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Geminin Has Dimerization, Cdt1-binding, and Destruction Domains That Are Required for Biological Activity

2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês

10.1074/jbc.m407726200

1083-351X

Jacqueline M. Benjamin, Susanna J. Torke, Borries Demeler, Thomas J. McGarry,

DNA Repair Mechanisms

Geminin is an unstable regulatory protein that affects both cell division and cell differentiation. Geminin inhibits a second round of DNA synthesis during S and G2 phase by binding the essential replication protein Cdt1. Geminin is also required for entry into mitosis, either by preventing replication abnormalities or by down-regulating the checkpoint kinase Chk1. Geminin overexpression during embryonic development induces ectopic neural tissue, inhibits eye formation, and perturbs the segmental patterning of the embryo. In order to define the structural and functional domains of the geminin protein, we generated over 40 missense and deletion mutations and tested their phenotypes in biological and biochemical assays. We find that geminin self-associates through the coiled-coil domain to form dimers and that dimerization is required for activity. Geminin contains a typical bipartite nuclear localization signal that is also required for its destruction during mitosis. Nondegradable mutants of geminin interfere with DNA replication in succeeding cell cycles. Geminin's Cdt1-binding domain lies immediately adjacent to the dimerization domain and overlaps it. We constructed two nonbinding mutants in this domain and found that they neither inhibited replication nor permitted entry into mitosis, indicating that this domain is necessary for both activities. We identified several missense mutations in geminin's Cdt1 binding domain that were deficient in their ability to inhibit replication yet were still able to allow mitotic entry, suggesting that these are separate functions of geminin. Geminin is an unstable regulatory protein that affects both cell division and cell differentiation. Geminin inhibits a second round of DNA synthesis during S and G2 phase by binding the essential replication protein Cdt1. Geminin is also required for entry into mitosis, either by preventing replication abnormalities or by down-regulating the checkpoint kinase Chk1. Geminin overexpression during embryonic development induces ectopic neural tissue, inhibits eye formation, and perturbs the segmental patterning of the embryo. In order to define the structural and functional domains of the geminin protein, we generated over 40 missense and deletion mutations and tested their phenotypes in biological and biochemical assays. We find that geminin self-associates through the coiled-coil domain to form dimers and that dimerization is required for activity. Geminin contains a typical bipartite nuclear localization signal that is also required for its destruction during mitosis. Nondegradable mutants of geminin interfere with DNA replication in succeeding cell cycles. Geminin's Cdt1-binding domain lies immediately adjacent to the dimerization domain and overlaps it. We constructed two nonbinding mutants in this domain and found that they neither inhibited replication nor permitted entry into mitosis, indicating that this domain is necessary for both activities. We identified several missense mutations in geminin's Cdt1 binding domain that were deficient in their ability to inhibit replication yet were still able to allow mitotic entry, suggesting that these are separate functions of geminin. Cell division and cell differentiation are tightly coupled during embryonic development. In many ways, cell division and differentiation are mutually exclusive; most differentiated cells in the adult are unable to divide, whereas relatively undifferentiated stem cells continue to divide throughout the life of an organism. Cancerous cells are characterized both by uncontrolled cell division and by a loss of differentiated function. The molecular pathways that link cell division and differentiation are poorly understood. Geminin is an unstable 25-kDa protein that has profound effects on both cell division and cell differentiation (1McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar, 2Kroll K.L. Salic A.N. Evans L.M. Kirschner M.W. Development. 1998; 125: 3247-3258Crossref PubMed Google Scholar, 3Del Bene F. Tessmar-Raible K. Wittbrodt J. Nature. 2004; 427: 745-749Crossref PubMed Scopus (211) Google Scholar, 4Luo L. Yang X. Takihara Y. Knoetgen H. Kessel M. Nature. 2004; 427: 749-753Crossref PubMed Scopus (181) Google Scholar). Geminin is a protein of complex multicellular organisms; it is found universally in vertebrates and in Drosophila but is absent from yeasts and the nematode Caenorhabditis elegans. Several different activities of geminin have been described. Geminin prevents a second round of DNA replication during S and G2 phase by inhibiting the reassembly of prereplication complex, a collection of essential replication factors that assembles on replication origins before DNA synthesis begins (1McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). Geminin binds and inhibits the protein Cdt1, an essential component of prereplication complex with an unknown biochemical function (5Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (584) Google Scholar, 6Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (390) Google Scholar). Geminin is destroyed by ubiquitin-dependent proteolysis during mitosis at the metaphase/anaphase transition, which allows a new round of DNA synthesis in the succeeding cell cycle (1McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). Geminin also induces entry into mitosis by antagonizing the checkpoint kinase Chk1. When geminin is depleted from Xenopus embryos or cultured somatic cells, Chk1 accumulates in its active phosphorylated form (7McGarry T.J. Mol. Biol. Cell. 2002; 13: 3662-3671Crossref PubMed Scopus (73) Google Scholar, 8Melixetian M. Ballabeni A. Masiero L. Gasparini P. Zamponi R. Bartek J. Lukas J. Helin K. J. Cell Biol. 2004; 165: 473-482Crossref PubMed Scopus (217) Google Scholar). Chk1 activation leads to phosphorylation and inhibition of the mitotic protein kinase Cdc2. The mechanism by which geminin influences Chk1 activity is unknown. It might affect Chk1 activity indirectly by preventing replication abnormalities, or it may be part of a regulatory pathway that directly down-regulates the kinase. Several groups have described specific effects of geminin on the development and differentiation of embryonic cells. In early Xenopus embryos, geminin induces uncommitted ectodermal cells to differentiate into nervous tissue (2Kroll K.L. Salic A.N. Evans L.M. Kirschner M.W. Development. 1998; 125: 3247-3258Crossref PubMed Google Scholar). The mechanism of this induction is unknown, but the activity is reproduced by a fragment of the protein consisting of amino acids 38–89. More recently, it has been reported that geminin can inhibit eye formation in medaka fish embryos by binding and inhibiting the transcription factor Six3 (3Del Bene F. Tessmar-Raible K. Wittbrodt J. Nature. 2004; 427: 745-749Crossref PubMed Scopus (211) Google Scholar). Geminin can also perturb the axial segmentation pattern of chick embryos by binding and inhibiting transcription factors in the hox gene family (4Luo L. Yang X. Takihara Y. Knoetgen H. Kessel M. Nature. 2004; 427: 749-753Crossref PubMed Scopus (181) Google Scholar). These same workers also reported that geminin binds to Scmh1, a protein in the polycomb gene family. The biological consequences of this interaction were not described, but the association suggests that geminin might modify chromatin structure. It has been difficult to understand how a small 25-kDa protein can have such diverse biological effects. Geminin is not homologous to any previously characterized protein. Sequence analysis indicates that geminin has an internal coiled-coil domain consisting of at least five heptad repeats (Figs. 1B and 10). A nine-amino acid destruction box located near the amino terminus is required for the ubiquitylation and destruction of geminin during mitosis (1McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). The region between the coiled-coil and the destruction sequence is rich in basic amino acids that could serve as nuclear localization signals or points of ubiquitin attachment. The carboxyl terminal region is rich in acidic amino acid residues but otherwise poorly conserved among species.Fig. 10Structural and functional domains of the geminin protein.D-Box, destruction box; NLS, nuclear localization signal.View Large Image Figure ViewerDownload (PPT) The purpose of this study was to define the structural and functional organization of the geminin protein. We found that geminin dimerizes through its internal coiled-coil domain with an association constant less than 100 nm. Only the dimerized form of the protein is biologically active. Geminin contains a typical bipartite nuclear localization signal (NLS) 1The abbreviations used are: NLS, nuclear localization sequence; WT, wild type; NTA, nitrilotriacetic acid. in the basic domain close to the destruction box. Surprisingly, the NLS sequence is required for the mitotic destruction of the protein. The binding site for Cdt1 is immediately adjacent to the coiledcoil domain and overlaps it slightly. We generated two non-binding mutants and showed that they are nonfunctional. Finally, we identified several missense mutants in the Cdt1-binding domain that inhibit replication poorly yet still promote entry into mitosis, indicating that these two functions are separable. Antibodies—Affinity-purified anti-geminin antibodies were raised by immunizing rabbits with full-length Xenopus geminin H (1McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). His Probe antibodies (sc-804) and agarose-conjugated anti-Myc antibody (9e10) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phosphospecific anti-phospho-Chk1 (Ser345) antibody was purchased from Cell Signaling Technology. For immunofluorescence studies, anti-Myc antibody (9e10) and Cy3-conjugated goat anti-mouse antibody were purchased from Zymed Laboratories Inc. Anti-Myc immunoblots were performed using polyclonal rabbit anti-human Myc antibody purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Plasmid Construction—For rescue studies, geminin deletion mutants were constructed by PCR amplification of geminin fragments using pCS2-gemininwobble as a template. pCS2-gemininwobble encodes wild-type geminin H with eight wobble mutations that preserve the amino acid sequence but make the RNA refractory to inhibition by anti-geminin morpholino oligonucleotides (7McGarry T.J. Mol. Biol. Cell. 2002; 13: 3662-3671Crossref PubMed Scopus (73) Google Scholar). Fragments amplified from pCS2-gemininwobble were inserted between the EcoRI and XhoI sites of pCS2. Geminin missense mutants were constructed by QuikChange site-directed mutagenesis (Stratagene) using pCS2-gemininwobble as a template. pCS2-gemininΔ100–117 was constructed by amplifying an EcoRI fragment of pCS2-gemininwobble encoding amino acids 1–100 and inserting it into the EcoRI site of pCS-2-gemininN117. Two extra bases were added during the PCR reaction in order to preserve the correct reading frame. A similar method was used to construct gemininΔ63–80, gemininΔ63–90, and gemininΔ63–100. The sequence of each mutant was confirmed by dideoxy sequencing. pET28-geminin clones were used to express geminin protein in bacteria as described (1McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). Geminin missense mutants were constructed by the QuikChange site-directed mutagenesis method (Stratagene) using pET28-gemininWT as a template. pCS2-MT-gemininWT, encoding the geminin H gene fused to a Myc tag, was constructed by amplifying the geminin H from the original Xenopus clone (6.42.152) and inserting it between the EcoRI and XhoI sites of pCS2-MT. Geminin deletion mutants were constructed in the same manner. pCS2-MT-gemininWT/NH has unique NdeI and HindIII sites flanking the region encoding amino acids 93–121. It was constructed from pCS2-MT-gemininWT by QuikChange mutagenesis. The NdeI site was generated by changing codon 93 from GCT to GCA, and the HindII site was generated by changing codon 121 from GCA to GCT. Neither mutation changes the amino acid sequence of the encoded protein. The mutation at codon 93 simultaneously destroys a second HindIII site found naturally in Xenopus geminin, making the engineered HindIII site unique. To make the NdeI site unique, we destroyed a second NdeI site found in the pCS-MT vector by QuikChange mutagenesis. Random missense mutations between amino acids 100 and 117 of geminin were constructed using a degenerate oligonucleotide approach (9Hill D.E. Chanda V.B. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1989: 8.2.1-8.2.7Google Scholar). We first synthesized a degenerate oligonucleotide that encoded this region flanked by an NdeI site and a HindIII site (CGACGACATATGACCTTATGGTGAAAgaAacAccAacTtgCctTtaCtgGaaGgGgtTgcAgaGgaAcgAagAaaGgcCCTCTATGAAGCTTCA). At each position denoted by a lowercase letter, a mixture of oligonucleotide precursors was used that contained 90% of the correct base and 3.3% of each of the three other bases. The 3′ end of the oligonucleotide was self-complementary. The oligonucleotide was hybridized to itself and made double-stranded using the large (Klenow) fragment of DNA polymerase I. It was then digested with NdeI and HindIII and inserted into pCS2-MTgemininWT/NH cut with the same enzymes. Individual mutant clones were screened for their ability to bind recombinant Cdt1 (see "Binding Assays") and transferred to pCS2-gemininwobble and pET28-gemininWT. pCS2-MTCdt1WT, encoding wild-type Xenopus Cdt1 fused to a Myc tag, was constructed by amplifying the Cdt1 gene from a cDNA clone of Xenopus Cdt1 (10Maiorano D. Moreau J. Mechali M. Nature. 2000; 404: 622-625Crossref PubMed Scopus (295) Google Scholar) and inserting it between the XbaI and XhoI sites of pCS2-MT. Protein Purification—Hexahistidine-tagged proteins were expressed in bacterial strain BL21 and purified using Ni2+-NTA-agarose according to standard techniques (Qiagen). For analytical ultracentrifuge analysis and importin-binding assays, the hexahistidine tag was removed by thrombin treatment, and contaminants were removed by passing the digest over Ni2+-NTA-agarose. For analytical ultracentrifuge analysis, geminin was further purified over Q-Sepharose and dialyzed against 50 mm sodium phosphate, pH 7.4, 300 mm NaCl. Analytical Ultracentrifugation—Sedimentation experiments were performed using a Beckman Optima XL-A centrifuge with an AN 60 Ti rotor. Samples were dissolved in 50 mm sodium phosphate, pH 7.4, 300 mm NaCl. The partial specific volume of geminin was estimated to be 0.7232 ml/g using the method of Cohn and Edsall (11Cohn E.J. Edsall J.T. Proteins, Amino Acids and Peptides as Ions and Dipole Ions. Reinhold, New York1943: 157Google Scholar). The molar extinction coefficient was estimated to be 18,140 at 280 nm using the method of Gill and von Hippel (12Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5060) Google Scholar). Molar extinction coefficients at other wavelengths were determined as previously described (13Russell T.R. Demeler B. Tu S.C. Biochemistry. 2004; 43: 1580-1590Crossref PubMed Scopus (14) Google Scholar). Sedimentation velocity experiments were performed at 45,000 rpm at 20 °C. The loading concentration was ∼8.6 μm. A total of 150 scans were collected and analyzed using the van Holde-Weischet method and the finite element method (14van Holde K.E. Weischet W.O. Biopolymers. 1978; 17: 1387-1403Crossref Scopus (318) Google Scholar, 15Demeler B. Saber H. Biophys. J. 1998; 74: 444-454Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 16.Deleted in proofGoogle Scholar). Sedimentation equilibrium experiments were performed at 4 °C at three different speeds (20,000, 26,600, and 33,300 rpm). Scans were collected at equilibrium at 230 and 280 nm. Multiple loading concentrations were measured at both wavelengths, and data points exceeding 0.9 OD were discarded. The protein concentration for the analyzed data points ranged between 80 nm and 40 μm. Data were fitted to multiple models. The most appropriate model was chosen based upon visual inspection of the residual run patterns and upon the best statistics. 95% confidence intervals were determined by Monte Carlo analysis. A minimum of 5000 Monte Carlo iterations were performed. All data analyses were performed using Ultrascan version 6.2. 2B. Demeler, University of Texas Health Science Center at San Antonio, Department of Biochemistry. Binding Assays—Proteins were either expressed in bacteria or produced by in vitro transcription and translation of plasmid DNA in reticulocyte lysate (Promega TNT system). Reactions typically contained 5 ng to 1.5 μg of recombinant protein or 10–25 μl of reticulocyte lysate. Proteins were mixed in a total volume of 25–50 μl and incubated at room temperature for 1 h. After binding, an aliquot was removed to be used as a loading control. Nickel-NTA or antibody-coated beads (2–5 μl of packed beads containing 1 μg of antibody/μl) were added, and the mixture was tumbled at room temperature for 1 h. The beads were recovered and washed with immunoprecipitation buffer (50 mm β-glycerol phosphate, pH 7.4, 5 mm EDTA, 0.1% Triton X-100, 1 mm dithiothreitol, 100–500 mm NaCl, and 10 μg/ml each leupeptin, pepstatin, and chymostatin). For importin binding assays, the wash buffer also contained 10% glycerol and 1 m NaCl, and the dithiothreitol was omitted. Proteins were separated on polyacrylamide gels and visualized by immunoblotting. For in vivo binding assays, Xenopus oocytes were injected with 16 ng of anti-geminin morpholino oligonucleotide and 200–400 pg of RNA encoding geminin or Cdt1. Our previous work has shown that this amount of RNA produces a physiological concentration of geminin (7McGarry T.J. Mol. Biol. Cell. 2002; 13: 3662-3671Crossref PubMed Scopus (73) Google Scholar). Immunofluorescence—BHK cells were cultured on coverslips in 1× Dulbecco's modified Eagle's medium, 10% calf serum, 10% tryptose phosphate broth. Cells were transiently transfected with plasmids encoding Myc-tagged geminin mutants using Lipofectamine™ (Invitrogen). Twenty-four hours after transfection, cells were fixed with 1× phosphate-buffered saline, 3.1% formaldehyde, permeabilized with 1× phosphate-buffered saline, 0.1% Triton X-100, and stained with 9e10 anti-Myc antibody (Zymed Laboratories Inc.) and CY3-conjugated goat anti-mouse antibody (Zymed Laboratories Inc.). Nuclei were counterstained with 1 μg/ml 4′,6-diamidino-2-phenylindole in phosphate-buffered saline. To visualize nuclei in Xenopus embryos, two-cell embryos were injected with fluoro-green (Amersham Biosciences) and allowed to develop to stage 9. Confocal images were taken at 10 μm intervals and projected onto a single plane. DNA Replication Assays—DNA replication assays were performed using cytostatic factor-arrested Xenopus egg extracts and demembranated sperm DNA template as described (1McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). Proteins were added at a concentration of 50 ng/μl. The negative controls were no template added and no calcium added. The positive controls were no addition and geminin dilution buffer only (10 mm HEPES, pH 7.7, 300 mm NaCl). Percentage replication was normalized to the positive controls. The average of at least two measurements is reported for each protein. Rescue Assays—Two-cell Xenopus embryos were injected with morpholino anti-geminin oligonucleotide and geminin RNA as described previously (7McGarry T.J. Mol. Biol. Cell. 2002; 13: 3662-3671Crossref PubMed Scopus (73) Google Scholar). For each mutant, a minimum of 18 two-cell embryos were injected on each side (36 injections total). Rescue efficiency was calculated as the percentage of rescue produced by the mutant divided by the percent of rescue produced by wild-type geminin, multiplied by 100%. Injection and scoring were performed blindly to avoid bias in the results. Degradation Assays—Geminin mutants were transcribed and translated from plasmid DNA using reticulocyte lysate (Promega TNT System) in the presence of [35S]methionine. Translation lysate was mixed with cytostatic factor-arrested Xenopus extract in a 1:4 volume ratio. An aliquot was withdrawn for the t = 0 sample. Degradation was initiated by adding calcium, and a second aliquot was taken after 1 h. The destruction of geminin was visualized by polyacrylamide gel electrophoresis and autoradiography. Geminin Dimerizes through the Coiled-coil Domain—Computer algorithms predict that the mid-portion of the geminin protein folds into a coiled-coil domain consisting of five or more heptad repeats (Fig. 1B). This observation suggests that geminin self-associates, since coiled-coil domains are frequently sites of homotypic protein-protein interactions. To test if geminin forms oligomers in solution, a plasmid encoding Myc-tagged geminin and a plasmid encoding His-tagged geminin were transcribed and translated together in reticulocyte lysate. When the Myc-tagged geminin was precipitated with anti-Myc antibodies, His-tagged geminin was detected in the precipitate by immunoblotting (Fig. 1A, lane 4). His-tagged geminin was not precipitated by anti-Myc antibodies when translated alone (lane 1). This indicates that the two tagged forms of geminin physically associate with each other. The association is greatly reduced if the proteins are translated separately and then mixed (lane 3), suggesting that oligomers of geminin are relatively stable once formed and do not readily exchange subunits. To determine whether oligomerization occurs through the coiled-coil domain, the binding experiment was repeated using full-length His-tagged geminin and a series of Myc-tagged geminin deletion mutants (Fig. 1, B and C). Removal of amino-terminal amino acids up to residue 117 did not affect the interaction between the two tagged proteins (Fig. 1C, lanes 1–4); nor did removal of carboxyl terminal amino acids beyond residue 180 (lanes 7–9). However, deletion of residues between positions 117 and 160 completely abolished the association (lanes 5 and 6). This indicates that the self-association domain lies between residues 120 and 160, which almost exactly corresponds the limits of the coiled-coil (residues 118–152). To see if geminin oligomerizes in vivo, mature stage VI oocytes were injected with 200 pg of RNA encoding Myc-tagged full-length geminin. Injection of this amount of RNA yields an amount of geminin protein that is similar to the endogenous level (7McGarry T.J. Mol. Biol. Cell. 2002; 13: 3662-3671Crossref PubMed Scopus (73) Google Scholar). The oocytes were then treated with progesterone to induce the translation of both the injected and the endogenous geminin RNA. To see if these two proteins associated, Myc-geminin was precipitated with 9e10 anti-Myc antibody, and the precipitate was blotted with anti-geminin antibody. Both Myc-tagged geminin (asterisk) and endogenous geminin (arrowheads) were detected in the precipitate (Fig. 1D, lane 2). The Myc antibody did not precipitate untagged geminin from uninjected eggs (lane 1). This indicates that geminin forms oligomers in vivo at physiological concentrations. To confirm that the interaction occurred through the coiled-coil, oocytes were injected with different Myc-tagged geminin deletion mutants. Mutants that included the coiled-coil domain associated with the endogenous geminin (lanes 3–6 and 9), and mutants that encroached upon this region did not bind (lanes 7 and 8). In this experiment, gemininC160, consisting of amino acids 1–160, associates less strongly with Myc-geminin than the wild-type. This suggests that the coiled-coil may extend past residue 160. To determine the number of geminin subunits in the multimer, we performed sedimentation velocity and sedimentation equilibrium analysis of highly purified bacterially expressed geminin. The velocity analysis indicated that ∼92% of the protein exhibited a sedimentation coefficient of 2.44 S (Fig. 2B). The equilibrium analysis was performed at several different protein concentrations in order to allow for the possibility that several different geminin species may be present in reversible equilibrium with each other. All experimental observations (18 scans total) could be fit to a model in which a single ideal species is present with a molecular mass of 52.69 ± 0.2 kDa. The variance was extremely low at 2.2 × 10–5. A plot of the residuals and overlays for this fit is shown in Fig. 2C. The measured molecular weight is in excellent agreement with that predicted for a geminin dimer based upon the protein sequence (51.31 kDa). Adding additional parameters to account for more than one ideal species (e.g. monomer + dimer or monomer + trimer) did not reduce the variance. We conclude that the data are best described by a single species consisting of geminin dimers. Because no monomer could be detected in the preparation, we estimate that the geminin-geminin association constant is less than 100 nm. Combining the data from the equilibrium and velocity analyses, we estimate that the frictional ratio for geminin (f/fo) is about 2.2. This value suggests that geminin assumes an elongated shape, consistent with the observation that geminin elutes from a gel filtration column at an apparent molecular weight that is markedly higher than the true molecular weight (Fig. 2A). Geminin Dimerization Is Required for Activity—We previously reported that the coiled-coil domain is required for geminin to inhibit replication (1McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). In that study, we found that the two deletion mutants that do not dimerize (gemininC120 and gemininC140) do not inhibit replication and that the C160 mutant, which dimerizes less efficiently, is somewhat reduced in its ability to inhibit replication (Table I and Fig. 4B, black bars). The phenotypes of these mutants indicate that only dimerized geminin protein is active as a replication inhibitor.Table IAbility of mutants to inhibit DNA replication and rescue geminin deficiencyMutantReplicationRescueMutantReplicationRescue%%%%Neg CO(100)0NDQ1.199WT2.1(100)LTS1.0100N305.695DPE1.0106N80<0.5ENK0.7100N100124EAY9.494N120145<3DL3.1104C12076<3KE0.497C140774.5A111G1.5106C1608.181Y106F1.995C1807.995AYPRV2.3124C2004.9119SS1.2112C12076<3AEERR6.3108LAP95FFK11.4109DEL0.2PTC12.3100T35A95KKFEV4685.5S42A107YWK93.325KRK1.6RTGG933RTK/KRK<0.5SAPD66<3RTK/KRK/KK0.8D100-11759<3 Open table in a new tab To see if dimerization is also required for geminin's biological activity, we tested the deletion mutants to see if they could rescue the lethal phenotype of geminin-deficient Xenopus embryos using a complementation assay that we developed previously (7McGarry T.J. Mol. Biol. Cell. 2002; 13: 3662-3671Crossref PubMed Scopus (73) Google Scholar). When geminin is depleted from Xenopus embryos with antisense oligonucleotides, the embryonic cells arrest in the G2 phase of the cell cycle after the 13th cell division. The phenotype is visually apparent under the dissecting microscope, because the arrested embryos have larger cells than control embryos (Fig. 3, compare A and B). At the arrest point, the checkpoint kinase Chk1 is found in the phosphorylated active form, which can be demonstrated by immunoblotting using a phosphospecific antibody raised against Chk1 phosphorylated on serine 345 (Ser345, Fig. 4A, lane 2). In contrast, control embryos have little phosphorylated Chk1 (lane 1). The arrest caused by geminin depletion can be rescued by injecting wild-type geminin RNA immediately after the antisense oligonucleotide. The rescuing RNA is mutated at eight "wobble" positions so that it does not hybridize to the antisense oligonucleotide, whereas the amino acid sequence is preserved. Suppression of the G2 arrest by wild-type geminin is accompanied by a reduction in the amount of Ser345-phosphorylated Chk1 down to the levels found in control embryos (Fig. 4A, lane 3). The degree of rescue is quantified by calculating the percentage of RNA injections that produce a sector of cells that continue dividing past the 13th division, as judged by visual inspection under a dissecting microscope (Fig. 2, compare B and C). Typically, 60–100% of wild-type geminin RNA injections produce a sector of rescued cells (Fig. 4B, gray bars). "Wobbled" RNAs encoding different geminin deletion mutants were injected after the antisense oligonucleotide to see if they could also rescue the G2 arrest phenotype. Carboxyl terminal deletion mutants truncated at amino acid positions 160, 180, or 200 are able to rescue geminin-deficient embryos nearly as well as wild-type geminin (Table I and Fig. 4B, gray bars). Embryos injected with these mutants also showed normal low levels of Chk1 phosphorylation on Ser345 (Fig. 4A, lanes 7–9), confirming that a G2 arrest had not occurred. These results indicate that the carboxyl-terminal amino acids from 160 to 219 are not required for biological activity. In contrast, the gemininC120 and gemininC140 are not able to rescue geminin-deficient embryos at all. Virtually all of the embryos injected with RNA encoding either of these mutants arres