A helicase-tethered ORC flip enables bidirectional helicase loading

  1. Shalini Gupta
  2. Larry J Friedman
  3. Jeff Gelles  Is a corresponding author
  4. Stephen P Bell  Is a corresponding author
  1. Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
  2. Brandeis University, United States

Abstract

Replication origins are licensed by loading two Mcm2‑7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2‑7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2‑7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first and second helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2‑7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

Data availability

Source data for the single-molecule experiments is provided as "intervals" files that can be read and manipulated by the Matlab program imscroll, which is publicly available: https://github.com/gelles-brandeis/CoSMoS_Analysis. These data can also be read directly in Matlab.The source data are archived at: https://doi.org/10.5061/dryad.547d7wm8z

The following data sets were generated

Article and author information

Author details

  1. Shalini Gupta

    Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5446-7912
  2. Larry J Friedman

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4946-8731
  3. Jeff Gelles

    Department of Biochemistry, Brandeis University, Waltham, United States
    For correspondence
    gelles@brandeis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7910-3421
  4. Stephen P Bell

    Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
    For correspondence
    spbell@mit.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2876-610X

Funding

Howard Hughes Medical Institute (Investigator Award)

  • Stephen P Bell

National Institute of General Medical Sciences (GM52339)

  • Stephen P Bell

National Institute of General Medical Sciences (GM81648)

  • Jeff Gelles

National Institute of General Medical Sciences (GM007287)

  • Shalini Gupta

National Cancer Institute (P30-CA14051)

  • Stephen P Bell

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Bruce Stillman, Cold Spring Harbor Laboratory, United States

Publication history

  1. Received: September 28, 2021
  2. Preprint posted: October 6, 2021 (view preprint)
  3. Accepted: December 8, 2021
  4. Accepted Manuscript published: December 9, 2021 (version 1)
  5. Accepted Manuscript updated: December 10, 2021 (version 2)
  6. Version of Record published: February 9, 2022 (version 3)

Copyright

© 2021, Gupta et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Shalini Gupta
  2. Larry J Friedman
  3. Jeff Gelles
  4. Stephen P Bell
(2021)
A helicase-tethered ORC flip enables bidirectional helicase loading
eLife 10:e74282.
https://doi.org/10.7554/eLife.74282

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