1. Biochemistry and Chemical Biology
Download icon

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
Research Advance
  • Cited 0
  • Views 336
  • Annotations
Cite this article as: eLife 2021;10:e70535 doi: 10.7554/eLife.70535

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.

Data availability

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

The following data sets were generated

Article and author information

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.

Reviewing Editor

  1. Stephen P Bell, Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States

Publication history

  1. Received: June 1, 2021
  2. Accepted: December 23, 2021
  3. Accepted Manuscript published: December 24, 2021 (version 1)

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.

Metrics

  • 336
    Page views
  • 96
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Thomas S McAlear, Susanne Bechstedt
    Research Article

    Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Andrea Loreto et al.
    Research Article Updated

    Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the NAD-consuming enzyme SARM1. Here, we report a novel activator of SARM1, a metabolite of the pesticide and neurotoxin vacor. Removal of SARM1 completely rescues mouse neurons from vacor-induced neuron and axon death in vitro and in vivo. We present the crystal structure of the Drosophila SARM1 regulatory domain complexed with this activator, the vacor metabolite VMN, which as the most potent activator yet known is likely to support drug development for human SARM1 and NMNAT2 disorders. This study indicates the mechanism of neurotoxicity and pesticide action by vacor, raises important questions about other pyridines in wider use today, provides important new tools for drug discovery, and demonstrates that removing SARM1 can robustly block programmed axon death induced by toxicity as well as genetic mutation.