RecA Activity Research Articles

Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

Induction of binding sites for RecA aptamers by differentiated-competition capillary Electrophoresis-SELEX

The binding site-oriented SELEX strategy is an effective way to promote the functional evolution of aptamers. Here we report a novel aptamer screening strategy (Differentiated-competition Capillary Electrophoresis-SELEX), which enables candidate aptamers to be directionally induced to bind to different active sites of RecA. In this strategy, we introduce two competing binding factors into the “binding - dissociation” dynamic equilibrium system of RecA and ssDNA libraries. Due to the completely different binding mechanism of the competitive factor and the ssDNA library, it exerts different interference on the binding of RecA and the ssDNA library, which directed the binding site of aptamer candidates during the SELEX process. Multifunctional aptamers with high affinity and specificity were found to inhibit RecA activity by binding to different active sites. In conclusion, the SELEX method developed in our current study have identified a variety of biologically functional aptamers with relatively well-defined binding sites that regulate RecA protein activity, which has potential applications and future prospects for accurate screening of biological functional aptamers.

A new insight into RecA filament regulation by RecX from the analysis of conformation-specific interactions.

RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on ssDNA in an ATP-dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (Escherichia coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

DNA Damage-Inducible Pyocin Expression Is Independent of RecA in xerC-Deleted Pseudomonas aeruginosa.

ABSTRACTPyocins are interbacterial killing complexes made by Pseudomonas aeruginosa primarily to enact intraspecific competition. DNA damage and the ensuing activation of RecA initiate canonical pyocin expression. We recently discovered that deletion of xerC, which encodes a tyrosine recombinase involved in chromosome decatenation, markedly elevates basal pyocin production independently of RecA. Interestingly, the already-elevated basal pyocin expression in ΔxerC cells is substantially further increased by ciprofloxacin treatment. Here, we asked whether this further increase is due to DNA damage additionally activating the canonical RecA-dependent pyocin expression pathway. We also interrogated the relationship between XerC recombinase activity and pyocin expression. Surprisingly, we find that DNA damage-induced pyocin stimulation in ΔxerC cells is independent of RecA but dependent on PrtN, implying a RecA-independent means of DNA damage sensing that activates pyocin expression via PrtN. In sharp contrast to the RecA independence of pyocin expression in ΔxerC strains, specific mutational inactivation of XerC recombinase activity (XerCY272F) caused modestly elevated basal pyocin expression and was further stimulated by DNA-damaging drugs, but both effects were fully RecA dependent. To test whether pyocins could be induced by chemically inactivating XerC, we deployed a previously characterized bacterial tyrosine recombinase inhibitor. However, the inhibitor did not activate pyocin expression even at growth-inhibitory concentrations, suggesting that its principal inhibitory activity resembles neither XerC absence nor enzymatic inactivation. Collectively, our results imply a second function of XerC, separate from its recombinase activity, whose absence permits RecA-independent but DNA damage-inducible pyocin expression.IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa produces pyocins—intraspecific, interbacterial killing complexes. The canonical pathway for pyocin production involves DNA damage and RecA activation. Pyocins are released by cell lysis, making production costly. We previously showed that cells lacking the tyrosine recombinase XerC produce pyocins independently of RecA. Here, we show that DNA-damaging agents stimulate pyocin expression in ΔxerC strains without involving RecA. However, strains mutated for XerC recombinase activity display strictly RecA-dependent pyocin production, and a known bacterial tyrosine recombinase inhibitor does not elicit pyocin expression. Our results collectively suggest that the use of XerC inhibition as an antipseudomonal strategy will require targeting the second function of XerC in regulating noncanonical pyocin production rather than targeting its recombinase activity.

PcrA Dissociates RecA Filaments and the SsbA and RecO Mediators Counterbalance Such Activity.

PcrA depletion is lethal in wild-type Bacillus subtilis cells. The PcrA DNA helicase contributes to unwinding RNA from the template strand, backtracking the RNA polymerase, rescuing replication-transcription conflicts, and disassembling RecA from single-stranded DNA (ssDNA) by poorly understood mechanisms. We show that, in the presence of RecA, circa one PcrA/plasmid-size circular ssDNA (cssDNA) molecule hydrolyzes ATP at a rate similar to that on the isolated cssDNA. PcrA K37A, which poorly hydrolyses ATP, fails to displace RecA from cssDNA. SsbA inhibits and blocks the ATPase activities of PcrA and RecA, respectively. RecO partially antagonizes and counteracts the negative effect of SsbA on PcrA- and RecA-mediated ATP hydrolysis, respectively. Conversely, multiple PcrA molecules are required to inhibit RecA·ATP-mediated DNA strand exchange (DSE). RecO and SsbA poorly antagonize the PcrA inhibitory effect on RecA·ATP-mediated DSE. We propose that two separable PcrA functions exist: an iterative translocating PcrA monomer strips RecA from cssDNA to prevent unnecessary recombination with the mediators SsbA and RecO balancing such activity; and a PcrA cluster that disrupts DNA transactions, as RecA-mediated DSE.

Bacillus subtilis RecA, DisA, and RadA/Sms Interplay Prevents Replication Stress by Regulating Fork Remodeling.

Reviving Bacillus subtilis spores require the recombinase RecA, the DNA damage checkpoint sensor DisA, and the DNA helicase RadA/Sms to prevent a DNA replication stress. When a replication fork stalls at a template lesion, RecA filaments onto the lesion-containing gap and the fork is remodeled (fork reversal). RecA bound to single-strand DNA (ssDNA) interacts with and recruits DisA and RadA/Sms on the branched DNA intermediates (stalled or reversed forks), but DisA and RadA/Sms limit RecA activities and DisA suppresses its c-di-AMP synthesis. We show that RecA, acting as an accessory protein, activates RadA/Sms to unwind the nascent lagging-strand of the branched intermediates rather than to branch migrate them. DisA limits the ssDNA-dependent ATPase activity of RadA/Sms C13A, and inhibits the helicase activity of RadA/Sms by a protein-protein interaction. Finally, RadA/Sms inhibits DisA-mediated c-di-AMP synthesis and indirectly inhibits cell proliferation, but RecA counters this negative effect. We propose that the interactions among DisA, RecA and RadA/Sms, which are mutually exclusive, contribute to generate the substrate for replication restart, regulate the c-di-AMP pool and limit fork restoration in order to maintain cell survival.

DinF Elicits Nitric Oxide Signaling Induced by Periplanetasin-4 from American Cockroach in Escherichia coli

Modern antibiotics have been developed with the aim of destroying cellular function; however, the risk of antibiotic-resistance is increasing continuously. As a result, antimicrobial peptide (AMP) is considered a novel strategy to substitute traditional drugs. This study focused on revealing the antibacterial mechanism(s) of periplanetasn-4, an AMP identified from Cockroach. To elucidate whether periplanetasin-4 generates reactive oxygen species (ROS), a crucial stress factor for cell death, intracellular ROS was measured in Escherichia coli. The degree of membrane and DNA damage was determined using the properties that ROS causes oxidative stress to cell components. Unlike normal cell death, membrane depolarization was observed but DNA fragmentation did not occur. In addition, accumulation of nitric oxide (NO), a free radical with high toxicity, was measured and the byproduct of NO also induced severe intracellular damage. Periplanetasin-4-induced NO also impacted on cytosol calcium levels and triggered lipid peroxidation and DNA oxidation. These features were weakened when NO synthesis was interrupted, and this data suggested that perplanetasin-4-induced NO participates in E. coli cell damage. Moreover, this AMP-induced NO stimulates expression of SOS repair proteins and activation of RecA, a bacterial caspase-like protein. Features of nitrosative damage did not occur especially without dinF gene which is associated with oxidative stress. Therefore, it was indicated that when there is a NO signal, dinF promotes cell death. In conclusion, the combined investigations demonstrated that the antibacterial mechanism(s) of periplanetasin-4 was a NO-induced cell death, and dinF gene is closely related to cell death pathway.

The regulation mechanism of the C-terminus of RecA proteins during DNA strand-exchange process

The C-terminus of Escherichia coli RecA protein can affect the DNA binding affinity, interact with accessory proteins, and regulate the RecA activity. A substantial upward shift in the pH-reaction profile of RecA-mediated DNA strand-exchange reactions was observed for C-terminal-truncated E. coli ΔC17 RecA, Deinococcus radiodurans RecA, and Deinococcus ficus RecA. Here, the process of RecA-mediated strand exchange from the beginning to the end was investigated with florescence resonance energy transfer and tethered particle motion experiments to determine the detailed regulation mechanism. RecA proteins with a shorter C-terminus possess more stable nuclei, higher DNA binding affinities, and lower protonation requirements for the formation of nucleoprotein filaments. Moreover, more stable synaptic complexes in the homologous sequence searching process were also observed for RecA proteins with a shorter C-terminus. Our results suggest that the C-terminus of RecA proteins regulates not only the formation of RecA nucleoprotein filaments but also the entrance of secondary DNA into RecA nucleoprotein filaments.

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 four out of nine 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.

Structural insights into the inhibition of bacterial RecA by naphthalene polysulfonated compounds.

SummaryAs a promising target for alternative antimicrobials, bacterial recombinase A (RecA) protein has attracted much attention for its roles in antibiotic-driven SOS response and mutagenesis. Naphthalene polysulfonated compounds (NPS) such as suramin have previously been explored as antibiotic adjuvants targeting RecA, although the underlying structural bases for RecA-ligand interactions remain obscure. Based on our in silico predictions and documented activity of NPS in vitro, we conclude that the analyzed NPS likely interact with Tyr103 (Y103) and other key residues in the ATPase activity center (pocket A). For validation, we generated recombinant RecA proteins (wild-type versus Y103 mutant) to determine the binding affinities for RecA protein interactions with suramin and underexamined NPS in isothermal titration calorimetry. The corresponding dissociation constants (Kd) ranged from 11.5 to 18.8 μM, and Y103 was experimentally shown to be critical to RecA-NPS interactions.

RecA and DNA recombination: a review of molecular mechanisms.

Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson-Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8-20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer-monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics.

MAW point mutation impairs H. Seropedicae RecA ATP hydrolysis and DNA repair without inducing large conformational changes in its structure.

RecA is a multifunctional protein that plays a central role in DNA repair in bacteria. The structural Make ATP Work motif (MAW) is proposed to control the ATPase activity of RecA. In the present work, we report the biochemical activity and structural effects of the L53Q mutation at the MAW motif of the RecA protein from H. seropedicae (HsRecA L53Q). In vitro studies showed that HsRecA L53Q can bind ADP, ATP, and ssDNA, as does wild-type RecA. However, the ATPase and DNA-strand exchange activities were completely lost. In vivo studies showed that the expression of HsRecA L53Q in E. coli recA1 does not change its phenotype when cells were challenged with MMS and UV. Molecular dynamics simulations showed the L53Q point mutation did not cause large conformational changes in the HsRecA structure. However, there is a difference on dynamical cross-correlation movements of the residues involved in contacts within the ATP binding site and regions that hold the DNA binding sites. Additionally, a new hydrogen bond, formed between Q53 and T49, was hypothesized to allow an independent motion of the MAW motif from the hydrophobic core, what could explain the observed loss of activity of HsRecA L53Q.

The effects of mCI inhibition on RecA activity

In E. coli, Recombinase A (RecA) is involved in the SOS response of DNA repair. RecA serves as the activator for the SOS response by cleaving the repressor LexA. The protein mCI is an inhibitor based on the bacteriophage □ repressor CI. mCI has been shown to bind to the helical groove of active RecA filaments which are formed after the cleavage of LexA. It is predicted that this binding results in the inhibition of RecA activity. Two types of in vitro experiments were carried out to test the effects of mCI on the overall catalytic activity of RecA. In the strand exchange assays, it was observed that mCI's inhibition on RecA was dependent on concentration and timing. The mCI protein blocked the initiation of strand exchange, but had relatively small effects once strand exchange was underway. In addition, mCI affected RecA‐mediated ATP hydrolysis. The implications of these results will be discussed.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bacillus subtilis DisA regulates RecA-mediated DNA strand exchange.

Bacillus subtilis diadenylate cyclase DisA converts two ATPs into c-di-AMP, but this activity is suppressed upon interaction with sites of DNA damage. DisA forms a rapid moving focus that pauses upon induction of DNA damage during spore development. We report that DisA pausing, however, was not observed in the absence of the RecO mediator or of the RecA recombinase, suggesting that DisA binds to recombination intermediates formed by RecA in concert with RecO. DisA, which physically interacts with RecA, was found to reduce its ATPase activity without competing for nucleotides or ssDNA. Furthermore, increasing DisA concentrations inhibit RecA-mediated DNA strand exchange, but this inhibition failed to occur when RecA was added prior to DisA, and was independent of RecA-mediated nucleotide hydrolysis or increasing concentrations of c-di-AMP. We propose that DisA may preserve genome integrity by downregulating RecA activities at several steps of the DNA damage tolerance pathway, allowing time for the repair machineries to restore genome stability. DisA might reduce RecA-mediated template switching by binding to a stalled or reversed fork.

Bacillus subtilis RecA interacts with and loads RadA/Sms to unwind recombination intermediates during natural chromosomal transformation.

During natural transformation Bacillus subtilis RecA, polymerized onto the incoming single-stranded (ss) DNA, catalyses DNA strand invasion resulting in a displacement loop (D-loop) intermediate. A null radA mutation impairs chromosomal transformation, and RadA/Sms unwinds forked DNA in the 5′→3′ direction. We show that in the absence of RadA/Sms competent cells require the RecG translocase for natural chromosomal transformation. RadA/Sms tetracysteine motif (C13A and C13R) variants, which fail to interact with RecA, are also deficient in plasmid transformation, but this defect is suppressed by inactivating recA. The RadA/Sms C13A and C13R variants bind ssDNA, and this interaction stimulates their ATPase activity. Wild-type (wt) RadA/Sms interacts with and inhibits the ATPase activity of RecA, but RadA/Sms C13A fails to do it. RadA/Sms and its variants, C13A and C13R, bound to the 5′-tail of a DNA substrate, unwind DNA in the 5′→3′ direction. RecA interacts with and loads wt RadA/Sms to promote unwinding of a non-cognate 3′-tailed or 5′-fork DNA substrate, but RadA/Sms C13A or C13R fail to do it. We propose that wt RadA/Sms interaction with RecA is crucial to recruit the former onto D-loop DNA, and both proteins in concert catalyse D-loop extension to favour integration of ssDNA during chromosomal transformation.

SOS genes contribute to Bac8c induced apoptosis-like death in Escherichia coli

Antimicrobial peptides are one of the promising substitutes for conventional antibiotics. To examine their potential usefulness for controlling bacterial infections, the present study aimed to determine the induction of the self-destruction of Escherichia coli mediated by bac8c, which was modified from template peptide bactenecin. The potential of bac8c (RIWVIWRR) was reflected by the change in physiological following exposure, which led to the accumulation of reactive oxygen species (ROS) with an imbalance in the antioxidant system and damage to the intracellular lipids. In addition, bac8c-mediated death exhibited typical features to bacterial apoptosis-like death (ALD). The pathway was not enhanced in the absence of autocleavage of RecA and LexA, the SOS response proteins. We observed that elevated levels of intracellular ROS induced ALD, but this effect was insufficient before preceding RecA activation and LexA autocleavage. Furthermore, dinF, which encodes a member of the toxic compound extrusion (MATE) family, was evaluated to examine the role of the ALD pathway. dinF protein was required to accelerate the ALD-mediated bac8c in combination with RecA. Taken together, bac8c-mediated cell death underlies the severe SOS response, leading to RecA activation, LexA proteolysis, and dinF overexpression. Since then, overproduction of intracellular ROS facilitated the cell death.

Arenicin-1-induced apoptosis-like response requires RecA activation and hydrogen peroxide against Escherichia coli.

Arenicin-1, a 21-mer antimicrobial peptide exerts significant broad-spectrum antimicrobial activity with membrane-active mechanisms. However, owing to multiple mechanisms of cell death, the antibacterial mechanism of arenicin-1 requires detailed analysis. In the present study, arenicin-1-treated bacteria underwent an apoptosis-like response, which was mechanistically and morphologically similar to eukaryotic apoptosis. Changes in the physiological status of arenicin-1-treated bacterial cells involved accumulation of reactive oxygen species, imbalance of intracellular calcium gradients, disruption of membrane potential, bacterial caspase-like protein activation, and DNA damage. In arenicin-1-induced apoptosis-like death, autocleavage of LexA was observed because of the activation of the caspase-like activity of RecA. Additionally, typical reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals, were scavenged in arenicin-1-treated cells to assess the role of specific reactive oxygen species. Scavenging of hydrogen peroxide interfered with the activity of arenicin-1 in Escherichia coli, whereas the superoxide and hydroxyl radicals level did not affect arenicin-1-induced apoptosis-like death activity. Furthermore, inhibition of Fenton reaction attenuated apoptosis-like response. In conclusion, arenicin-1-induced apoptosis like death requires SOS response proteins and is mediated by hydrogen peroxide and Fenton reaction in E. coli. Arenicin-1 may be a representative antimicrobial peptide with potent apoptotic response against E. coli.

Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E.coli through Transient Inhibition of Host RecA Activity.

Phage-derived "recombineering" methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes, and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well as mutation rates through induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting the SOS response and RecA activity through the expression of a mutant dominant negative form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using a plasmid-based system in which Cas9 and recA mutants are coexpressed, we can achieve increased efficiency and consistency of CRISPR-Cas9-mediated selection and recombineering in E.coli, while reducing the induction of the SOS response. To date, this approach has been shown to be independent of recA genotype and host strain lineage. Using this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10 000 guides covering the E.coli chromosome. The use of dominant negative RecA or homologues may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS pathways are present.

A Small-Molecule Inducible Synthetic Circuit for Control of the SOS Gene Network without DNA Damage.

The bacterial SOS stress-response pathway is a pro-mutagenic DNA repair system that mediates bacterial survival and adaptation to genotoxic stressors, including antibiotics and UV light. The SOS pathway is composed of a network of genes under the control of the transcriptional repressor, LexA. Activation of the pathway involves linked but distinct events: an initial DNA damage event leads to activation of RecA, which promotes autoproteolysis of LexA, abrogating its repressor function and leading to induction of the SOS gene network. These linked events can each independently contribute to DNA repair and mutagenesis, making it difficult to separate the contributions of the different events to observed phenotypes. We therefore devised a novel synthetic circuit to unlink these events and permit induction of the SOS gene network in the absence of DNA damage or RecA activation via orthogonal cleavage of LexA. Strains engineered with the synthetic SOS circuit demonstrate small-molecule inducible expression of SOS genes as well as the associated resistance to UV light. Exploiting our ability to activate SOS genes independently of upstream events, we further demonstrate that the majority of SOS-mediated mutagenesis on the chromosome does not readily occur with orthogonal pathway induction alone, but instead requires DNA damage. More generally, our approach provides an exemplar for using synthetic circuit design to separate an environmental stressor from its associated stress-response pathway.

Blocking the RecA activity and SOS-response in bacteria with a short α-helical peptide.

The RecX protein, a very active natural RecA protein inhibitor, can completely disassemble RecA filaments at nanomolar concentrations that are two to three orders of magnitude lower than that of RecA protein. Based on the structure of RecX protein complex with the presynaptic RecA filament, we designed a short first in class α-helical peptide that both inhibits RecA protein activities in vitro and blocks the bacterial SOS-response in vivo. The peptide was designed using SEQOPT, a novel method for global sequence optimization of protein α-helices. SEQOPT produces artificial peptide sequences containing only 20 natural amino acids with the maximum possible conformational stability at a given pH, ionic strength, temperature, peptide solubility. It also accounts for restrictions due to known amino acid residues involved in stabilization of protein complexes under consideration. The results indicate that a few key intermolecular interactions inside the RecA protein presynaptic complex are enough to reproduce the main features of the RecX protein mechanism of action. Since the SOS-response provides a major mechanism of bacterial adaptation to antibiotics, these results open new ways for the development of antibiotic co-therapy that would not cause bacterial resistance.