Redox controls RecA protein activity via reversible oxidation of its methionine residues
- University of Wisconsin-Madison, United States
- Aix-Marseille University, CNRS, France
- Université Catholique de Louvain, Belgium
- Universidad de Sevilla, Spain
- Institut Pasteur, France
Abstract
Reactive oxygen species (ROS) cause damage to DNA and proteins. Here we report that the RecA recombinase is itself oxidized by ROS. Genetic and biochemical analyses revealed that oxidation of RecA altered its DNA repair and DNA recombination activities. Mass spectrometry analysis showed that exposure to ROS converted 4 out of 9 Met residues of RecA to methionine sulfoxide. Mimicking oxidation of Met35 by changing it for Gln caused complete loss of function whereas mimicking oxidation of Met164 resulted in constitutive SOS activation and loss of recombination activity. Yet, all ROS-induced alterations of RecA activity were suppressed by methionine sulfoxide reductases MsrA and MsrB. These findings indicate that under oxidative stress, MsrA/B is needed for RecA homeostasis control. The implication is that, besides damaging DNA structure directly, ROS prevent repair of DNA damage by hampering RecA activity.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided in Dryad (doi:10.5061/dryad.zpc866t78).
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Redox controls RecA protein activity via reversible oxidation of its methionine residuesDryad Digital Repository, doi:10.5061/dryad.zpc866t78.
Article and author information
Author details
Angela Mérida-Floriano
Funding
Agence Nationale de la Recherche (ANR-METOXIC)
- Benjamin Ezraty
Centre National de la Recherche Scientifique (PICS-PROTOX)
- Benjamin Ezraty
Agence Nationale de la Recherche (ANR-10-LABX-62-IBEID)
- Frédéric Barras
Fondation pour la Recherche Médicale
- Camille Henry
Aix-Marseille Université (AMidex)
- Camille Henry
National Institute of General Medical Sciences (GM32335)
- Michael M Cox
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Henry 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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Redox controls RecA protein activity via reversible oxidation of its methionine residues
eLife 10:e63747.
https://doi.org/10.7554/eLife.63747
Further reading
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- Biochemistry and Chemical Biology
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
