Redox controls RecA protein activity via reversible oxidation of its methionine residues
- University of Wisconsin-Madison, United States
- CNRS, France
- Université Catholique de Louvain, Belgium
- Universidad de Sevilla, Spain
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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Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.
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Chlamydia trachomatis is an obligate intracellular bacterial pathogen with a unique developmental cycle. It differentiates between two functional and morphological forms: the elementary body (EB) and the reticulate body (RB). The signals that trigger differentiation from one form to the other are unknown. EBs and RBs have distinctive characteristics that distinguish them, including their size, infectivity, proteome, and transcriptome. Intriguingly, they also differ in their overall redox status as EBs are oxidized and RBs are reduced. We hypothesize that alterations in redox may serve as a trigger for secondary differentiation. To test this, we examined the function of the primary antioxidant enzyme alkyl hydroperoxide reductase subunit C (AhpC), a well-known member of the peroxiredoxins family, in chlamydial growth and development. Based on our hypothesis, we predicted that altering the expression of ahpC would modulate chlamydial redox status and trigger earlier or delayed secondary differentiation. Therefore, we created ahpC overexpression and knockdown strains. During ahpC knockdown, ROS levels were elevated, and the bacteria were sensitive to a broad set of peroxide stresses. Interestingly, we observed increased expression of EB-associated genes and concurrent higher production of EBs at an earlier time in the developmental cycle, indicating earlier secondary differentiation occurs under elevated oxidation conditions. In contrast, overexpression of AhpC created a resistant phenotype against oxidizing agents and delayed secondary differentiation. Together, these results indicate that redox potential is a critical factor in developmental cycle progression. For the first time, our study provides a mechanism of chlamydial secondary differentiation dependent on redox status.
