1. Biochemistry and Chemical Biology

Molecular determinants of phase separation for Drosophila DNA replication licensing factors

  1. Matthew W Parker  Is a corresponding author
  2. Jonchee A Kao
  3. Alvin Huang
  4. James M Berger
  5. Michael R Botchan
  1. The University of Texas Southwestern Medical Center, United States
  2. University of California, Berkeley, United States
  3. Johns Hopkins University School of Medicine, United States
https://doi.org/10.7554/eLife.70535
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  1. Matthew W Parker
  2. Jonchee A Kao
  3. Alvin Huang
  4. James M Berger
  5. Michael R Botchan
(2021)
Molecular determinants of phase separation for Drosophila DNA replication licensing factors
eLife 10:e70535.
https://doi.org/10.7554/eLife.70535
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Abstract

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.

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All data generated during this study is included in the manuscript and supporting files.

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Author details

  1. Matthew W Parker

    Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    matthew.parker@utsouthwestern.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7571-0010

  2. Jonchee A Kao

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1701-3265

  3. Alvin Huang

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  4. James M Berger

    Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    James M Berger, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0666-1240

  5. Michael R Botchan

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Michael R Botchan, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0459-5518

Funding

National Institute of General Medical Sciences (R01GM141045-01)

  • James M Berger
  • Michael R Botchan

National Cancer Institute (R01CA030490)

  • James M Berger
  • Michael R Botchan

National Institute of General Medical Sciences (F32GM116393)

  • Matthew W Parker

UC Berkeley Jessie Rabinowitz Award

  • Jonchee A Kao

Cancer Prevention and Research Institute of Texas (RR200070)

  • Matthew W Parker

Welch Foundation (I-2074-20210327)

  • Matthew W Parker

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

Copyright

© 2021, Parker 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. Matthew W Parker
  2. Jonchee A Kao
  3. Alvin Huang
  4. James M Berger
  5. Michael R Botchan
(2021)
Molecular determinants of phase separation for Drosophila DNA replication licensing factors
eLife 10:e70535.
https://doi.org/10.7554/eLife.70535

Share this article

https://doi.org/10.7554/eLife.70535

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Further reading

    1. Biochemistry and Chemical Biology

    A new class of disordered elements controls DNA replication through initiator self-assembly

    Matthew W Parker, Maren Bell ... James M Berger
    Research Article

    The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.