DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

  1. Lorraine De Jesus Kim
  2. Larry J Friedman
  3. Marko Looke
  4. Christian K Ramsoomair
  5. Jeff Gelles  Is a corresponding author
  6. Stephen P Bell  Is a corresponding author
  1. Massachusetts Institute of Technology, United States
  2. Brandeis University, United States
  3. Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States

Abstract

The committed step of eukaryotic DNA replication occurs when the pairs of Mcm2-7 replicative helicases that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45, GINS, and likely additional proteins are recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). DDK phosphorylation of multiple phosphorylation sites on the Mcm2‑7 tails modulates the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity increases the frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for control of replication origin efficiency and timing.

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.The source data are archived as doi: 10.5281/zenodo.4309997The sequencing data from the in vivo replication assay is archived as DOI: 10.5281/zenodo.4507881

The following data sets were generated

Article and author information

Author details

  1. Lorraine De Jesus Kim

    Biology, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  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. Marko Looke

    Biology, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Christian K Ramsoomair

    Biology, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. 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
  6. 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 Institutes of Health (R01 GM52339)

  • Stephen P Bell

National Institutes of Health (R01 GM81648)

  • Jeff Gelles

National Institutes of Health (T32 GM007287)

  • Lorraine De Jesus Kim

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: December 4, 2020
  2. Accepted: February 21, 2021
  3. Accepted Manuscript published: February 22, 2021 (version 1)
  4. Version of Record published: March 12, 2021 (version 2)

Copyright

© 2021, De Jesus Kim 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. Lorraine De Jesus Kim
  2. Larry J Friedman
  3. Marko Looke
  4. Christian K Ramsoomair
  5. Jeff Gelles
  6. Stephen P Bell
(2021)
DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7
eLife 10:e65471.
https://doi.org/10.7554/eLife.65471

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