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ATPase-dependent quality control of DNA replication origin licensing

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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Figure 1: Mcm3 is necessary and sufficient for Mcm2–7 recruitment.
Figure 2: The C terminus of Mcm3 is required for Mcm2–7 recruitment.
Figure 3: Both Mcm2–7 hexamers must interact with ORC–Cdc6 through Mcm3.
Figure 4: Mcm3 binding activates the ORC–Cdc6 ATPase.
Figure 5: ATP hydrolysis by ORC–Cdc6 can promote Mcm2–7 release.

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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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Correspondence to John F. X. Diffley.

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The authors declare no competing financial interests.

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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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