A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding

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

Abstract

Licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential Mcm4 motif that permit loading of two Mcm2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant Mcm2-7 forms initial hexamer-hexamer interactions, however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant Mcm2-7 reveal a limited time window for double-hexamer formation following second Mcm2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, Mcm10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation.

Data availability

Source data for the plots in Figs. 3, 4, and 7 are found in the file "Champasa_Band_Analysis.xlsx".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.The source data are archived as doi:10.5281/zenodo.2556799.All remaining data generated or analyzed during this study are included in the manuscript.

Article and author information

Author details

  1. Kanokwan Champasa

    Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Caitlin Blank

    Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. 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
  4. 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
  5. 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

National Institute of General Medical Sciences (GM52339)

  • Stephen P Bell

Howard Hughes Medical Institute (Investigator)

  • Stephen P Bell

G Harold and Leila Y Matthews Foundation

  • Jeff Gelles

National Cancer Institute (P30-CA14051)

  • Stephen P Bell

National Institute of General Medical Sciences (GM81648)

  • Jeff Gelles

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

Reviewing Editor

  1. Michael R Botchan, University of California, Berkeley, United States

Publication history

  1. Received: January 25, 2019
  2. Accepted: August 5, 2019
  3. Accepted Manuscript published: August 6, 2019 (version 1)
  4. Version of Record published: August 20, 2019 (version 2)

Copyright

© 2019, Champasa 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. Kanokwan Champasa
  2. Caitlin Blank
  3. Larry J Friedman
  4. Jeff Gelles
  5. Stephen P Bell
(2019)
A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding
eLife 8:e45538.
https://doi.org/10.7554/eLife.45538

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