. is supported by the Institut National de la Santé et de la Recherche Médicale. M.L. is supported by a grant from the Liebig-Stipendium des Fonds der chemischen Industrie Deutschland. This work was supported by grants from the Human Frontier Science Programme, Ligue Contre le Cancer, and Association pour la Recherche contre le Cancer.

Received: September 9, 2004

Revised: October 28, 2004

Accepted: November 10, 2004

Published online: December 2, 2004




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Figure 1. Recombinant Cdt1 Can Cross the Nuclear Membrane in G2 (A) Binding of Cdt1 and Cdc45 to early and late S phase chromatin. Nuclei formed after 30 min (early) or 90 min (late) incubation in egg extracts were isolated and fractionated to obtain insoluble (chr) and soluble (nuc) nuclear fractions (see experimental procedures). Proteins were detected by Western blot with the indicated antibodies. (B) Coomassie blue staining of purified Xenopus Cdt1 protein expressed in bacteria. (C) Activity assay of recombinant Cdt1. DNA replication of sperm chromatin incubated in egg extracts supplemented with 100 nM BSA, 100 nM Geminin, or 100 nM Geminin and 200 nM recombinant Cdt1 (+ Cdt1) was measured after 90 min incubation at room temperature by incorporation of a radioactive precursor. (D) Kinetics of DNA synthesis of sperm chromatin (140 ng) incubated in egg extracts, to which recombinant Cdt1 is added at the end of S phase (RecCdt1 addition) and from which G2 nuclei are isolated (nuclei isolation). DNA synthesis stopped by 60 min, and G2 nuclei did not contain condensed chromatin (see Figure 3A). (E) Recombinant Cdt1 binds to G2 chromatin. Western blot of chromatin isolated upon incubation in egg extracts (D) in the presence (+ Cdt1) or absence (control) of recombinant Cdt1. One aliquot of purified, recombinant Cdt1 was included as a control (RecCdt1).




Figure 2. Recombinant Cdt1 Induces Reinitiation of DNA Synthesis in G2 (A) Kinetics of DNA replication of G2 nuclei upon transfer to fresh egg extracts. Nuclei replicated in egg extracts (G2 nuclei) were exposed (+ Cdt1) or not (− Cdt1) to recombinant Cdt1 (Figure 1D) and transferred to fresh egg extracts containing α-[32P] dATP. DNA replication was measured as in Figure 1D and expressed as a percent of newly replicated DNA upon transfer to fresh extracts compared to the total input DNA (80 ng). A shaded line represents one round of DNA synthesis relative to the input DNA upon transfer. (B) Determination of the integrity of the nuclear membrane after addition of recombinant Cdt1. G2 nuclei exposed (+ Cdt1) or not (− Cdt1) to recombinant Cdt1 were isolated and incubated on ice with texas-red conjugated IgGs in the presence (+ NP-40) or absence (− NP-40) of 0.25% Nonidet P-40. Nuclei were purified and stained with both the membrane-specific dye DIOC (0.5 µg/ml) and Hoechst (1 µg/ml). (C) Analysis of rereplicated DNA by density substitution. Nuclei replicated in egg extracts in the presence of BrdUTP and α-[32P] dATP (G2 nuclei) and exposed (+ Cdt1, diamonds) or not (− Cdt1, squares) to recombinant Cdt1 were transferred to fresh egg extracts containing BrdUTP and α-[32P] dATP. After 170 min incubation, total DNA was purified and fractionated by centrifugation through a CsCl density gradient. Fractions were collected and the radioactivity of each sample (cpm) was measured. The position of heavy-heavy (HH) and heavy-light (HL) DNA species is indicated by the arrows. The position of the light-light (LL) peak was determined by running a parallel gradient containing salmon sperm DNA and analysis of its position by agarose gel electrophoresis followed by ethidium bromide staining. The refractory index (RI, triangles) of gradient fractions is given on the right ordinate axis. (D) Analysis of replicated DNA by alkaline gel electrophoresis. DNA replicated in the presence (+ Cdt1) or absence (− Cdt1) of recombinant Cdt1 (Figure 2A) at the indicated time points was fractionated by 1% agarose gel electrophoresis in the presence of 30 mM NaOH. Replicated DNA was revealed by autoradiography. DNA standard size markers were run on the same gel (Kbp).




Figure 3. Recombinant Cdt1 Reloads MCM3 on G2 Chromatin (A) Indirect immunofluorescence of detergent-extracted G2 nuclei exposed (+ Cdt1) or not (− Cdt1) to recombinant Cdt1 and stained with Cdc6 and MCM3 antibodies. DNA was visualized with Hoechst staining (DNA). (B) Recombinant Cdc6 crosses the nuclear membrane but does not induce rereplication. Replication of G2 nuclei exposed to the indicated proteins upon transfer to fresh egg extracts. DNA synthesis was measured as in Figure 1C upon 150 min incubation. The binding of Cdc6 to G2 chromatin was determined by Western blot (inset). (C) Experimental setup. In the first experimental protocol (I), G2 nuclei are incubated with both recombinant Cdt1 and geminin proteins (Cdt1 + Gem) before nuclear transfer. In the second experimental protocol (II), G2 nuclei are incubated with recombinant Cdt1 and then transferred to fresh extract supplemented with recombinant geminin. (D) Recombinant Cdt1 induces licensing in G2. The extent of rereplication of G2 nuclei in the presence of the indicated proteins added in G2 (protocol I) or that of G2 nuclei transferred to fresh egg extracts and supplemented with geminin (protocol II) is shown. DNA synthesis was measured after 90 min incubation upon transfer of G2 nuclei to fresh egg extracts.




Figure 4. Recombinant Cdt1 Induces Rereplication without Nuclear Transfer, in G2 (A) Recombinant Cdt1 was added to egg extracts at the end of S phase (+ Cdt1 at 100 min, arrow), and DNA synthesis was measured as shown in Figure 1D and compared to a control reaction (− Cdt1). (B) Analysis of rereplicated DNA by density substitution. DNA replicated in the presence of recombinant Cdt1 (+ Cdt1, squares) or without Cdt1 (− Cdt1, diamonds) was fractionated by centrifugation through a cesium chloride gradient and analyzed as in Figure 2C. The radioactivity of each fraction was determined by scintillation counting (cpm). (C) Cdt1 induces rereplication only when added at the end of S phase in egg extracts. Recombinant Cdt1 (+ Cdt1) was added to egg extracts synchronized in very early interphase (G1), during DNA synthesis (S), at the end of S phase (G2), or to G2 egg extracts driven into mitosis by the addition of recombinant cyclin B∆90 (M). DNA synthesis was measured after a 210 min incubation at room temperature and compared to a control reaction without recombinant Cdt1.




Supplemental Data

Recombinant Cdt1 Induces Rereplication

of G2 Nuclei in Xenopus Egg Extracts Domenico Maiorano, Liliana Krasinska, Malik Lutzmann, and Marcel Mechali

Figure S1. Purification and Activity of Cdt1 Proteins (A) Coomassie blue stain of the Cdt1 N-terminal deletion mutant (amino acids 141–621, lane 1) and that of the Cdt1 C-terminal fragment (amino acids 372–621, lane 2) after 10% SDS-PAGE. (B) Licensing activity assay of Cdt1 mutants. Replication of sperm chromatin incubated in egg extracts supplemented with either BSA, or Geminin in combination with the indicated Cdt1 proteins. DNA replication was measured by incorporation of α-[32P] dATP after 90 min incubation at room temperature. (C) Wild-type recombinant Cdt1 rescues DNA replication in Cdt1-depleted egg extracts. Egg extracts were mock-depleted (Mock) or depleted with Cdt1-specific antibodies (Cdt1). Cdt1-depleted extracts were supplemented with recombinant wild-type Cdt1 (RecCdt1) and DNA synthesis was measured after 90 min incubation.




Figure S2. Recombinant Cdc6 Does Not Induce Rereplication in Egg Extracts (A) DNA replicated in the presence (+ Cdc6) or absence (− Cdc6) of recombinant Cdc6, was fractionated by centrifugation through a CsCl density gradient and analyzed as in Figure 2C. (B) Recombinant Cdc6 rescues DNA synthesis in Cdc6-depleted egg extracts. DNA synthesis of mock-depleted (Mock-depleted), Cdc6-depleted egg extracts (Cdc6-depleted) reconstituted with either dialysis buffer (+ buffer) or recombinant Cdc6 (+ Cdc6) is shown.

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Recombinant Cdt1 Induces Rereplication of G2 Nuclei in Xenopus Egg Extracts

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