Redox controls reca protein activity via reversible oxidation of its methionine residues

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.Agence Nationale de la Re-cherche ANR-10-LABX-62-IBEIDFondation pour la Recherche Medicale FRM - FDT20150532554National Institute of General Medical Sciences GM3233

Frequent template switching in postreplication gaps: suppression of deleterious consequences by the Escherichia coli Uup and RadD proteins

The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. When replication forks encounter template DNA lesions, the lesion is simply skipped in some cases. The resulting lesion-containing gap must be converted to duplex DNA to permit repair. Some gap filling occurs via template switching, a process that generates recombination-like branched DNA intermediates. The Escherichia coli Uup and RadD proteins function in different pathways to process the branched intermediates. Uup is a UvrA-like ABC family ATPase. RadD is a RecQ-like SF2 family ATPase. Loss of both functions uncovers frequent and RecA-independent deletion events in a plasmid-based assay. Elevated levels of crossing over and repeat expansions accompany these deletion events, indicating that many, if not most, of these events are associated with template switching in postreplication gaps as opposed to simple replication slippage. The deletion data underpin simulations indicating that multiple postreplication gaps may be generated per replication cycle. Both Uup and RadD bind to branched DNAs in vitro. RadD protein suppresses crossovers and Uup prevents nucleoid mis-segregation. Loss of Uup and RadD function increases sensitivity to ciprofloxacin. We present Uup and RadD as genomic guardians. These proteins govern two pathways for resolution of branched DNA intermediates such that potentially deleterious genome rearrangements arising from frequent template switching are averted

Structure and cellular dynamics of Deinococcus radiodurans single-stranded DNA (ssDNA)-binding protein (SSB)-DNA complexes.

International audienceThe single-stranded DNA (ssDNA)-binding protein from the radiation-resistant bacterium Deinococcus radiodurans (DrSSB) functions as a homodimer in which each monomer contains two oligonucleotide-binding (OB) domains. This arrangement is exceedingly rare among bacterial SSBs, which typically form homotetramers of single-OB domain subunits. To better understand how this unusual structure influences the DNA binding and biological functions of DrSSB in D. radiodurans radiation resistance, we have examined the structure of DrSSB in complex with ssDNA and the DNA damage-dependent cellular dynamics of DrSSB. The x-ray crystal structure of the DrSSB-ssDNA complex shows that ssDNA binds to surfaces of DrSSB that are analogous to those mapped in homotetrameric SSBs, although there are distinct contacts in DrSSB that mediate species-specific ssDNA binding. Observations by electron microscopy reveal two salt-dependent ssDNA-binding modes for DrSSB that strongly resemble those of the homotetrameric Escherichia coli SSB, further supporting a shared overall DNA binding mechanism between the two classes of bacterial SSBs. In vivo, DrSSB levels are heavily induced following exposure to ionizing radiation. This accumulation is accompanied by dramatic time-dependent DrSSB cellular dynamics in which a single nucleoid-centric focus of DrSSB is observed within 1 h of irradiation but is dispersed by 3 h after irradiation. These kinetics parallel those of D. radiodurans postirradiation genome reconstitution, suggesting that DrSSB dynamics could play important organizational roles in DNA repair

Electron microscopy of RecA variant protein filaments on cssDNA, with and without treatment by RecX protein.

<p>Electron micrographs show filament formation of wild type RecA and RecA variant proteins on M13mp18 ssDNA (A) without RecX protein and (B) with 100 nM RecX protein. RecA filaments were placed into 5 different categories based upon the size and completeness of filaments. (C) The composition of filaments in the various categories without RecX protein and (D) with RecX protein.</p

Cell growth competition assays.

<p>Assays were carried out as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005278#sec016" target="_blank">Materials and Methods</a>. (A) Two trial competitions. The top trial shows a competition between two wild type cultures, one of which carries the Ara^– mutation. The lower one shows a competition between wild type cells and cells expressing RecA E38K. As is the case for the RecA variants studied here, RecA E38K also confers a growth disadvantage on cells in which it is expressed. Colony counts revealing the % of cells expressing the mutant RecA proteins with enhanced conjugational function are plotted as a function of the daily growth cycle of the experiment. (B) Competitions between cells expressing each of the three variant proteins and wild type cells. Two competitions are shown for each, with the Ara^– mutation either in the mutant or wild type cells. (C) Competitions between wild type cells (red) and cells with a gene expressing NgRecX protein from the normal <i>recX</i> locus on the <i>E</i>. <i>coli</i> chromosome. The cells expressed either the wild type RecA or RecA V79L from the <i>recA</i> locus as indicated. In two cases, a plasmid also expressing the NgRecX protein at higher levels (pEAW947) was included (NgRecX++). (D) Examples of competition plates from an earlier trial, showing the mixtures of red and white colonies before and after the three days of growth cycles.</p

SOS response of RecA variant proteins.

<p>RecA variant strains containing a plasmid expressing GFP under SOS control (utilizing the <i>recN</i> promoter) were grown in LB. Specific fluorescence, defined as measured fluorescence divided by the OD₆₀₀, is shown. (A) The SOS response in cells expressing RecA variant proteins without treatment with any DNA damaging agent. (B) The SOS response in cells expressing RecA variant proteins after induction by adding 0.005 μg/ml ciprofloxacin at 180 minutes. Due to the error inherent in dividing very small numbers, specific fluorescence is not displayed for times prior to 200 and 230 min in panels A and B, respectively.</p

Effect of EcRecX on RecA and RecA variant filaments.

<p>Quantitation of electron microscopy results. The fraction of observed molecules in each category described in the text and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005278#pgen.1005278.g004" target="_blank">Fig 4</a> are summarized.</p><p>Effect of EcRecX on RecA and RecA variant filaments.</p

Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination

<div><p>The recombination activity of <i>Escherichia coli</i> (<i>E</i>. <i>coli</i>) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A <i>recA</i> gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 <i>recA</i> gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism <i>in vivo</i>. The growth deficiency is alleviated by expression of the functionally robust RecX protein from <i>Neisseria gonorrhoeae</i>. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism.</p></div

Measurements of RecA filaments of different size categories.

<p>Measurements of RecA filament lengths for each category were carried out as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005278#sec016" target="_blank">Materials and Methods</a>. Lengths are reported in μm.</p><p>Measurements of RecA filaments of different size categories.</p

Active displacement of RecA filaments by UvrD translocase activity

The UvrD helicase has been implicated in the disas-sembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an ac-tive mechanism to remove RecA from the DNA. Ef-ficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibil-ity of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with en-hanced DNA-binding properties. The ATPase rate of UvrD during RecA removal is much slower than the ATPase activity of UvrD when it is functioning either as a translocase or a helicase on DNA in the absence of RecA. Thus, in this context UvrD may operate in a specialized disassembly mode