Rad52-Rad51 association is essential to protect Rad51 filaments against Srs2, but facultative for filament formation

  1. Emilie Ma
  2. Pauline Dupaigne
  3. Laurent Maloisel
  4. Raphaël Guerois
  5. Eric Le Cam
  6. Eric Coïc  Is a corresponding author
  1. CEA-Université Paris Saclay, France
  2. Institut Gustave Roussy, France

Abstract

Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51. However, we report here that Rad52 mutations that disrupt this interaction do not affect γ-ray- or HO endonuclease-induced gene conversion frequencies. In vivo and in vitro studies confirmed that Rad51 filaments formation is not affected by these mutations. Instead, we found that Rad52-Rad51 association makes Rad51 filaments toxic in Srs2-deficient cells after exposure to DNA damaging agents, independently of Rad52 role in Rad51 filament assembly. Importantly, we also demonstrated that Rad52 is essential for protecting Rad51 filaments against dissociation by the Srs2 DNA translocase. Our findings open new perspectives in the understanding of the role of Rad52 in eukaryotes.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Emilie Ma

    Institut de Biologie François Jacob, IRCM, CEA-Université Paris Saclay, Fontenay-aux-Roses, France
    Competing interests
    The authors declare that no competing interests exist.
  2. Pauline Dupaigne

    Signalisation, Noyaux et Innovation en Cancérologie, Institut Gustave Roussy, Villejuif, France
    Competing interests
    The authors declare that no competing interests exist.
  3. Laurent Maloisel

    Institut de Biologie François Jacob, IRCM, CEA-Université Paris Saclay, Fontenay-aux-Roses, France
    Competing interests
    The authors declare that no competing interests exist.
  4. Raphaël Guerois

    CEA-Université Paris Saclay, Gif-sur-Yvette, France
    Competing interests
    The authors declare that no competing interests exist.
  5. Eric Le Cam

    Signalisation, Noyaux et Innovation en Cancérologie, Institut Gustave Roussy, Villejuif, France
    Competing interests
    The authors declare that no competing interests exist.
  6. Eric Coïc

    Institut de Biologie François Jacob, IRCM, CEA-Université Paris Saclay, Fontenay-aux-Roses, France
    For correspondence
    eric.coic@cea.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9549-8969

Funding

Commissariat à l'Énergie Atomique et aux Énergies Alternatives (Recurrent funding)

  • Raphaël Guerois
  • Eric Coïc

Centre National de la Recherche Scientifique (Recurrent funding)

  • Eric Le Cam

Fondation ARC pour la Recherche sur le Cancer (SFI20121205689)

  • Eric Coïc

Ligue Contre le Cancer (2015-16)

  • Eric Coïc

Agence Nationale de la Recherche (ANR-15-CE11-0008-01)

  • Raphaël Guerois

Region Ile-de-France (DIM Nano-K No F13012333)

  • Eric Le Cam

Fondation ARC pour la Recherche sur le Cancer (PJA 20141201772)

  • Eric Coïc

Ligue Contre le Cancer (2016-2017)

  • Eric Le Cam

Agence Nationale de la Recherche (ANR-13-BSV8-0022)

  • Eric Le Cam

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

Reviewing Editor

  1. Andrés Aguilera, CABIMER, Universidad de Sevilla, Spain

Version history

  1. Received: October 12, 2017
  2. Accepted: June 30, 2018
  3. Accepted Manuscript published: July 9, 2018 (version 1)
  4. Version of Record published: July 23, 2018 (version 2)

Copyright

© 2018, Ma 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

  • 2,885
    views
  • 392
    downloads
  • 19
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Emilie Ma
  2. Pauline Dupaigne
  3. Laurent Maloisel
  4. Raphaël Guerois
  5. Eric Le Cam
  6. Eric Coïc
(2018)
Rad52-Rad51 association is essential to protect Rad51 filaments against Srs2, but facultative for filament formation
eLife 7:e32744.
https://doi.org/10.7554/eLife.32744

Share this article

https://doi.org/10.7554/eLife.32744

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Natalia Dolgova, Eva-Maria E Uhlemann ... Oleg Y Dmitriev
    Research Article

    Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Isabelle Petit-Hartlein, Annelise Vermot ... Franck Fieschi
    Research Article

    NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.