RecA finds homologous DNA by reduced dimensionality search.

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3' single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.

Mechanism of strand exchange from RecA-DNA synaptic and D-loop structures.

The strand-exchange reaction is central to homologous recombination. It is catalysed by the RecA family of ATPases, which form a helical filament with single-stranded DNA (ssDNA) and ATP. This filament binds to a donor double-stranded DNA (dsDNA) to form synaptic filaments, which search for homology and then catalyse the exchange of the complementary strand, forming either a new heteroduplex or-if homology is limited-a D-loop1,2. How synaptic filaments form, search for homology and catalyse strand exchange is poorly understood. Here we report the cryo-electron microscopy analysis of synaptic mini-filaments with both non-complementary and partially complementary dsDNA, and structures of RecA-D-loop complexes containing a 10- or a 12-base-pair heteroduplex. The C-terminal domain of RecA binds to dsDNA and directs it to the RecA L2 loop, which inserts into and opens up the duplex. The opening propagates through RecA sequestering the homologous strand at a secondary DNA-binding site, which frees the complementary strand to sample pairing with the ssDNA. At each RecA step, there is a roughly 20% probability that duplex opening will terminate and the as-yet-unopened dsDNA portion will bind to another C-terminal domain. Homology suppresses this process, through the cooperation of heteroduplex pairing with the binding of ssDNA to the secondary site, to extend dsDNA opening. This mechanism locally limits the length of ssDNA sampled for pairing if homology is not encountered, and could allow for the formation of multiple, widely separated synapses on the donor dsDNA, which would increase the likelihood of encountering homology. These findings provide key mechanistic insights into homologous recombination.

Homology recognition without double-stranded DNA-strand separation in D-loop formation by RecA.

RecA protein and RecA/Rad51 orthologues are required for homologous recombination and DNA repair in all living creatures. RecA/Rad51 catalyzes formation of the D-loop, an obligatory recombination intermediate, through an ATP-dependent reaction consisting of two phases: homology recognition between double-stranded (ds)DNA and single-stranded (ss)DNA to form a hybrid-duplex core of 6-8 base pairs and subsequent hybrid-duplex/D-loop processing. How dsDNA recognizes homologous ssDNA is controversial. The aromatic residue at the tip of the ß-hairpin loop (L2) was shown to stabilize dsDNA-strand separation. We tested a model in which dsDNA strands were separated by the aromatic residue before homology recognition and found that the aromatic residue was not essential to homology recognition, but was required for D-loop processing. Contrary to the model, we found that the double helix was not unwound even a single turn during search for sequence homology, but rather was unwound only after the homologous sequence was recognized. These results suggest that dsDNA recognizes its homologous ssDNA before strand separation. The search for homologous sequence with homologous ssDNA without dsDNA-strand separation does not generate stress within the dsDNA; this would be an advantage for dsDNA to express homology-dependent functions in vivo and also in vitro.

Trans-complementation by the RecB nuclease domain of RecBCD enzyme reveals new insight into RecA loading upon χ recognition.

The loading of RecA onto ssDNA by RecBCD is an essential step of RecBCD-mediated homologous recombination. RecBCD facilitates RecA-loading onto ssDNA in a χ-dependent manner via its RecB nuclease domain (RecBn). Before recognition of χ, RecBn is sequestered through interactions with RecBCD. It was proposed that upon χ-recognition, RecBn undocks, allowing RecBn to swing out via a contiguous 70 amino acid linker to reveal the RecA-loading surface, and then recruit and load RecA onto ssDNA. We tested this hypothesis by examining the interactions between RecBn (RecB928-1180) and truncated RecBCD (RecB1-927CD) lacking the nuclease domain. The reconstituted complex of RecB1-927CD and RecBn is functional in vitro and in vivo. Our results indicate that despite being covalently severed from RecB1-927CD, RecBn can still load RecA onto ssDNA, establishing that RecBn does not function while only remaining tethered to the RecBCD complex via the linker. Instead, RecBCD undergoes a χ-induced intramolecular rearrangement to reveal the RecA-loading surface.

RecA-dependent or independent recombination of plasmid DNA generates a conflict with the host EcoKI immunity by launching restriction alleviation.

Bacterial defence systems are tightly regulated to avoid autoimmunity. In Type I restriction-modification (R-M) systems, a specific mechanism called restriction alleviation (RA) controls the activity of the restriction module. In the case of the Escherichia coli Type I R-M system EcoKI, RA proceeds through ClpXP-mediated proteolysis of restriction complexes bound to non-methylated sites that appear after replication or reparation of host DNA. Here, we show that RA is also induced in the presence of plasmids carrying EcoKI recognition sites, a phenomenon we refer to as plasmid-induced RA. Further, we show that the anti-restriction behavior of plasmid-borne non-conjugative transposons such as Tn5053, previously attributed to their ardD loci, is due to plasmid-induced RA. Plasmids carrying both EcoKI and Chi sites induce RA in RecA- and RecBCD-dependent manner. However, inactivation of both RecA and RecBCD restores RA, indicating that there exists an alternative, RecA-independent, homologous recombination pathway that is blocked in the presence of RecBCD. Indeed, plasmid-induced RA in a RecBCD-deficient background does not depend on the presence of Chi sites. We propose that processing of random dsDNA breaks in plasmid DNA via homologous recombination generates non-methylated EcoKI sites, which attract EcoKI restriction complexes channeling them for ClpXP-mediated proteolysis.

The RecA-directed recombination pathway of natural transformation initiates at chromosomal replication forks in the pneumococcus.

Homologous recombination (HR) is a crucial mechanism of DNA strand exchange that promotes genetic repair and diversity in all kingdoms of life. Bacterial HR is driven by the universal recombinase RecA, assisted in the early steps by dedicated mediators that promote its polymerization on single-stranded DNA (ssDNA). In bacteria, natural transformation is a prominent HR-driven mechanism of horizontal gene transfer specifically dependent on the conserved DprA recombination mediator. Transformation involves internalization of exogenous DNA as ssDNA, followed by its integration into the chromosome by RecA-directed HR. How DprA-mediated RecA filamentation on transforming ssDNA is spatiotemporally coordinated with other cellular processes remains unknown. Here, we tracked the localization of fluorescent fusions to DprA and RecA in Streptococcus pneumoniae and revealed that both accumulate in an interdependent manner with internalized ssDNA at replication forks. In addition, dynamic RecA filaments were observed emanating from replication forks, even with heterologous transforming DNA, which probably represent chromosomal homology search. In conclusion, this unveiled interaction between HR transformation and replication machineries highlights an unprecedented role for replisomes as landing pads for chromosomal access of tDNA, which would define a pivotal early HR step for its chromosomal integration.

Structural basis for regulation of SOS response in bacteria.

In response to DNA damage, bacterial RecA protein forms filaments with the assistance of DinI protein. The RecA filaments stimulate the autocleavage of LexA, the repressor of more than 50 SOS genes, and activate the SOS response. During the late phase of SOS response, the RecA filaments stimulate the autocleavage of UmuD and λ repressor CI, leading to mutagenic repair and lytic cycle, respectively. Here, we determined the cryo-electron microscopy structures of Escherichia coli RecA filaments in complex with DinI, LexA, UmuD, and λCI by helical reconstruction. The structures reveal that LexA and UmuD dimers bind in the filament groove and cleave in an intramolecular and an intermolecular manner, respectively, while λCI binds deeply in the filament groove as a monomer. Despite their distinct folds and oligomeric states, all RecA filament binders recognize the same conserved protein features in the filament groove. The SOS response in bacteria can lead to mutagenesis and antimicrobial resistance, and our study paves the way for rational drug design targeting the bacterial SOS response.

PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps.

Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA-bound proteins, and basic properties like the elementary step size remain controversial. Using single-molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single-stranded loop. Static disorder limited previous ensemble studies of a PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations, suggesting a mode of action for eliminating potentially deleterious recombination intermediates.

RecA and SSB genome-wide distribution in ssDNA gaps and ends in Escherichia coli.

Single-stranded DNA (ssDNA) gapped regions are common intermediates in DNA transactions. Using a new non-denaturing bisulfite treatment combined with ChIP-seq, abbreviated 'ssGap-seq', we explore RecA and SSB binding to ssDNA on a genomic scale in E. coli in a wide range of genetic backgrounds. Some results are expected. During log phase growth, RecA and SSB assembly profiles coincide globally, concentrated on the lagging strand and enhanced after UV irradiation. Unexpected results also abound. Near the terminus, RecA binding is favored over SSB, binding patterns change in the absence of RecG, and the absence of XerD results in massive RecA assembly. RecA may substitute for the absence of XerCD to resolve chromosome dimers. A RecA loading pathway may exist that is independent of RecBCD and RecFOR. Two prominent and focused peaks of RecA binding revealed a pair of 222 bp and GC-rich repeats, equidistant from dif and flanking the Ter domain. The repeats, here named RRS for replication risk sequence, trigger a genomically programmed generation of post-replication gaps that may play a special role in relieving topological stress during replication termination and chromosome segregation. As demonstrated here, ssGap-seq provides a new window on previously inaccessible aspects of ssDNA metabolism.

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

Heterogeneity of SOS response expression in clinical isolates of Escherichia coli influences adaptation to antimicrobial stress.

In recent years, new evidence has shown that the SOS response plays an important role in the response to antimicrobials, with involvement in the generation of clinical resistance. Here we evaluate the impact of heterogeneous expression of the SOS response in clinical isolates of Escherichia coli on response to the fluoroquinolone, ciprofloxacin. In silico analysis of whole genome sequencing data showed remarkable sequence conservation of the SOS response regulators, RecA and LexA. Despite the genetic homogeneity, our results revealed a marked differential heterogeneity in SOS response activation, both at population and single-cell level, among clinical isolates of E. coli in the presence of subinhibitory concentrations of ciprofloxacin. Four main stages of SOS response activation were identified and correlated with cell filamentation. Interestingly, there was a correlation between clinical isolates with higher expression of the SOS response and further progression to resistance. This heterogeneity in response to DNA damage repair (mediated by the SOS response) and induced by antimicrobial agents could be a new factor with implications for bacterial evolution and survival contributing to the generation of antimicrobial resistance.

SMC protein RecN drives RecA filament translocation for in vivo homology search.

While the molecular repertoire of the homologous recombination pathways is well studied, the search mechanism that enables recombination between distant homologous regions is poorly understood. Earlier work suggests that the recombinase RecA, an essential component for homology search, forms an elongated filament, nucleating at the break site. How this RecA structure carries out long-distance search remains unclear. Here, we follow the dynamics of RecA after induction of a single double-strand break on the Caulobacter chromosome. We find that the RecA-nucleoprotein filament, once formed, rapidly translocates in a directional manner in the cell, undergoing several pole-to-pole traversals, until homology search is complete. Concomitant with translocation, we observe dynamic variation in the length of the filament. Importantly in vivo, the RecA filament alone is incapable of such long-distance movement; both translocation and associated length variations are contingent on action of structural maintenance of chromosome (SMC)-like protein RecN, via its ATPase cycle. In summary, we have uncovered the three key elements of homology search driven by RecN: mobility of a finite segment of RecA, changes in filament length, and ability to conduct multiple pole-to-pole traversals, which together point to an optimal search strategy.

Single strand gap repair: The presynaptic phase plays a pivotal role in modulating lesion tolerance pathways.

During replication, the presence of unrepaired lesions results in the formation of single stranded DNA (ssDNA) gaps that need to be repaired to preserve genome integrity and cell survival. All organisms have evolved two major lesion tolerance pathways to continue replication: Translesion Synthesis (TLS), potentially mutagenic, and Homology Directed Gap Repair (HDGR), that relies on homologous recombination. In Escherichia coli, the RecF pathway repairs such ssDNA gaps by processing them to produce a recombinogenic RecA nucleofilament during the presynaptic phase. In this study, we show that the presynaptic phase is crucial for modulating lesion tolerance pathways since the competition between TLS and HDGR occurs at this stage. Impairing either the extension of the ssDNA gap (mediated by the nuclease RecJ and the helicase RecQ) or the loading of RecA (mediated by RecFOR) leads to a decrease in HDGR and a concomitant increase in TLS. Hence, we conclude that defects in the presynaptic phase delay the formation of the D-loop and increase the time window allowed for TLS. In contrast, we show that a defect in the postsynaptic phase that impairs HDGR does not lead to an increase in TLS. Unexpectedly, we also reveal a strong genetic interaction between recF and recJ genes, that results in a recA deficient-like phenotype in which HDGR is almost completely abolished.

Beneficial and detrimental genes in the cellular response to replication arrest.

DNA replication is essential for all living organisms. Several events can disrupt replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and so-called "roadblocks" (e.g., DNA-binding proteins or transcription). Bacteria have several well-characterized mechanisms for repairing damaged DNA and then restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of chemical alterations to DNA. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive exposure to an inhibitor that arrests replication elongation, but does not cause chemical alteration of the DNA. Genes identified include those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by chemical damage to DNA and loss of either nuclease causes increased sensitivity to DNA damaging agents. These nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single-stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that in the absence of chemical damage to DNA, RecJ is detrimental to survival. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ appeared to promote too much assembly of RecA filaments. Our results indicate that in the absence of chemical damage to DNA, RecA is dispensable for cells to survive replication arrest and that the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.

RecN spatially and temporally controls RecA-mediated repair of DNA double-strand breaks.

RecN, a bacterial structural maintenance of chromosomes-like protein, plays an important role in maintaining genomic integrity by facilitating the repair of DNA double-strand breaks (DSBs). However, how RecN-dependent chromosome dynamics are integrated with DSB repair remains unclear. Here, we investigated the dynamics of RecN in response to DNA damage by inducing RecN from the PBAD promoter at different time points. We found that mitomycin C (MMC)-treated ΔrecN cells exhibited nucleoid fragmentation and reduced cell survival; however, when RecN was induced with arabinose in MMC-exposed ΔrecN cells, it increased a level of cell viability to similar extent as WT cells. Furthermore, in MMC-treated ΔrecN cells, arabinose-induced RecN colocalized with RecA in nucleoid gaps between fragmented nucleoids and restored normal nucleoid structures. These results suggest that the aberrant nucleoid structures observed in MMC-treated ΔrecN cells do not represent catastrophic chromosome disruption but rather an interruption of the RecA-mediated process. Thus, RecN can resume DSB repair by stimulating RecA-mediated homologous recombination, even when chromosome integrity is compromised. Our data demonstrate that RecA-mediated presynapsis and synapsis are spatiotemporally separable, wherein RecN is involved in facilitating both processes presumably by orchestrating the dynamics of both RecA and chromosomes, highlighting the essential role of RecN in the repair of DSBs.

Exploring the links between SOS response, mutagenesis, and resistance during the recovery period.

Although the mechanistic connections between SOS-induced mutagenesis and antibiotic resistance are well established, our current understanding of the impact of SOS response levels, recovery durations, and transcription/translation activities on mutagenesis remains relatively limited. In this study, when bacterial cells were exposed to mutagens like ultraviolet light for defined time intervals, a compelling connection between the rate of mutagenesis and the RecA-mediated SOS response levels became evident. Our observations also indicate that mutagenesis primarily occurs during the subsequent recovery phase following the removal of the mutagenic agent. When transcription/translation was inhibited or energy molecules were depleted at the onset of treatment or during the early recovery phase, there was a noticeable decrease in SOS response activation and mutagenesis. However, targeting these processes later in the recovery phase does not have the same effect in reducing mutagenesis, suggesting that the timing of inhibiting transcription/translation or depleting energy molecules is crucial for their efficacy in reducing mutagenesis. Active transcription, translation, and energy availability within the framework of SOS response and DNA repair mechanisms appear to be conserved attributes, supported by their consistent manifestation across diverse conditions, including the use of distinct mutagens such as fluoroquinolones and various bacterial strains.

Plasmid expression of Deinococcus radiodurans RecA confers UV-A protection to Escherichia coli with an inverse protein dose dependence, which does not exceed conspecific RecA protection.

Low level expression in Escherichia coli of the RecA protein from the radiation resistant bacterium Deinococcus radiodurans protects a RecA deficient strain of E. coli from UV-A irradiation by up to ∼160% over basal UV-A resistance. The protection effect is inverse protein dose dependent: increasing the expression level of the D. radiodurans RecA (DrRecA) protein decreases the protection factor. This inverse protein dose dependence effect helps resolve previously conflicting reports of whether DrRecA expression is protective or toxic for E. coli. In contrast to the D. radiodurans protein effect, conspecific plasmid expression of E. coli RecA protein in RecA deficient E. coli is consistently protective over several protein expression levels, as well as consistently more protective to higher levels of UV-A exposure than that provided by the D. radiodurans protein. The results indicate that plasmid expression of D. radiodurans RecA can modestly enhance the UV resistance of living E. coli, but that the heterospecific protein shifts from protective to toxic as expression is increased.

Features of the DNA Escherichia coli RecN interaction revealed by fluorescence microscopy and single-molecule methods.

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.

RAD51 supports DMC1 by inhibiting the SMC5/6 complex during meiosis.

Meiosis is a fundamental process for sexual reproduction in most eukaryotes and the evolutionarily conserved recombinases RADiation sensitive51 (RAD51) and Disrupted Meiotic cDNA1 (DMC1) are essential for meiosis and thus fertility. The mitotic function of RAD51 is clear, but the meiotic function of RAD51 remains largely unknown. Here we show that RAD51 functions as an interacting protein to restrain the Structural Maintenance of Chromosomes5/6 (SMC5/6) complex from inhibiting DMC1. We unexpectedly found that loss of the SMC5/6 partially suppresses the rad51 knockout mutant in terms of sterility, pollen inviability, and meiotic chromosome fragmentation in a DMC1-dependent manner in Arabidopsis thaliana. Biochemical and cytological studies revealed that the DMC1 localization in meiotic chromosomes is inhibited by the SMC5/6 complex, which is attenuated by RAD51 through physical interactions. This study not only identified the long-sought-after function of RAD51 in meiosis but also discovered the inhibition of SMC5/6 on DMC1 as a control mechanism during meiotic recombination.

Recombination and replication in DNA repair of heavily irradiated Deinococcus radiodurans.

Deinococcus radiodurans' extreme resistance to ionizing radiation, desiccation, and DNA-damaging chemicals involves a robust DNA repair that reassembles its shattered genome. The repair process requires diploidy and commences with an extensive exonucleolytic erosion of DNA fragments. Liberated single-stranded overhangs prime strand elongation on overlapping fragments and the elongated complementary strands reestablish chromosomal contiguity by annealing. We explored the interdependence of the DNA recombination and replication processes in the reconstitution of the D. radiodurans genome disintegrated by ionizing radiation. The priming of extensive DNA repair synthesis involves RecA and RadA proteins. DNA polymerase III is essential for the initiation of repair synthesis, whereas efficient elongation requires DNA polymerases I and III. Inactivation of both polymerases leads to degradation of DNA fragments and rapid cell death. The present in vivo characterization of key recombination and replication processes dissects the mechanism of DNA repair in heavily irradiated D. radiodurans.