Cdt1 Self-associates via the Winged-Helix Domain of the Central Region during the Licensing Reaction, Which Is Inhibited by Geminin

Abstract

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein–protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein–protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two β-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.

INTRODUCTION

The initiation of DNA replication is tightly regulated by the licensing system, which controls the chromatin loading of mini-chromosome maintenance (MCM) complexes that function as replicative DNA helicases. From the late M phase to the early G1 phase of the cell cycle, origin recognition complexes (ORC) that consist of Orc1–6 subunits bind to the replication origins, followed by Cdc6 and Cdt1 recruitment to the origin in an ORC-dependent manner. The MCM complexes are eukaryotic replicative DNA helicases composed of Mcm2–7 subunits, which are loaded through direct interaction with Cdt1 to form pre-RCs on the origins of DNA replication¹⁾ (Fig. 1A, “late M–early G1 phase”). The MCM complexes loaded onto the origins are activated in the S phase and unwind the parental strands to move the DNA replication forks forward. After the onset of the S phase, pre-RCs formation is suppressed until the late M phase to prevent excessive DNA replication. In higher eukaryotes, Cdt1 is the major target for pre-RC formation suppression, which is inhibited by cell cycle-dependent proteolysis or the binding of the endogenous inhibitor, geminin²⁾ (Fig. 1A, “S phase”). Geminin forms dimers and stably binds to the middle domain of monomeric Cdt1, resulting in the inhibition of licensing activity. The coiled-coil domain of geminin is essential for inhibition of the licensing reaction,^3–5) and this domain hinders the interaction between Cdt1 and the MCM complex.⁶⁾ Moreover, geminin binds to Cdt1 to form an inhibitory heterohexamer.⁷⁾ Despite these structural findings, the mechanical basis for licensing activity inhibition by geminin has not yet been fully elucidated. In addition, the molecular mechanism of Cdt1 function in the licensing reaction and the significance of its functional interactions with licensing proteins have not yet been addressed.

Fig. 1. Self-association of Cdt1 Requires Its Central Region

(A) Schematic representation of the licensing reaction. (B) GST-tagged Cdt1 (GST-Cdt1), His-tagged Cdt1 (His-Cdt1), and GST were added to the reaction mixture and incubated. GST pull-down fractions from the reaction mixtures were electrophoresed and immunoblotted to detect Cdt1 or GST. (C) Schematic representation of the protein structure of Xenopus laevis (X. l) Cdt1. “WHD” indicates the winged-helix domains of Cdt1. (D) GST-Cdt1, His-Cdt1, and His-FLAG-tagged Cdt1 fragments (1–243, 244–447, and 448–620) were added to the reaction mixture, followed by GST pull-down assay and immunoblot detection of Cdt1 and FLAG.

To investigate the detailed function of Cdt1, we used recombinant proteins and a Xenopus egg extract cell-free system and observed that Cdt1 self-associates through its central region. The Cdt1 mutant protein that carries a reduced ability for self-association showed decreased licensing activity compared to that in the wild-type protein, suggesting that the self-association of Cdt1 is important in the licensing reaction.

MATERIALS AND METHODS

Recombinant Proteins and Antibodies

Glutathione S-transferase (GST)-fused proteins were expressed in BL21-CodonPlus-RIL (Agilent Technologies, CA, U.S.A.), into which the cDNA for the recombinant proteins in the pGEX-6P-3 vector (GE Healthcare, IL, U.S.A.) was introduced. The expressed proteins were purified using Glutathione Sepharose 4B (GE Healthcare). For untagged Cdt1 and Cdt1-S1S2, the GST-tags fused with the proteins were removed using PreScission Protease (GE Healthcare) as described previously.⁸⁾ Hexahistidine (His)- and FLAG-tagged proteins were expressed from the cDNA subcloned into the pET-15b vector (Merck KGaA, Darmstadt, Germany) in BL21-CodonPlus-RIL and purified using Ni-NTA agarose (Invitrogen, MA, U.S.A.). Anti-Xenopus Cdt1 rabbit polyclonal antibody was raised against the purified Xenopus Cdt1 protein expressed in BL21-CodonPlus-RIL. Anti-FLAG M2 monoclonal and anti-GST antibodies were purchased from Sigma-Aldrich (MO, U.S.A.) and MBL (Tokyo, Japan), respectively. Anti-human Cdc6 polyclonal antibody and anti-human Mcm4 polyclonal antibody were purchased from Proteintech (IL, U.S.A.).

GST Pull-Down Assay

GST-fused bait (100 nM) and prey (100 nM) proteins were incubated for 20 min at 23 °C in 40 µL binding buffer (40 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)–KOH, pH 7.6, 120 mM KCl, 0.25% Triton X-100, 10% sucrose, and 2 mM ATP-Mg). For the pull-down assay using Cdt1 fragment proteins, the following binding buffer was used: 40 mM HEPES–KOH, pH 8.0, 20 mM K₂HPO₄/KH₂PO₄, 2 mM MgCl₂, 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 2 mM dithiothreitol, 10% (w/v) sucrose, 1 µg/mL each of leupeptin, pepstatin, and aprotinin, 50 mM KCl, 2 mM ATP-Mg, and 0.1% Triton X-100. After incubation, the mixture was added to 15 µL Glutathione Sepharose 4B beads and incubated for 60 min at 4 °C. Then, the beads were washed three times with 200 µL binding buffer, and the proteins adsorbed on the beads were eluted with 15 µL elution buffer composed of 40 mM HEPES–KOH, pH 7.6, 120 mM KCl, 0.25% Triton X-100, 10% sucrose, and 40 mM glutathione. Proteins in the reaction mixture (i.e., input proteins) and the eluted fraction from the glutathione Sepharose beads were detected by immunoblotting.

DNA-Binding Assay

Wild-type Cdt1 (WT, 200 nM) or Cdt1-S1S2 (200 nM) was mixed with double-stranded DNA cellulose (dsDNA cellulose; Sigma-Aldrich) in binding buffer (20 mM Tris–HCl, pH 7.5, 50 mM NaCl, and 0.1% Triton X-100) and incubated for 1 h at 4 °C. After washing with binding buffer, the bound proteins were eluted sequentially with binding buffer supplemented with increasing concentrations of NaCl. Proteins in the eluted fractions were detected using immunoblotting.

Preparation of Xenopus Egg Extracts and Demembranated Sperm Nuclei

Metaphase-arrested Xenopus egg extracts and Xenopus sperm nuclei were prepared as previously described.⁹⁾ The egg extracts were released into the interphase by adding CaCl₂ (0.3 mM final concentration) and incubated for 20 min at 23 °C before use.

Immunodepletion

The rProtein A Sepharose Fast Flow (GE Healthcare) was incubated with three times the volume of anti-Xenopus Cdt1 rabbit antiserum for 60 min at room temperature. After washing with buffer (40 mM HEPES-KOH, pH 8.0, 20 mM K₂HPO₄/KH₂PO₄, 2 mM MgCl₂, 1 mM EGTA, 2 mM dithiothreitol, 10% (w/v) sucrose, 1 µg/mL each leupeptin, pepstatin, and aprotinin, 50 mM KCl, and 2 mM ATP-Mg), the beads were incubated with the same volume of Xenopus egg extracts for 60 min at 4 °C. The mixture was then passed through a nylon filter (30 µm mesh, Merck KGaA) to remove the beads. Mock-treated extracts were prepared following the same procedure using pre-immune rabbit serum instead of anti-Cdt1 serum.

Chromatin Isolation

Sperm nuclei (3000 nuclei/µL) were incubated in Xenopus egg extracts supplemented with the indicated proteins for 20 min at 23 °C. After incubation, the chromatin fraction was isolated and proteins in the fraction were detected by immunoblotting, as described previously.⁹⁾

Data Collection and Presentation

All experiments described in this manuscript were repeated at least three times and representative data are shown.

Statistics

Quantification of the Western blot band intensities are shown as means  ±  standard error (n = 3) and the unpaired Student’s two-tailed t-test or Dunnett’s multiple comparison test was used to determine the statistical significance (* p < 0.05).

RESULTS

Xenopus Cdt1 Self-associated through Its Central Region

The ORC, Cdc6, and MCM complexes self-associate when they function in licensing reactions.^10–12) However, the self-association of Cdt1 is not clearly described. To clarify the function of Cdt1 in the licensing reaction, we first investigated whether Cdt1 formed a self-associated complex using bacterially expressed recombinant proteins. The results of the pull-down assay showed that GST-tagged Xenopus Cdt1 was associated with His-tagged Xenopus Cdt1, indicating that Xenopus Cdt1 was self-associated in solution (Fig. 1B). Next, we explored which region of Cdt1 is required for self-association. The structure of Xenopus Cdt1 was divided into N-terminal, central, and C-terminal functional regions^2,13) (Fig. 1C). The N-terminal region is a regulatory domain involved in protein degradation or phosphorylation of Cdt1, whereas the central and C-terminal regions contain geminin- and MCM-binding domains and are sufficient for the licensing activity of Cdt1.^2,13–16) We produced three protein fragments of Xenopus Cdt1 tagged with His and FLAG, each of which contained N-terminal 1–243 aa, central 244–447 aa, or C-terminal 448–620 aa, to test the ability of these fragments to associate with GST-tagged full-length Xenopus Cdt1. We observed that only the central fragment efficiently coprecipitated with full-length Cdt1 (Fig. 1D), suggesting that the self-association of Cdt1 is mediated by the central region.

Self-association of Cdt1 Is Inhibited by Geminin

Since the central region of Xenopus Cdt1 contains a geminin-binding domain,^6,13,14) we next examined whether geminin inhibited the association of GST-Cdt1 with untagged Cdt1 and found that Cdt1 co-precipitation with the GST-tagged protein significantly decreased when geminin was added to the reaction (Figs. 2A, B). Inhibition of the licensing activity of Cdt1 by geminin requires a coiled-coil domain and truncation of the C-terminal extension of the coiled-coil domain significantly attenuates the inhibitory activity without eliminating geminin binding to Cdt1^3,4,6,7) (Fig. 2C). When the C-terminal region was truncated from the recombinant geminin (1–150 aa region: Gem1–150), which can still form geminin dimers,⁶⁾ Gem1–150 bound to Cdt1 to a comparable extent to that of wild-type geminin in pull-down assays. The band intensity of Cdt1 relative to GST-Cdt1 decreased with the addition of wild-type geminin. In contrast, the addition of Gem1-150 did not decrease the band intensity of Cdt1, suggesting that the inhibitory effect of geminin on Cdt1 self-association was significantly reduced by truncating the C-terminal region of geminin (Figs. 2D, E). These results suggested that geminin requires its C-terminal region to inhibit the self-association of Cdt1.

Fig. 2. Geminin Inhibited the Self-association of Cdt1

(A) GST-tagged and untagged Cdt1 were incubated with or without His-FLAG-geminin (600 nM) for use in a GST pull-down assay. (B) Quantification of the Cdt1 band intensity of the Western blot shown in panel (A). The intensity of the bands relative to that of GST-Cdt1 is plotted. The graph depicts the results from three independent experiments. Error bars represent standard error. * p < 0.05, Student’s two-tailed t-test. (C) Schematic representation of the protein structure of Xenopus laevis (X.l.) geminin. The coiled-coil domain and the regions required for dimerization, Cdt1 binding, and replication inhibition are indicated. (D) GST-tagged and untagged Cdt1 were incubated without or with the indicated concentrations of His-FLAG-Geminin (Geminin-WT) or His-FLAG-Gem1-150 (Gem1-150), followed by a GST pull-down assay and immunoblotting of Cdt1 and FLAG. (E) Quantification of the Cdt1 band intensity from the Western blot presented in panel (D). The intensity of the bands relative to that of GST-Cdt1 is plotted. The graph depicts the results from three independent experiments. Error bars represent standard error. * p < 0.05, Dunnett’s test.

Mutation of the Winged-Helix Domain in the Central Region of Cdt1 Attenuates Self-association and Licensing Activity in Xenopus Egg Extracts

As shown in Fig. 1C, Cdt1 has two sequential winged-helix domains (WHD) in its central (PDB ID: 2ZXX, Fig. 3A) and C-terminal regions (PDB ID: 3A4C).^2,6,16) Although WHD participates in DNA binding, the β-sheet structure of the C-terminal WHD of Cdt1 is involved in Mcm6-binding.¹⁶⁾ Thus, it is possible that the WHD in the central region of Cdt1 is also involved in protein–protein interactions. Structural analysis of Cdt1 revealed that the central WHD has six α-helices (H1–H6) and two β-strands (S1, S2).⁶⁾ To explore whether the central WHD is involved in the self-association of Cdt1, four and seven amino acids located in the two β-strands were substituted to alanine (Cdt1-S1S2, Fig. 3B). The results of the pull-down assay showed an attenuated interaction between wild-type Cdt1 and Cdt1-S1S2, suggesting that either or both β-strands of the central WHD are required for the self-association of Cdt1 (Figs. 3C, D).

Fig. 3. The Central Winged-Helix Domain Is Involved in the Self-association of Cdt1

(A) Crystral structure of the central region of mouse Cdt1 (PDB ID: 2ZXX) in ribbon representation. Alpha-helices and β-strands are colored red and blue, respectively. (B) Secondary structure of the central winged-helix domain of Cdt1 (upper) and sequence alignment of the two β-strands from human (H.s.), mouse (M.m.), and Xenopus (X.l.) Cdt1 proteins (middle). Amino acid residues mutated in Cdt1-S1S2 are framed by a box in the alignment. The amino acid sequence of the two β-strands of Cdt1-S1S2 are also indicated (lower). (C) GST-Cdt1, untagged wild-type Cdt1 (Cdt1-WT), or untagged Cdt1-S1S2 were mixed as indicated before GST pull-down fractions were immunoblotted to detect Cdt1. (D) Quantification of the Cdt1-WT and Cdt1-S1S2 band intensity from the Western blot shown in panel (C). The plotted intensity of the bands is relative to that of GST-Cdt1. The graph depicts the results from three independent experiments. Error bars represent standard error. * p < 0.05, Student’s two-tailed t-test. (E) Cdt1- and mock-depleted Xenopus egg extracts were electrophoresed and immunoblotted to detect Mcm4, Cdt1, and Cdc6 (left panel). The Cdt1-depleted extracts were supplemented with 10 nM untagged Cdt1-WT or Cdt1-S1S2 and incubated with sperm nuclei for 20 min. After the incubation, the chromatin fraction was isolated and immunoblotted to detect Mcm4, Cdt1, and Cdc6 (right panel).

To investigate the significance of the self-association of Cdt1 in the licensing reaction, we examined the ability of Cdt1-S1S2 to load MCM complexes onto chromatin in Xenopus egg extracts. Sperm nuclei were incubated in Cdt1-depleted egg extracts supplemented with wild-type Cdt1 or Cdt1-S1S2, and Mcm4 was detected in the isolated chromatin fractions (Fig. 3E). These results showed that the addition of Cdt1-S1S2 did not restore the decreased chromatin binding of Mcm4 in Cdt1-depleted extracts. We also observed a marked reduction in chromatin binding of Cdt1-S1S2 when compared to that of wild-type Cdt1, which further indicated that the amino acid substitutions of S1 and S2 in Cdt1 abolished the licensing activity.

Cdt1-S1S2 Associates with Proteins Involved in the Licensing Reaction and DNA

In higher eukaryotes, Cdt1 interacts with multiple proteins of the pre-RC component, including Orc2, Cdc6, Mcm2, and Mcm6.^{14,15,17–20)} We tested whether Cdt1-S1S2 associates with these pre-RC components to ask whether the self-association of Cdt1 is required for these interactions (Fig. 4). Consistent with the results of previous studies,^6,14) geminin suppressed the interaction between wild-type Cdt1 and Mcm6 (Fig. 4A). Geminin also inhibited the interaction of Cdt1 with Mcm2, Orc2, and Cdc6, while Cdt1-S1S2 bound to these proteins to a similar extent as wild-type Cdt1 (Figs. 4B–E).

Fig. 4. Cdt1-S1S2 Interacts with Pre-RC Components, Geminin, and DNA

(A–E) GST-Cdt1 wild type (GST-Cdt1-WT) or GST-Cdt1-S1S2 was incubated with the indicated recombinant proteins without or with 600 nM His-FLAG-Geminin (A–D). GST pull-down fractions were immunoblotted to detect Cdt1, geminin (A–E), Mcm6 (A), Mcm2 (B), Orc2 (C), and Cdc6 (D). (F) Untagged Cdt1-WT or Cdt1-S1S2 were loaded onto double-stranded DNA cellulose and bound proteins were eluted with NaCl to detect Cdt1 with immunoblotting. Asterisks in panels B and F indicate non-specific bands. (G) Possible function of Cdt1 self-association in the loading of MCM complex onto replication origins (see Discussion).

Cdt1 binds to DNA in a sequence- and strand-independent manner.¹⁴⁾ Cdt1-S1S2 bound to double-stranded DNA to a level similar to that of wild-type Cdt1 (Fig. 4F). These results suggested that the ability of Cdt1 to interact with other pre-RC components and DNA does not require the central WHD and that Cdt1 self-association is not required for these interactions.

DISCUSSION

In this study, we investigated the importance of the self-association of Cdt1 in licensing reactions. We observed that Cdt1 was self-associated with the central region and that mutations in the two β-strands of WHD in the central region of Cdt1 (Cdt1-S1S2) reduced its self-association activity. Although Cdt1-S1S2 interacted with geminin, other pre-RC components and DNA similarly to wild-type Cdt1 (Fig. 4), the licensing activity of Cdt1-S1S2 in Xenopus egg extracts was hardly detected (Fig. 3E) and we observed decreased chromatin binding of Cdt1-S1S2 in Cdt1-depleted egg extracts. These results suggest that self-association is important for the chromatin binding of Cdt1 during the licensing reaction, which promotes the loading of MCM complexes onto the origin region. It has been suggested that multiple Cdt1 molecules associate with the origin DNA to load MCM complexes onto the DNA in a yeast model,²¹⁾ which is consistent with our findings.

We observed that geminin inhibited the self-association of Cdt1 and the interaction of Cdt1 with Orc2, Cdc6, Mcm2, and Mcm6 (Figs. 2A, B, 4A–D). The inhibition of Cdt1 self-association requires the C-terminal extension of geminin (Figs. 2D, E), which is also necessary for geminin to inhibit licensing reactions.^3–6) These results suggest that the inhibition of pre-RC-related protein–protein interactions contributes to the licensing inhibition activity of geminin.^3–5) In contrast, Cdt1-S1S2 could interact with Orc2, Cdc6, Mcm2, and Mcm6, suggesting that the self-association of Cdt1 is not required for these interactions.

The central region of Cdt1 is required for its licensing activity,¹³⁾ but the detailed role of this region has not been characterized. Here, we show that the central region participates in the self-association of Cdt1. The self-association of ORC, Cdc6, and MCM complexes has also been reported to be important for pre-RC formation.^10–12) Our results suggest that the self-association of Cdt1 is involved in the licensing reaction. It is possible that Cdt1 self-associates and forms a scaffold on chromatin to recruit MCM complexes to the origin (Fig. 4G). Moreover, Cdt1 self-association might recruit two MCM complexes to a replication origin in a single reaction. These findings provide novel insights into the molecular mechanism of DNA replication initiation.

Acknowledgments

We thank Reo Sato and Sara Kaneko for their technical assistance. This work was supported by JSPS KAKENHI (Grant No. 21K06551).

Conflict of Interest

The authors declare no conflict of interest.

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