Ctf4 organizes sister replisomes and Pol α into a replication factory

  1. Zuanning Yuan
  2. Roxana Georgescu
  3. Ruda de Luna Almeida Santos
  4. Daniel Zhang
  5. Lin Bai
  6. Nina Y Yao
  7. Gongpu Zhao
  8. Michael E O'Donnell  Is a corresponding author
  9. Huilin Li  Is a corresponding author
  1. Van Andel Institute, United States
  2. Howard Hughes Medical Institute, The Rockefeller University, United States
  3. The Rockefeller University, United States

Abstract

The current view is that eukaryotic replisomes are independent. Here we show that Ctf4 tightly dimerizes CMG helicase, with an extensive interface involving Psf2, Cdc45, and Sld5. Interestingly, Ctf4 binds only one Pol α-primase. Thus, Ctf4 may have evolved as a trimer to organize two helicases and one Pol α-primase into a replication factory. In the 2CMG-Ctf43-1Pol α-primase factory model, the two CMGs nearly face each other, placing the two lagging strands toward the center and two leading strands out the sides. The single Pol α-primase is centrally located and may prime both sister replisomes. The Ctf4-coupled-sister replisome model is consistent with cellular microscopy studies revealing two sister forks of an origin remain attached and are pushed forward from a protein platform. The replication factory model may facilitate parental nucleosome transfer during replication.

Data availability

The 3D cryo-EM maps of Ctf43-CMG1, Ctf43-CMG2, and Ctf43-CMG3 at 3.8-Å, 5.8-Å and 7.0-Å resolution have been deposited in the Electron Microscopy Data Bank under accession codes EMD-20471, EMD-20472 and EMD-20473, respectively. The corresponding atomic models have been deposited in the Protein Data Bank under accession codes PDB 6PTJ, PDB 6PTN, PDB 6PTO, respectively.

The following data sets were generated

Article and author information

Author details

  1. Zuanning Yuan

    Structural Biology Program, Van Andel Institute, Grand Rapids, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Roxana Georgescu

    Howard Hughes Medical Institute, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1882-2358
  3. Ruda de Luna Almeida Santos

    Structural Biology Program, Van Andel Institute, Grand Rapids, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Daniel Zhang

    DNA Replication Laboratory, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Lin Bai

    Structural Biology Program, Van Andel Institute, Grand Rapids, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Nina Y Yao

    DNA Replication Laboratory, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Gongpu Zhao

    David Van Andel Advanced Cryo-EM Suite, Van Andel Institute, Grand Rapids, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Michael E O'Donnell

    Howard Hughes Medical Institute, The Rockefeller University, New York, United States
    For correspondence
    odonnel@rockefeller.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9002-4214
  9. Huilin Li

    Structural Biology Program, Van Andel Institute, Grand Rapids, United States
    For correspondence
    Huilin.Li@vai.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8085-8928

Funding

National Institutes of Health (GM115809)

  • Michael E O'Donnell

National Institutes of Health (GM131754)

  • Huilin Li

Howard Hughes Medical Institute

  • Michael E O'Donnell

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

Reviewing Editor

  1. James M Berger, Johns Hopkins University School of Medicine, United States

Publication history

  1. Received: April 4, 2019
  2. Accepted: October 4, 2019
  3. Accepted Manuscript published: October 7, 2019 (version 1)
  4. Version of Record published: October 18, 2019 (version 2)

Copyright

© 2019, Yuan 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

  • 2,347
    Page views
  • 459
    Downloads
  • 27
    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)

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

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

  1. Zuanning Yuan
  2. Roxana Georgescu
  3. Ruda de Luna Almeida Santos
  4. Daniel Zhang
  5. Lin Bai
  6. Nina Y Yao
  7. Gongpu Zhao
  8. Michael E O'Donnell
  9. Huilin Li
(2019)
Ctf4 organizes sister replisomes and Pol α into a replication factory
eLife 8:e47405.
https://doi.org/10.7554/eLife.47405

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Morgane Boone et al.
    Research Advance Updated

    In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Florian Bleffert et al.
    Research Article Updated

    Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF-mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.