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Mechanism and timing of Mcm2–7 ring closure during DNA replication origin licensing

Nature Structural & Molecular Biology volume 24, pages 309315 (2017) | Download Citation

Abstract

The opening and closing of two ring-shaped Mcm2–7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2–7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2–7 during origin licensing. The two Mcm2–7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2–7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2–7 prevented recruitment of the second Mcm2–7. Our findings identify key mechanisms controlling the Mcm2–7 DNA-entry gate during origin licensing, and reveal that the two Mcm2–7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.

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Acknowledgements

We are grateful to the members of the Bell laboratory for useful discussions, B. Sauer for comments on the manuscript, and L.D. Lavis (Janelia Research Campus, Ashburn, Virginia, USA) for providing Janelia fluorophores. This work was supported by the NIH (grants R01 GM52339 (to S.P.B.) and R01 GM81648 (to J.G.); pre-doctoral training grant GM007287 to S.T.) and the G. Harold and Leila Y. Matthews Foundation (grant to J.G.). S.P.B. is an investigator with the Howard Hughes Medical Institute. This work was supported in part by the Koch Institute (support grant P30-CA14051 from the NCI to T. Jacks, Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, USA). We thank the Koch Institute Swanson Biotechnology Center for technical support, specifically the Biopolymers and Genomics cores.

Author information

Author notes

    • Simina Ticau

    Present address: VL34 Inc., Cambridge, Massachusetts, USA.

Affiliations

  1. Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Simina Ticau
    • , Kanokwan Champasa
    •  & Stephen P Bell
  2. Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.

    • Larry J Friedman
    •  & Jeff Gelles
  3. New England BioLabs, Ipswich, Massachusetts, USA.

    • Ivan R Corrêa Jr

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Contributions

S.T. performed all experiments with feedback from J.G., L.J.F. and S.P.B., except for the ensemble FRET studies, which were performed by K.C. I.R.C. prepared essential reagents. S.T., L.J.F. and J.G. analyzed the data. S.P.B. wrote the paper with input from all other authors. S.P.B. and J.G. directed the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jeff Gelles or Stephen P Bell.

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DOI

https://doi.org/10.1038/nsmb.3375

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