Abstract
The mouse int6 gene is a frequent integration site of the mouse mammary tumor virus and INT6 silencing by RNA interference in HeLa cells causes an increased number of cells in the G2/M phases of the cell cycle, along with mitotic defects. In this report, we investigated the functional significance of the interaction between INT6 and MCM7, which was observed in a two-hybrid screen performed with INT6 as bait. It was found that proteasome inhibition strengthens interaction between both proteins and that INT6 stabilizes MCM7. Removal of MCM7 from chromatin as replication proceeds was accelerated in INT6-silenced cells and reduced amounts of protein were transiently observed, followed by a correction resulting from stimulation of mcm7 gene expression. Synchronized cells depleted for either INT6 or MCM7 display a reduction in thymidine incorporation and a reinforced association of RPA and claspin with chromatin. These data show that INT6 stabilizes chromatin-bound MCM7 and that alteration of this effect is associated with replication deficiency.
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References
Akiyoshi Y, Clayton J, Phan L, Yamamoto M, Hinnebusch AG, Watanabe Y et al. (2001). Fission yeast homolog of murine Int-6 protein, encoded by mouse mammary tumor virus integration site, is associated with the conserved core subunits of eukaryotic translation initiation factor 3. J Biol Chem 276: 10056–10062.
Asano K, Merrick WC, Hershey JW . (1997). The translation initiation factor eIF3-p48 subunit is encoded by int-6, a site of frequent integration by the mouse mammary tumor virus genome. J Biol Chem 272: 23477–23480.
Bandyopadhyay A, Matsumoto T, Maitra U . (2000). Fission yeast INT6 is not essential for global translation initiation, but deletion of INT6(+) causes hypersensitivity to caffeine and affects spore formation. Mol Biol Cell 11: 4005–4018.
Blow JJ, Dutta A . (2005). Preventing re-replication of chromosomal DNA. Nat Rev Mol Cell Biol 6: 476–486.
Burks EA, Bezerra PP, Le H, Gallie DR, Browning KS . (2001). Plant initiation factor 3 subunit composition resembles mammalian initiation factor 3 and has a novel subunit. J Biol Chem 276: 2122–2131.
Buttitta F, Martella C, Barassi F, Felicioni L, Salvatore S, Rosini S et al. (2005). INT6 expression can predict survival in early-stage non-small cell lung cancer patients. Clin Cancer Res 11: 3198–3204.
Cook JG, Chasse DA, Nevins JR . (2004). The regulated association of Cdt1 with minichromosome maintenance proteins and Cdc6 in mammalian cells. J Biol Chem 279: 9625–9633.
Crane R, Craig R, Murray R, Dunand-Sauthier I, Humphrey T, Norbury C . (2000). A fission yeast homolog of Int-6, the mammalian oncoprotein and eIF3 subunit, induces drug resistance when overexpressed. Mol Biol Cell 11: 3993–4003.
Desbois C, Rousset R, Bantignies F, Jalinot P . (1996). Exclusion of Int-6 from PML nuclear bodies by binding to the HTLV-I Tax oncoprotein. Science 273: 951–953.
Durfee T, Becherer K, Chen P-L, Yeh S-H, Yang Y, Kilburn AE et al. (1993). The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev 7: 555–569.
Fitch MJ, Donato JJ, Tye BK . (2003). Mcm7, a subunit of the presumptive MCM helicase, modulates its own expression in conjunction with Mcm1. J Biol Chem 278: 25408–25416.
Fujita M, Kiyono T, Hayashi Y, Ishibashi M . (1996). hCDC47, a human member of the MCM family. Dissociation of the nucleus-bound form during S phase. J Biol Chem 271: 4349–4354.
Gillespie PJ, Li A, Blow JJ . (2001). Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins. BMC Biochem 2: 15.
Guo J, Sen GC . (2000). Characterization of the interaction between the interferon-induced protein P56 and the INT6 protein encoded by a locus of insertion of the mouse mammary tumor virus. J Virol 74: 1892–1899.
Harlow E, Lane D . (1988). Immunoprecipitation. In: CSH Laboratory CSH (ed). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, pp 447–470.
Hoareau Alves K, Bochard V, Rety S, Jalinot P . (2002). Association of the mammalian proto-oncoprotein Int-6 with the three protein complexes eIF3, COP9 signalosome and 26S proteasome. FEBS Lett 527: 15–21.
Holthoff HP, Baack M, Richter A, Ritzi M, Knippers R . (1998). Human protein MCM6 on HeLa cell chromatin. J Biol Chem 273: 7320–7325.
Hwang GW, Sasaki D, Naganuma A . (2005). Overexpression of Rad23 confers resistance to methylmercury in Saccharomyces cerevisiae via inhibition of the degradation of ubiquitinated proteins. Mol Pharmacol 68: 1074–1078.
Ishimi Y . (1997). A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex. J Biol Chem 272: 24508–24513.
Kimura H, Takizawa N, Nozaki N, Sugimoto K . (1995). Molecular cloning of cDNA encoding mouse Cdc21 and CDC46 homologs and characterization of the products: physical interaction between P1(MCM3) and CDC46 proteins. Nucleic Acids Res 23: 2097–2104.
Kuhne C, Banks L . (1998). E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G box. J Biol Chem 273: 34302–34309.
Kuo M-L, Kinsella T . (1997). Overexpression of a hexa-histidine and T7 peptide-tagged human ribonucleotide reductase small subunit, R2 in Escherichia coli and the generation of human R2 antibodies. Intl J Oncol 10: 515–520.
Lee JK, Hurwitz J . (2001). Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures. Proc Natl Acad Sci USA 98: 54–59.
Lin SY, Li K, Stewart GS, Elledge SJ . (2004). Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation. Proc Natl Acad Sci USA 101: 6484–6489.
Maiorano D, Cuvier O, Danis E, Mechali M . (2005). MCM8 is an MCM2-7-related protein that functions as a DNA helicase during replication elongation and not initiation. Cell 120: 315–328.
Marchetti A, Buttitta F, Miyazaki S, Gallahan D, Smith GH, Callahan R . (1995). Int-6, a highly conserved, widely expressed gene, is mutated by mouse mammary tumor virus in mammary preneoplasia. J Virol 69: 1932–1938.
Mayeur GL, Hershey JW . (2002). Malignant transformation by the eukaryotic translation initiation factor 3 subunit p48 (eIF3e). FEBS Lett 514: 49–54.
Morris C, Jalinot P . (2005). Silencing of human Int-6 impairs mitosis progression and inhibits cyclin B-Cdk1 activation. Oncogene 24: 1203–1211.
Morris-Desbois C, Bochard V, Reynaud C, Jalinot P . (1999). Interaction between the Ret finger protein and the Int-6 gene product and co-localisation into nuclear bodies. J Cell Sci 112: 3331–3342.
Raasi S, Pickart CM . (2003). Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains. J Biol Chem 278: 8951–8959.
Rasmussen SB, Kordon E, Callahan R, Smith GH . (2001). Evidence for the transforming activity of a truncated INT6 gene, in vitro. Oncogene 20: 5291–5301.
Sar F, Lindsey-Boltz LA, Subramanian D, Croteau DL, Hutsell SQ, Griffith JD et al. (2004). Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures. J Biol Chem 279: 39289–39295.
Wang Y, Xu F, Hall FL . (2000). The MAT1 cyclin-dependent kinase-activating kinase (CAK) assembly/targeting factor interacts physically with the MCM7 DNA licensing factor. FEBS Lett 484: 17–21.
Yahalom A, Kim TH, Winter E, Karniol B, von Arnim AG, Chamovitz DA . (2001). Arabidopsis eIF3e (INT-6) associates with both eIF3c and the COP9 signalosome subunit CSN7. J Biol Chem 276: 334–340.
Yanagi K, Mizuno T, You Z, Hanaoka F . (2002). Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity. J Biol Chem 277: 40871–40880.
Yen HC, Chang EC . (2000). Yin6, a fission yeast INT6 homolog, complexes with Moe1 and plays a role in chromosome segregation. Proc Natl Acad Sci USA 97: 14370–14375.
Yen HC, Gordon C, Chang EC . (2003). Schizosaccharomyces pombe INT6 and Ras homologs regulate cell division and mitotic fidelity via the proteasome. Cell 112: 207–217.
You Z, Ishimi Y, Masai H, Hanaoka F . (2002). Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex. J Biol Chem 277: 42471–42479.
Zhang X, Turnell AS, Gorbea C, Mymryk JS, Gallimore PH, Grand RJ . (2004). The targeting of the proteasomal regulatory subunit S2 by adenovirus E1A causes inhibition of proteasomal activity and increased p53 expression. J Biol Chem 279: 25122–25133.
Zhou C, Arslan F, Wee S, Krishnan S, Ivanov AR, Oliva A et al. (2005). PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes. BMC Biol 3: 14.
Zhou H, Wertz I, O'Rourke K, Ultsch M, Seshagiri S, Eby M et al. (2004). Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO. Nature 427: 167–171.
Acknowledgements
We thank V Dixit, M Fujita, H Kimura and R Knippers for the generous gifts of plasmids and antibodies. We are also grateful to A Roisin for help with cell culture. This work was supported by a grant from the ‘Fondation pour la Recherche Médicale’ and by a fellowship from the ‘Ligue Nationale contre le cancer’ (SB).
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).
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Buchsbaum, S., Morris, C., Bochard, V. et al. Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Oncogene 26, 5132–5144 (2007). https://doi.org/10.1038/sj.onc.1210314
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DOI: https://doi.org/10.1038/sj.onc.1210314
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