Nucleosomes influence multiple steps during replication initiation

  1. Ishara F Azmi
  2. Shinya Watanabe
  3. Michael F Maloney
  4. Sukhyun Kang
  5. Jason A Belsky
  6. David M MacAlpine
  7. Craig L Peterson
  8. Stephen P Bell  Is a corresponding author
  1. Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
  2. University of Massachusetts Medical School, United States
  3. Duke University Medical Center, United States

Abstract

Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin.

Article and author information

Author details

  1. Ishara F Azmi

    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. Shinya Watanabe

    Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Michael F Maloney

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

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

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2945-6282
  6. David M MacAlpine

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Craig L Peterson

    Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. 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)

  • Stephen P Bell

National Institute of General Medical Sciences (GM54096 and GM104097)

  • David M MacAlpine
  • Craig L Peterson

American Cancer Society (123700-PF-13-071-01-DM)

  • Ishara F Azmi

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

Reviewing Editor

  1. Robert Sclafani, University of Colorado School of Medicine, United States

Publication history

  1. Received: October 19, 2016
  2. Accepted: March 20, 2017
  3. Accepted Manuscript published: March 21, 2017 (version 1)
  4. Version of Record published: April 21, 2017 (version 2)

Copyright

© 2017, Azmi 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. Ishara F Azmi
  2. Shinya Watanabe
  3. Michael F Maloney
  4. Sukhyun Kang
  5. Jason A Belsky
  6. David M MacAlpine
  7. Craig L Peterson
  8. Stephen P Bell
(2017)
Nucleosomes influence multiple steps during replication initiation
eLife 6:e22512.
https://doi.org/10.7554/eLife.22512
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