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• W2028331361 abstract "Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development. Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development. Development and homeostasis of multicellular organisms depends on the generation of the appropriate number of cells through cell proliferation and the acquisition of specialized cell functions through cell differentiation. Geminin has been suggested to regulate proliferation-differentiation decisions through balanced interactions with the DNA replication licensing factor Cdt1 and factors regulating transcription during development (1Seo S. Kroll K.L. Cell Cycle. 2006; 5: 374-379Crossref PubMed Scopus (50) Google Scholar). Geminin, initially identified as a substrate of the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase during mitosis (2McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar), was shown to regulate DNA replication by directly binding to and inhibiting the DNA replication licensing factor Cdt1 (3Wohlschlegel 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, 4Tada S. Li A. Maiorano D. Méchali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (390) Google Scholar, 5Nishitani H. Taraviras S. Lygerou Z. Nishimoto T. J. Biol. Chem. 2001; 276: 44905-44911Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Geminin binding to Cdt1 during the S and G2 phases of the cell cycle ensures that licensing of a further round of DNA replication is inhibited until the end of mitosis (6Blow J.J. Dutta A. Nat. Rev. Mol. Cell Biol. 2005; 6: 476-486Crossref PubMed Scopus (533) Google Scholar, 7Lygerou Z. Nurse P. Science. 2000; 290: 2271-2273PubMed Google Scholar). Geminin depletion causes over-replication, DNA damage, G2/M arrest (8Zhu W. Chen Y. Dutta A. Mol. Cell. Biol. 2004; 24: 7140-7150Crossref PubMed Scopus (205) Google Scholar, 9Melixetian 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, 10Lin J.J. Dutta A. J. Biol. Chem. 2007; 282: 30357-30362Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 11McGarry T.J. Mol. Biol. Cell. 2002; 13: 3662-3671Crossref PubMed Scopus (73) Google Scholar), and centrosome over-duplication (12Tachibana K.E. Gonzalez M.A. Guarguaglini G. Nigg E.A. Laskey R.A. EMBO Rep. 2005; 6: 1052-1057Crossref PubMed Scopus (58) Google Scholar) in a cell type-specific manner (13Zhu W. Depamphilis M.L. Cancer Res. 2009; 69: 4870-4877Crossref PubMed Scopus (97) Google Scholar). Geminin overexpression in human cell lines is also cell type-specific, leading to arrest at the G1/S phase transition or a defective S-phase followed by apoptosis (14Shreeram S. Sparks A. Lane D.P. Blow J.J. Oncogene. 2002; 21: 6624-6632Crossref PubMed Scopus (152) Google Scholar). Geminin was also identified as a putative regulator of differentiation, as its overexpression promoted the expansion of the neural plate in Xenopus embryos (15Kroll K.L. Salic A.N. Evans L.M. Kirschner M.W. Development. 1998; 125: 3247-3258Crossref PubMed Google Scholar). Geminin was shown to interact with transcriptional regulators of differentiation, such as Six3 (16Del Bene F. Tessmar-Raible K. Wittbrodt J. Nature. 2004; 427: 745-749Crossref PubMed Scopus (213) Google Scholar), Hox and Polycomb family members (17Luo L. Yang X. Takihara Y. Knoetgen H. Kessel M. Nature. 2004; 427: 749-753Crossref PubMed Scopus (182) Google Scholar), the catalytic subunit of the SWI/SNF chromatin remodeling complex, Brg1/Brm (18Seo S. Herr A. Lim J.W. Richardson G.A. Richardson H. Kroll K.L. Genes Dev. 2005; 19: 1723-1734Crossref PubMed Scopus (162) Google Scholar), and regulators of Sox2 gene transcription (19Papanayotou C. Mey A. Birot A.M. Saka Y. Boast S. Smith J.C. Samarut J. Stern C.D. PLoS Biol. 2008; 6: e2Crossref PubMed Scopus (88) Google Scholar), thereby modulating proliferation-differentiation decisions during development. Geminin balanced interactions with its multiple binding partners are central to its function in the coordination of proliferation and differentiation. The central region of Geminin, containing the Geminin coiled-coil, is sufficient for interactions with Cdt1 and inhibition of licensing and mediates homodimerization of Geminin (20Lee C. Hong B. Choi J.M. Kim Y. Watanabe S. Ishimi Y. Enomoto T. Tada S. Kim Y. Cho Y. Nature. 2004; 430: 913-917Crossref PubMed Scopus (119) Google Scholar, 21Saxena S. Yuan P. Dhar S.K. Senga T. Takeda D. Robinson H. Kornbluth S. Swaminathan K. Dutta A. Mol. Cell. 2004; 15: 245-258Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 22Thépaut M. Maiorano D. Guichou J.F. Augé M.T. Dumas C. Méchali M. Padilla A. J. Mol. Biol. 2004; 342: 275-287Crossref PubMed Scopus (28) Google Scholar, 23De Marco V. Gillespie P.J. Li A. Karantzelis N. Christodoulou E. Klompmaker R. van Gerwen S. Fish A. Petoukhov M.V. Iliou M.S. Lygerou Z. Medema R.H. Blow J.J. Svergun D.I. Taraviras S. Perrakis A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19807-19812Crossref PubMed Scopus (62) Google Scholar). In this study we introduce a previously uncharacterized human protein that is similar to Geminin in its central coiled-coil region. We name this protein Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology). We show that Idas binds to the Geminin coiled-coil region and can modulate Geminin ability to bind Cdt1. Our data highlight Idas as a novel Geminin binding partner and regulator. Tblastn (NCBI) was used to search expressed sequences from the human genome using the hGeminin protein sequence as query. mRNAs deriving from 5q11.2, on chromosome 5, were identified as encoding a protein with significant similarity to Geminin. This region is currently annotated as LOC345643. Gene2EST was used to identify Expressed Sequence Tags from this locus. The expression of LOC345643 is supported by 1 full-length cDNA (clone CS0DK002YL21) and 10 Expressed Sequence Tags that represent sequence reads from 5 cDNAs from HeLa cells (clone CS0DK002YL21), Jurkat cells (clone CS0DJ001YB09), melanotic melanoma (clone IMAGE:3916292) and mammary adenocarcinoma cell lines (clone IMAGE:5406358), and pooled primary tissues. (DR007866.1). Alignment of available Expressed Sequence Tags and cDNA sequences was used to generate a predicted mRNA of 2087 nucleotides. This is longer than the automatically predicted mRNA present in the databases (Locus XR_040412, 1158 nucleotides) because of the presence of 5′-UTR (178 nucleotides) and 3′-UTR (929 nucleotides) sequences. The intron-exon boundaries were defined by aligning the predicted mRNA to human genomic sequences. Fragments of the predicted mRNA were amplified by polymerase chain reaction (PCR) and sequenced to verify expression of this locus in HeLa cells. Real time PCR was used to detect expression of Idas in different human cell lines. The full predicted open reading frame (ORF) was amplified from HeLa cDNA and sequenced. The hIdas protein sequence was derived from the ORF. Idas orthologues in mouse (LOC622408) and Xenopus laevis (LOC100158359) were identified by Blast using the human Idas protein sequence as query. For sequence analysis and alignments, the following programs were used: Gene2EST (24Gemünd C. Ramu C. Altenberg-Greulich B. Gibson T.J. Nucleic Acids Res. 2001; 29: 1272-1277Crossref PubMed Scopus (28) Google Scholar), Coils (25Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3483) Google Scholar), ELM (Eukaryotic Linear Motif) resource for functional sites in proteins (26Puntervoll P. Linding R. Gemünd C. Chabanis-Davidson S. Mattingsdal M. Cameron S. Martin D.M. Ausiello G. Brannetti B. Costantini A. Ferrè F. Maselli V. Via A. Cesareni G. Diella F. Superti-Furga G. Wyrwicz L. Ramu C. McGuigan C. Gudavalli R. Letunic I. Bork P. Rychlewski L. Küster B. Helmer-Citterich M. Hunter W.N. Aasland R. Gibson T.J. Nucleic Acids Res. 2003; 31: 3625-3630Crossref PubMed Scopus (520) Google Scholar), and ClustalW (27Larkin M.A. Blackshields G. Brown N.P. Chenna R. McGettigan P.A. McWilliam H. Valentin F. Wallace I.M. Wilm A. Lopez R. Thompson J.D. Gibson T.J. Higgins D.G. Bioinformatics. 2007; 23: 2947-2948Crossref PubMed Scopus (22841) Google Scholar). Total HeLa cDNA was used to amplify the hIdas ORF by nested PCR, introducing NheI and KpnI sites at the ends of the predicted hIdas ORF. The PCR product was cloned into the mammalian expression vector cDNA3.1EGFP (Invitrogen) at the NheI and KpnI restriction sites to produce a protein C-terminally fused to green fluorescent protein (GFP) under the control of the constitutive CMV promoter. N-Idas (amino acids 1–127) and C-Idas (amino acids 131–385) were cloned into the NheI and HindIII sites of pcDNA3.1EGFP (Invitrogen) after PCR amplification from full-length Idas to introduce restriction sites. IdasHA and Idas-Cherry were generated by replacing GFP from the IdasGFP pcDNA3.1 construct with three repeats of the human influenza hemagglutinin epitope (HA) and sequences coding for Cherry (Clontech), respectively. All products produced by PCR were fully sequenced. For in vitro experiments, the predicted folded domain (101–284, dIdas) and the coiled-coil domain (173–245, tIdas) were cloned for expression in the NKI-His-3C-LIC (for cleavable His-tag expression) and the NKI-LIC plasmids (for native versions). Because these plasmids are resistant to kanamycin and ampicillin, respectively, they allow for efficient co-expression experiments. The Idas sequence was analyzed using the ProteinCCD server (28Mooij W.T. Mitsiki E. Perrakis A. Nucleic Acids Res. 2009; 37: W402-W405Crossref PubMed Scopus (38) Google Scholar), which was also used for the automated design of oligonucleotides suitable for PCR-based restriction and ligase-free cloning to the NKI-LIC vectors. A construct expressing full-length Geminin was cloned in the pET22b vector from Novagen. A construct for expressing human Cdt1 harboring residues (158–396, mini-Cdt1) was cloned in the pET28a vector (Novagen) with an N-terminal His tag. For antibody production, N-Idas was cloned into the NheI and HindIII sites of pET28a vector (Novagen) with the N-terminal His tag using PCR amplification to introduce sites. A mouse Idas cDNA (IMAGE: 5830438-C23) in vector pFLCI was obtained (Geneservice Ltd). A vector suitable for generation of antisense probes for in situ hybridization was constructed by removing the 3′-untranslated region of the cDNA using an internal KpnI site. A second vector for in situ hybridization was generated by subcloning the mIdas open reading frame to pBluescript between SacI and KpnI sites. The following plasmids have been described elsewhere: GemininGFP (29Roukos V. Iliou M.S. Nishitani H. Gentzel M. Wilm M. Taraviras S. Lygerou Z. J. Biol. Chem. 2007; 282: 9346-9357Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), GemininΔ90–120GFP (30Ballabeni A. Melixetian M. Zamponi R. Masiero L. Marinoni F. Helin K. EMBO J. 2004; 23: 3122-3132Crossref PubMed Scopus (116) Google Scholar, 31Xouri G. Squire A. Dimaki M. Geverts B. Verveer P.J. Taraviras S. Nishitani H. Houtsmuller A.B. Bastiaens P.I. Lygerou Z. EMBO J. 2007; 26: 1303-1314Crossref PubMed Scopus (66) Google Scholar), Cdt1dhcRed (31Xouri G. Squire A. Dimaki M. Geverts B. Verveer P.J. Taraviras S. Nishitani H. Houtsmuller A.B. Bastiaens P.I. Lygerou Z. EMBO J. 2007; 26: 1303-1314Crossref PubMed Scopus (66) Google Scholar), GemininHA (29Roukos V. Iliou M.S. Nishitani H. Gentzel M. Wilm M. Taraviras S. Lygerou Z. J. Biol. Chem. 2007; 282: 9346-9357Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), plasmids for mouse Geminin in situ probes (32Xouri G. Lygerou Z. Nishitani H. Pachnis V. Nurse P. Taraviras S. Eur. J. Biochem. 2004; 271: 3368-3378Crossref PubMed Scopus (101) Google Scholar, 33Spella M. Britz O. Kotantaki P. Lygerou Z. Nishitani H. Ramsay R.G. Flordellis C. Guillemot F. Mantamadiotis T. Taraviras S. Neuroscience. 2007; 147: 373-387Crossref PubMed Scopus (36) Google Scholar). Idas mRNA expression levels were assessed by quantitative real time PCR (LightCycler 02, Roche Applied Science) using the QuantiFast SYBR Green PCR kit (204052, Qiagen). The primer sequences for Idas are 5′-GACGCGCTTGTTGAGAATAA-3′ and 5′-CACGTTCCGCTCCTTGAG-3′. Total mRNA was isolated using the RNeasy mini kit (74104,Qiagen), and 800 ng of total RNA were converted to cDNA using Superscript II (18064-014, Invitrogen). A final dilution of 1:5 from the resulting cDNA was amplified for 40 cycles. All samples were normalized to the YWHAZ (tryptophan S-monooxygenase activation protein, ζ polypeptide) gene. The primer sequences for YWHAZ gene amplification are 5′-GATCCCCAATGCTTCACAAG-3′ and 5′-TGCTTGTTGTGACTGATCGAC-3′. HeLa and 293T cells were grown in DMEM (Invitrogen) with 10% fetal bovine serum (Invitrogen). MCF7 cells were grown in DMEM (Invitrogen) with 20% fetal bovine serum (Invitrogen). MCF7 (1–3 × 105 cells) were transfected with a total of 1 μg of plasmid DNA using Genejammer (Stratagene) following the manufacturer's instructions, and cells were analyzed 22 h post-transfection. 293T cells were transfected using the calcium phosphate method. For the construction of stable cell lines expressing IdasGFP, HeLa cells were transfected with IdasGFP vector using Genejammer (Stratagene) and grown in the presence of Geneticin (500 μg/ml, 10131-019, Invitrogen). Resistant colonies were selected, screened for expression, and amplified. For RNAi experiments, the following STEALTH (Invitrogen) siRNAs were used for Idas: 5′-CCACCAAACGGAAGCAGACTTCAAT-3′ and 5′-GAGACGCGCTTGTTGAGAATAATCA-3′. The negative control was 5′-CCAAACAGGAACGGACATTCCCAAT-3′. For Idas silencing, HeLa cells were treated with 50 nm concentrations of each Idas siRNA or 100 nm control siRNA twice (at 0 and 24 h) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and were analyzed 24 h after the second transfection. For mitotic synchronization, HeLa cells and 293T cells were incubated with nocodazole (50 ng/ml, Sigma) for 16 h. When synchronization was combined with transient transfection in HeLa or 293T cells, nocodazole was added 5 or 18 h after transfection, respectively. Cells grown on poly-lysine-covered coverslips were fixed in 4% paraformaldehyde for 10 min, washed twice with phosphate-buffered saline (PBS), permeabilized with 0.3% Triton X-100 in PBS, and blocked in 3% bovine serum albumin and 10% fetal bovine serum in PBS (blocking solution) for 1 h. Coverslips were incubated with primary antibody in blocking solution overnight at 4 °C. Cells were washed with PBS, 0.1% Tween and incubated for 1 h with the following secondary antibodies in blocking solution: Alexa 488-conjugated goat anti-rabbit (A11034, Molecular Probes) and Alexa 568-conjugated goat anti-mouse (A11004, Molecular Probes). After washing, DNA was stained with DAPI or Hoechst. For double immunostaining with BrdU, cells were first immunostained for Geminin or Cdt1 as above, fixed with 4% paraformaldehyde, and washed twice with PBS, 0.1% Tween followed by 1 h of incubation with 2 n HCl, washing with 0.1 m Tris-HCl, pH 8.8, and 1 h of blocking. Coverslips were incubated with anti-BrdU (1:80) (Oxford Biotechnology, OBT 00305) in blocking solution overnight at 4 °C, washed with PBS, 0.1% Tween, and incubated for 1 h with Alexa 568-conjugated goat anti-rat (A11077, Molecular Probes) secondary antibody in blocking solution. DNA was stained with Hoechst. Fluorescence immunohistochemistry was performed as described previously (33Spella M. Britz O. Kotantaki P. Lygerou Z. Nishitani H. Ramsay R.G. Flordellis C. Guillemot F. Mantamadiotis T. Taraviras S. Neuroscience. 2007; 147: 373-387Crossref PubMed Scopus (36) Google Scholar) on mouse embryos at E12.5 dpc. 2The abbreviations used are: dpcdays post-coitusNLSnuclear localization signalGEMGemininD-boxdestruction boxAPC/CAnaphase Promoting Complex/Cyclosome. days post-coitus nuclear localization signal Geminin destruction box Anaphase Promoting Complex/Cyclosome. Non-radioactive in situ hybridization was performed as described in Spella et al. (33Spella M. Britz O. Kotantaki P. Lygerou Z. Nishitani H. Ramsay R.G. Flordellis C. Guillemot F. Mantamadiotis T. Taraviras S. Neuroscience. 2007; 147: 373-387Crossref PubMed Scopus (36) Google Scholar) on 10-μm sections of E12.5-dpc mouse embryos. For the generation of Idas antisense RNA probes, the pFLCI vector containing the mIdas open reading frame was first linearized with NotI and in vitro transcribed with T3 RNA polymerase. For control sense probe generation, the pBluescript vector containing the mIdas open reading frame was linearized with KpnI and transcribed with T7 RNA polymerase. Geminin sense and antisense probes were as described previously (32Xouri G. Lygerou Z. Nishitani H. Pachnis V. Nurse P. Taraviras S. Eur. J. Biochem. 2004; 271: 3368-3378Crossref PubMed Scopus (101) Google Scholar, 33Spella M. Britz O. Kotantaki P. Lygerou Z. Nishitani H. Ramsay R.G. Flordellis C. Guillemot F. Mantamadiotis T. Taraviras S. Neuroscience. 2007; 147: 373-387Crossref PubMed Scopus (36) Google Scholar). Sense probes were used as the control in all experiments. All animal-related procedures conformed to the international guidelines on the ethical use of animals. Images were recorded on a Nikon Eclipse TE 2000-U inverted microscope using 60×/1.40 oil and 40×/0.75 lenses. MCF7 cells were transfected with the indicated plasmids and collected 22 h post-transfection. For immunoprecipitation of the endogenous proteins, total cell extracts were collected from HeLa cells. Total extracts were prepared by lysing cells in buffer containing 50 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA, 0,5% Triton X-100, 5 mm MgCl2, and protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 2 mm benzamidine, 1 μg/μl leupeptin, 1.4 μg/μl pepstatin,1 μg/μl aprotinin, 1 μg/μl chymostatin, 0.1 m sodium vanadate). 1.5-μg antibodies bound to protein A-agarose beads (Upstate) were incubated with cell extracts for 3 h at 4 °C. After 3 washes with lysis buffer, immunoprecipitates and total cell extracts corresponding to 10% of immunoprecipitates were analyzed by Western blotting. The following primary antibodies were used at the indicated dilutions. For immunofluorescence: rabbit anti-hIdas (hIdasAb2) (1:500), mouse monoclonal anti-Geminin (1:50) (32Xouri G. Lygerou Z. Nishitani H. Pachnis V. Nurse P. Taraviras S. Eur. J. Biochem. 2004; 271: 3368-3378Crossref PubMed Scopus (101) Google Scholar), rabbit anti-GFP (Molecular Probes, 1:2,000), mouse monoclonal anti-cyclin A (6E6,MS-1061–5, Neomarkers, 1:40), mouse monoclonal anti-cyclin B1 (GNS1, Santa Cruz, 1:200), and mouse monoclonal anti-α-tubulin (T5168, Sigma, 1:20,000). For immunohistochemistry: rabbit anti-hIdas (1:200) rabbit anti-Geminin (Ref. 32Xouri G. Lygerou Z. Nishitani H. Pachnis V. Nurse P. Taraviras S. Eur. J. Biochem. 2004; 271: 3368-3378Crossref PubMed Scopus (101) Google Scholar, 1:1500), mouse monoclonal anti-Pax6 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, 1:1,000). For immunoprecipitation: mouse monoclonal geminin (32Xouri G. Lygerou Z. Nishitani H. Pachnis V. Nurse P. Taraviras S. Eur. J. Biochem. 2004; 271: 3368-3378Crossref PubMed Scopus (101) Google Scholar), mouse monoclonal anti-GFP (3E6, 1181460001, Roche Applied Science), mouse monoclonal anti-HA (12CA5, sc-57592 Santa Cruz), and total mouse IgGs (Sigma). For Western blotting: rabbit anti-hIdas (hIdasAb1) (1:1000), rabbit anti-hIdas (hIdasAb2) (1:500) mouse monoclonal anti-actin (C4, Millipore, 1:3000), rabbit anti-Cdt1 (1:1000) (32Xouri G. Lygerou Z. Nishitani H. Pachnis V. Nurse P. Taraviras S. Eur. J. Biochem. 2004; 271: 3368-3378Crossref PubMed Scopus (101) Google Scholar), rabbit anti-Geminin (1:500) (32Xouri G. Lygerou Z. Nishitani H. Pachnis V. Nurse P. Taraviras S. Eur. J. Biochem. 2004; 271: 3368-3378Crossref PubMed Scopus (101) Google Scholar), rabbit anti-GFP (Santa Cruz, 1:500), and monoclonal anti-HA (12ca5, sc-57592 Santa Cruz, 1:500). Total human IgGs (Sigma) at 2 μg/ml were added to the blocking buffer to reduce binding of secondary antibodies to IgG chains and protein A/G present in immunoprecipitates. N-Idas in pET28 vector was transformed in Rossetta cells (Novagen). Protein production was induced with isopropyl 1-thio-β-d-galactopyranoside (1 mm) at 37 °C at an optical density between 0.7–0.9 for 5 h. Cell pellets were sonicated in sonication buffer (300 mm NaCl, 50 mm Na2HPO4/H2NaPO4, 5 mm imidazole, 5 mm β-mercaptoethanol). After centrifugation, extracts were loaded on a nickel-nitrilotriacetic acid (Qiagen) column. N-Idas protein was eluted from the column at 500 mm imidazole, and dialyzed against 25 mm Tris, 50 mm NaCl buffer. Different Idas constructs were designed in ProteinCCD (28Mooij W.T. Mitsiki E. Perrakis A. Nucleic Acids Res. 2009; 37: W402-W405Crossref PubMed Scopus (38) Google Scholar, 34Luna-Vargas M.P. Christodoulou E. Alfieri A. van Dijk W.J. Stadnik M. Hibbert R.G. Sahtoe D.D. Clerici M. Marco V.D. Littler D. Celie P.H. Sixma T.K. Perrakis A. J. Struct Biol. 2011; 10.1016/j.jsb.2011.03.017PubMed Google Scholar), amplified by PCR, and inserted to the pETNKI-His-3C-LIC-kan vector. Vectors were transformed to Rosetta-2 cells (Novagen) and grown in the presence of a suitable antibiotic. For co-expression experiments, Idas plasmids were simultaneously transformed together with Geminin plasmids (23De Marco V. Gillespie P.J. Li A. Karantzelis N. Christodoulou E. Klompmaker R. van Gerwen S. Fish A. Petoukhov M.V. Iliou M.S. Lygerou Z. Medema R.H. Blow J.J. Svergun D.I. Taraviras S. Perrakis A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19807-19812Crossref PubMed Scopus (62) Google Scholar) in the presence of suitable antibiotics. Protein production was induced with isopropyl 1-thio-β-d-galactopyranoside at 15 °C at an optical density of ∼0.8, and cells were left to grow for an additional 16–18 h at 15 °C. Cell pellets were obtained by centrifugation at 4000 rpm and lysed by sonication in 20 mm Hepes, pH 7.5, 200 mm NaCl, 5 mm β-mercaptoethanol (buffer A) plus a tablet of Complete, EDTA-free Protease Inhibitor Cocktail (Roche Applied Science) at 4 °C. After clearing the supernatant by centrifugation, the proteins were bound in a His-trap column (GE Healthcare) and eluted with a linear gradient of buffer A containing 20–500 mm imidazole. Fractions were analyzed by SDS-PAGE, and pure fractions were pooled. Yields were between 5–10 mg/liter of culture depending on the construct. All proteins were more than 95% pure as judged by SDS-PAGE (Fig. 3). All Idas-Geminin co-expression experiments were done with immobilized metal ion affinity chromatography purification mediated by the N-terminal histidine tag on Idas; in all purified complexes, stoichiometric amounts of Geminin were co-purified with Idas. All His tags were cleaved with 3C-protease before the characterization experiments. We were unable to obtain full-length Idas or its complexes in a soluble form suitable for biophysical experiments. dIdas and N-Idas recombinant proteins were used for rabbit immunizations (Harlan Sera-Lab) to generate antibodies hIdasAb1 and hIdasAb2, respectively. Crude sera were affinity-purified against the recombinant protein used for immunization. The specificity of the affinity purified antibodies were tested by Western blot analysis and shown to specifically recognize the hIdas protein in HeLa cells. Experiments were performed in a Superdex 75 HR 10/30 column attached to an Akta FPLC and coupled to a miniDAWN light scattering detector (Wyatt Technology) and a Dn-1000 differential refractive index detector (WGE Dr. Bures). 100 μl of purified tIdas at a concentration of 0.3 mg/ml were injected to the column. Data analysis was carried out with the program Astra using a differential index of refraction value of 0.185. Surface plasmon resonance spectroscopy was preformed at 25 °C on a Biacore T100. About 6000 relative units of mini-Cdt1 and mini-Cdt1 Y170A were immobilized on a CM5 Chip (Biacore) at pH 5.5 using amino coupling of lysine residues. Concentrations series of all described Geminin and Idas variants and their complexes were injected across the chip in a buffer of 20 mm Hepes, pH 7.5, 200 mm NaCl, Tween 20 0.01% at a flow rate of 30 μl/min. The concentrations used were: 68, 200, 610, 1830, and 5500 nm for dIdas; 50, 151, 456, 1370, and 4100 nm for tIdas; 6, 19, 57, 170, 511, 1530, and 4600 nm for Geminin-dIdas; 13, 38, 113, 340, 102, 3060, and 9200 nm for Geminin-tIdas; 12, 36, 107, 322, and 967 nm for Geminin. Binding curves were recorded for each protein using the empty flow cell as reference. All experiments were repeated at least two times in a non-sequential manner to exclude systematic errors. The Biacore T100 evaluation software was used for the initial data analysis. All binding curves are shown in supplemental Fig. 2. We searched human databases for Geminin homologues and identified the gene product of LOC345643, on chromosome 5q11.2, as being similar to Geminin in its central coiled-coil region. We named this gene Idas. Idas has 7 exons (supplemental Fig. 1A) and codes for a previously uncharacterized protein of 385 amino acids containing a putative coiled-coil region. Idas orthologues can be detected in different vertebrates. A multiple sequence alignment of Geminin orthologues with Idas orthologues from human, mouse, and Xenopus is shown in Fig. 1A. Geminin and Idas exhibit low overall similarity but significant conservation in their central coiled-coil regions, where the human Idas and Geminin proteins share 53% identity (Fig. 1A, black box). The central region of Geminin, which is conserved in Idas, mediates Geminin homodimerization and binding to Cdt1 (20Lee C. Hong B. Choi J.M. Kim Y. Watanabe S. Ishimi Y. Enomoto T. Tada S. Kim Y. Cho Y. Nature. 2004; 430: 913-917Crossref PubMed Scopus (119) Google Scholar, 21Saxena S. Yuan P. Dhar S.K. Senga T. Takeda D. Robinson H. Kornbluth S. Swaminathan K. Dutta A. Mol. Cell. 2004; 15: 245-258Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 22Thépaut M. Maiorano D. Guichou J.F. Augé M.T. Dumas C. Méchali M. Padilla A. J. Mol. Biol. 2004; 342: 275-287Crossref PubMed Scopus (28) Google Scholar, 23De Marco V. Gillespie P.J. Li A. Karantzelis N. Christodoulou E. Klompmaker R. van Gerwen S. Fish A. Petoukhov M.V. Iliou M.S. Lygerou Z. Medema R.H. Blow J.J. Svergun D.I. Taraviras S. Perrakis A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19807-19812Crossref PubMed Scopus (62) Google Scholar). The Geminin coiled-coil has unique properties, with three polar residues in a and d positions of the heptad repeats, which are also present in Idas (Fig. 1A, arrowheads), suggesting that Idas may both homodimerize and heterodimerize with Geminin. Geminin-Cdt1 interactions were analyzed by PISA (35Krissinel E. Henrick K. J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (6929) Google Scholar) bas" @default.
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• W2028331361 title "Idas, a Novel Phylogenetically Conserved Geminin-related Protein, Binds to Geminin and Is Required for Cell Cycle Progression" @default.
• W2028331361 cites W1510905532 @default.
• W2028331361 cites W1535571384 @default.
• W2028331361 cites W1967048572 @default.
• W2028331361 cites W1979481942 @default.
• W2028331361 cites W1985608714 @default.
• W2028331361 cites W1985874860 @default.
• W2028331361 cites W1988153215 @default.
• W2028331361 cites W1995767984 @default.
• W2028331361 cites W2001108757 @default.
• W2028331361 cites W2007028310 @default.
• W2028331361 cites W2015141765 @default.
• W2028331361 cites W2024645073 @default.
• W2028331361 cites W2029925140 @default.
• W2028331361 cites W2033661691 @default.
• W2028331361 cites W2035503835 @default.
• W2028331361 cites W2036920293 @default.
• W2028331361 cites W2039252915 @default.
• W2028331361 cites W2045665377 @default.
• W2028331361 cites W2048476437 @default.
• W2028331361 cites W2055631421 @default.
• W2028331361 cites W2057896961 @default.
• W2028331361 cites W2058953495 @default.
• W2028331361 cites W2060088582 @default.
• W2028331361 cites W2061145246 @default.
• W2028331361 cites W2071685764 @default.
• W2028331361 cites W2072472133 @default.
• W2028331361 cites W2077460505 @default.
• W2028331361 cites W2086535617 @default.
• W2028331361 cites W2092109854 @default.
• W2028331361 cites W2097088500 @default.
• W2028331361 cites W2112809461 @default.
• W2028331361 cites W2114070867 @default.
• W2028331361 cites W2115438940 @default.
• W2028331361 cites W2121058712 @default.
• W2028331361 cites W2121936339 @default.
• W2028331361 cites W2122073313 @default.
• W2028331361 cites W2128056082 @default.
• W2028331361 cites W2128875977 @default.
• W2028331361 cites W2134629510 @default.
• W2028331361 cites W2135138200 @default.
• W2028331361 cites W2137015675 @default.
• W2028331361 cites W2139808477 @default.
• W2028331361 cites W2142732735 @default.
• W2028331361 cites W2143705913 @default.
• W2028331361 cites W2153045920 @default.
• W2028331361 cites W2163375855 @default.
• W2028331361 cites W2163701261 @default.
• W2028331361 cites W2164599679 @default.
• W2028331361 cites W2168323994 @default.
• W2028331361 cites W2168514491 @default.
• W2028331361 cites W346313106 @default.
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