Abstract
The regulated loading of the Mcm2–7 DNA helicase (comprising six related subunits, Mcm2 to Mcm7) into pre-replicative complexes at multiple replication origins ensures precise once per cell cycle replication in eukaryotic cells. The origin recognition complex (ORC), Cdc6 and Cdt1 load Mcm2–7 into a double hexamer bound around duplex DNA in an ATP-dependent reaction, but the molecular mechanism of this origin ‘licensing’ is still poorly understood. Here we show that both Mcm2–7 hexamers in Saccharomyces cerevisiae are recruited to origins by an essential, conserved carboxy-terminal domain of Mcm3 that interacts with and stimulates the ATPase activity of ORC–Cdc6. ATP hydrolysis can promote Mcm2–7 loading, but can also promote Mcm2–7 release if components are missing or if ORC has been inactivated by cyclin-dependent kinase phosphorylation. Our work provides new insights into how origins are licensed and reveals a novel ATPase-dependent mechanism contributing to precise once per cell cycle replication.
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References
Boos, D., Frigola, J. & Diffley, J. F. X. Activation of the replicative DNA helicase: breaking up is hard to do. Curr. Opin. Cell Biol. 24, 423–430 (2012)
Tanaka, S. & Araki, H. Regulation of the initiation step of DNA replication by cyclin-dependent kinases. Chromosoma 119, 565–574 (2010)
Méchali, M. Eukaryotic DNA replication origins: many choices for appropriate answers. Nature Rev. Mol. Cell Biol. 11, 728–738 (2010)
Masai, H., Matsumoto, S., You, Z., Yoshizawa-Sugata, N. & Oda, M. Eukaryotic chromosome DNA replication: where, when, and how? Annu. Rev. Biochem. 79, 89–130 (2010)
Bell, S. P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002)
Blow, J. J. & Dutta, A. Preventing re-replication of chromosomal DNA. Nature Rev. Mol. Cell Biol. 6, 476–486 (2005)
Remus, D. et al. Concerted loading of Mcm2–7 double hexamers around DNA during DNA replication origin licensing. Cell 139, 719–730 (2009)
Evrin, C. et al. A double-hexameric MCM2–7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. Proc. Natl Acad. Sci. USA 106, 20240–20245 (2009)
Gambus, A., Khoudoli, G. A., Jones, R. C. & Blow, J. J. MCM2–7 form double hexamers at licensed origins in Xenopus egg extract. J. Biol. Chem. 286, 11855–11864 (2011)
Speck, C., Chen, Z., Li, H. & Stillman, B. ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. Nature Struct. Mol. Biol. 12, 965–971 (2005)
Tye, B. K. & Sawyer, S. The hexameric eukaryotic MCM helicase: building symmetry from nonidentical parts. J. Biol. Chem. 275, 34833–34836 (2000)
Bochman, M. L. & Schwacha, A. The Mcm complex: unwinding the mechanism of a replicative helicase. Microbiol. Mol. Biol. Rev. 73, 652–683 (2009)
Labib, K., Tercero, J. A. & Diffley, J. F. X. Uninterrupted MCM2–7 function required for DNA replication fork progression. Science 288, 1643–1647 (2000)
Lee, D. G. & Bell, S. P. Architecture of the yeast origin recognition complex bound to origins of DNA. Mol. Cell. Biol. 17, 7159–7168 (1997)
Nguyen, V. Q., Co, C. & Li, J. J. Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature 411, 1068–1073 (2001)
Wilmes, G. M. et al. Interaction of the S-phase cyclin Clb5 with an ‘RXL’ docking sequence in the initiator protein Orc6 provides an origin-localized replication control switch. Genes Dev. 18, 981–991 (2004)
Chen, S. & Bell, S. P. CDK prevents Mcm2–7 helicase loading by inhibiting Cdt1 interaction with Orc6. Genes Dev. 25, 363–372 (2011)
Chen, S., de Vries, M. A. & Bell, S. P. Orc6 is required for dynamic recruitment of Cdt1 during repeated Mcm2–7 loading. Genes Dev. 21, 2897–2907 (2007)
Takara, T. J. & Bell, S. P. Multiple Cdt1 molecules act at each origin to load replication-competent Mcm2–7 helicases. EMBO J. 30, 4885–4896 (2011)
Duderstadt, K. E. & Berger, J. M. AAA+ ATPases in the initiation of DNA replication. Crit. Rev. Biochem. Mol. Biol. 43, 163–187 (2008)
Fujita, M. Cdt1 revisited: complex and tight regulation during the cell cycle and consequences of deregulation in mammalian cells. Cell Div. 1, 22 (2006)
Havens, C. G. & Walter, J. C. Mechanism of CRL4Cdt2, a PCNA-dependent E3 ubiquitin ligase. Genes Dev. 25, 1568–1582 (2011)
Diffley, J. F. X. Quality control in the initiation of eukaryotic DNA replication. Phil. Trans. R. Soc. B 366, 3545–3553 (2011)
Diffley, J. F. X. The many faces of redundancy in DNA replication control. Cold Spring Harb. Symp. Quant. Biol. 75, 135–142 (2010)
Pasion, S. G. & Forsburg, S. L. Nuclear localization of Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM complex assembly. Mol. Biol. Cell 10, 4043–4057 (1999)
Remus, D., Beall, E. L. & Botchan, M. R. DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding. EMBO J. 23, 897–907 (2004)
Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989)
Gelbart, M. E., Rechsteiner, T., Richmond, T. J. & Tsukiyama, T. Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates. Mol. Cell. Biol. 21, 2098–2106 (2001)
Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001)
Mochida, S., Ikeo, S., Gannon, J. & Hunt, T. Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts. EMBO J. 28, 2777–2785 (2009)
Davey, M. J., Indiani, C. & O’Donnell, M. Reconstitution of the Mcm2-7p heterohexamer, subunit arrangement, and ATP site architecture. J. Biol. Chem. 278, 4491–4499 (2003)
Tsakraklides, V. & Bell, S. P. Dynamics of pre-replicative complex assembly. J. Biol. Chem. 285, 9437–9443 (2010)
Tanaka, S. & Diffley, J. F. X. Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2–7 during G1 phase. Nature Cell Biol. 4, 198–207 (2002)
Gambus, A. et al. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nature Cell Biol. 8, 358–366 (2006)
Acknowledgements
We are grateful to N. Cook for help with protein purifications, A. Early and L. Drury for help with strain constructions, G. Coster for help with Mcm3 complementation, S. Mochida and B. Pfander for vectors and K. Labib for antibodies. We also thank members of the Diffley laboratory for critical reading of the manuscript. This work was funded by Cancer Research UK and grants from the Association for International Cancer Research (10-0270) and the European Research Council (249883 – EUKDNAREP).
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J.F., D.R., A.M. and J.F.X.D. conceived the experiments and wrote the paper. J.F., D.R. and A.M. performed all experiments.
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Frigola, J., Remus, D., Mehanna, A. et al. ATPase-dependent quality control of DNA replication origin licensing. Nature 495, 339–343 (2013). https://doi.org/10.1038/nature11920
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DOI: https://doi.org/10.1038/nature11920
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