RESUMO
AIM: Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores. METHODS AND RESULTS: We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' α/ß-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50 V/cm. CONCLUSIONS: In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Temperatura Alta , Recombinases Rec A , Esporos Bacterianos , Esporos Bacterianos/efeitos da radiação , Esporos Bacterianos/genética , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Bacillus subtilis/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Calefação , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
Curcumin is a natural compound that is considered safe and may have potential health benefits; however, its poor stability and water insolubility limit its therapeutic applications. Different strategies aim to increase its water solubility. Here, we tested the compound PVP-curcumin as a photosensitizer for antimicrobial photodynamic therapy (aPDT) as well as its potential to act as an adjuvant in antibiotic drug therapy. Gram-negative E. coli K12 and Gram-positive S. capitis were subjected to aPDT using various PVP-curcumin concentrations (1-200 µg/mL) and 475 nm blue light (7.5-45 J/cm2). Additionally, results were compared to aPDT using 415 nm blue light. Gene expression of recA and umuC were analyzed via RT-qPCR to assess effects on the bacterial SOS response. Further, the potentiation of Ciprofloxacin by PVP-curcumin was investigated, as well as its potential to prevent the emergence of antibiotic resistance. Both bacterial strains were efficiently reduced when irradiated with 415 nm blue light (2.2 J/cm2) and 10 µg/mL curcumin. Using 475 nm blue light, bacterial reduction followed a biphasic effect with higher efficacy in S. capitis compared to E. coli K12. PVP-curcumin decreased recA expression but had limited effect regarding enhancing antibiotic treatment or impeding resistance development. PVP-curcumin demonstrated effectiveness as a photosensitizer against both Gram-positive and Gram-negative bacteria but did not modulate the bacterial SOS response.
Assuntos
Antibacterianos , Ciprofloxacina , Curcumina , Fármacos Fotossensibilizantes , Recombinases Rec A , Curcumina/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Recombinases Rec A/metabolismo , Recombinases Rec A/genética , Ciprofloxacina/farmacologia , Antibacterianos/farmacologia , Fotoquimioterapia/métodos , Resposta SOS em Genética/efeitos dos fármacos , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Povidona/química , Povidona/farmacologia , Testes de Sensibilidade Microbiana , Escherichia coli/efeitos dos fármacos , Luz , Proteínas de Ligação a DNARESUMO
In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the "translocatable unit (TU)", but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria.
Assuntos
Elementos de DNA Transponíveis , Farmacorresistência Bacteriana Múltipla , Klebsiella pneumoniae , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Elementos de DNA Transponíveis/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Plasmídeos/genética , Antibacterianos/farmacologiaRESUMO
The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reÑover 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.
Assuntos
DNA Bacteriano , Escherichia coli , Microscopia de Fluorescência , Imagem Individual de Molécula , Microscopia de Fluorescência/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Imagem Individual de Molécula/métodos , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Resposta SOS em Genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ligação Proteica , Recombinases Rec A/metabolismo , Recombinases Rec A/genética , Pinças ÓpticasRESUMO
The replicative helicase, DnaB, is a central component of the replisome and unwinds duplex DNA coupled with immediate template-dependent DNA synthesis by the polymerase, Pol III. The rate of helicase unwinding is dynamically regulated through structural transitions in the DnaB hexamer between dilated and constricted states. Site-specific mutations in DnaB enforce a faster more constricted conformation that dysregulates unwinding dynamics, causing replisome decoupling that generates excess ssDNA and induces severe cellular stress. This surplus ssDNA can stimulate RecA recruitment to initiate recombinational repair, restart, or activation of the transcriptional SOS response. To better understand the consequences of dysregulated unwinding, we combined targeted genomic dnaB mutations with an inducible RecA filament inhibition strategy to examine the dependencies on RecA in mitigating replisome decoupling phenotypes. Without RecA filamentation, dnaB:mut strains had reduced growth rates, decreased mutagenesis, but a greater burden from endogenous damage. Interestingly, disruption of RecA filamentation in these dnaB:mut strains also reduced cellular filamentation but increased markers of double strand breaks and ssDNA gaps as detected by in situ fluorescence microscopy and FACS assays, TUNEL and PLUG, respectively. Overall, RecA plays a critical role in strain survival by protecting and processing ssDNA gaps caused by dysregulated helicase activity in vivo.
Assuntos
Replicação do DNA , DNA de Cadeia Simples , DnaB Helicases , Mutação , Recombinases Rec A , Recombinases Rec A/metabolismo , Recombinases Rec A/genética , DNA de Cadeia Simples/metabolismo , DnaB Helicases/metabolismo , DnaB Helicases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Polimerização , Proteínas de Ligação a DNARESUMO
Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single-strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single-strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non-transformable bacteria.
Assuntos
Acinetobacter , Proteínas de Bactérias , Mutação , Recombinases Rec A , Recombinação Genética , Acinetobacter/genética , Acinetobacter/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Exodesoxirribonuclease V/metabolismo , Exodesoxirribonuclease V/genética , DNA Bacteriano/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de MembranaRESUMO
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 â¼160% 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.
Assuntos
Deinococcus , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Deinococcus/genética , Deinococcus/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Plasmídeos/genética , Raios Ultravioleta , Reparo do DNA , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
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.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Plasmídeos , Recombinases Rec A , Plasmídeos/genética , Escherichia coli/genética , Recombinases Rec A/metabolismo , Recombinases Rec A/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Recombinação Genética , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Endopeptidase Clp/metabolismo , Endopeptidase Clp/genética , Exodesoxirribonuclease V/metabolismo , Exodesoxirribonuclease V/genética , DNA Bacteriano/metabolismo , Elementos de DNA Transponíveis/genética , Enzimas de Restrição do DNA , Proteínas de Ligação a DNARESUMO
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.
Assuntos
Antibacterianos , Ciprofloxacina , Proteínas de Escherichia coli , Escherichia coli , Testes de Sensibilidade Microbiana , Recombinases Rec A , Resposta SOS em Genética , Resposta SOS em Genética/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Ciprofloxacina/farmacologia , Humanos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Antibacterianos/farmacologia , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Farmacorresistência Bacteriana/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA/efeitos dos fármacos , Sequenciamento Completo do Genoma , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Adaptação Fisiológica , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNARESUMO
Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.
Assuntos
Sistemas CRISPR-Cas , Reparo do DNA , Deinococcus , Edição de Genes , Deinococcus/genética , Edição de Genes/métodos , Reparo do DNA/genética , Genoma Bacteriano , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plasmídeos/genética , Mutagênese , Instabilidade Genômica , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Dano ao DNARESUMO
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.
Assuntos
Escherichia coli , Mutagênese , Recombinases Rec A , Resposta SOS em Genética , Raios Ultravioleta , Resposta SOS em Genética/efeitos dos fármacos , Resposta SOS em Genética/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Antibacterianos/farmacologia , Reparo do DNA , Mutagênicos/farmacologia , Proteínas de Escherichia coli/genética , Farmacorresistência Bacteriana/genética , Transcrição GênicaRESUMO
Homologous recombination plays a key role in double-strand break repair, stalled replication fork repair, and meiosis. The RecA/Rad51 family recombinases catalyze the DNA strand invasion reaction that occurs during homologous recombination. However, the high sequence differences between homologous groups have hindered the thoroughly studies of this ancient protein family. The dynamic mechanisms of the family, particularly at the residual level, remain poorly understood. In this work, five representative RecA/Rad51 recombinase family members from all major kingdoms of living organisms: prokaryotes, eukaryotes, archaea, and viruses, were selected to explore the molecular mechanisms behind their conserved biological significance. A variety of techniques, including all-atom molecular dynamics simulation, perturbation response scanning, and protein structure network analysis, were used to examine the flexibility and correlation of protein domains, distribution of sensors and effectors and conserved hub residues. Furthermore, the potential communication routes between the ATP-binding region and the DNA-binding region of each recombinase were identified. Our results demonstrate the conserved molecular dynamics of these recombinases in the early stage of homologous recombination, including cooperative motions between regions, conserved sensing and effecting functional residue distribution, and conserved hub residues. Meanwhile, the unique ATP-DNA communication routes of each recombinase was also revealed. These results provide new insights into the mechanism of RecA/Rad51 family proteins, and provide new theoretical guidance for the development of allosteric inhibitors and the application of RecA/Rad51 family proteins.
Assuntos
Rad51 Recombinase , Recombinases Rec A , Rad51 Recombinase/genética , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/química , Recombinases Rec A/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA de Cadeia Simples , DNA/química , Recombinases/genética , Recombinases/metabolismo , Trifosfato de AdenosinaRESUMO
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.
Assuntos
Proteínas de Escherichia coli , Exodesoxirribonuclease V , Recombinases Rec A , DNA de Cadeia Simples/genética , Endonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonuclease V/metabolismo , Exodesoxirribonucleases/metabolismo , Recombinases Rec A/metabolismoRESUMO
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.
Assuntos
DNA de Cadeia Simples , Recombinação Homóloga , Rad51 Recombinase , Pareamento de Bases , DNA/química , DNA de Cadeia Simples/genética , Recombinases Rec A/metabolismoRESUMO
Linear dichroism (LD) is a differential polarized light absorption spectroscopy used for studying filamentous molecules such as DNA and protein filaments. In this study, we review the applications of LD for the analysis of DNA-protein interactions. LD signals can be measured in a solution by aligning the sample using flow-induced shear force or a strong electric field. The signal generated is related to the local orientation of chromophores, such as DNA bases, relative to the filament axis. LD can thus assess the tilt and roll of DNA bases and distinguish intercalating from groove-binding ligands. The intensity of the LD signal depends upon the degree of macroscopic orientation. Therefore, DNA shortening and bending can be detected by a decrease in LD signal intensity. As examples of LD applications, we present a kinetic study of DNA digestion by restriction enzymes and structural analyses of homologous recombination intermediates, i.e., RecA and Rad51 recombinase complexes with single-stranded DNA. LD shows that the DNA bases in these complexes are preferentially oriented perpendicular to the filament axis only in the presence of activators, suggesting the importance of organized base orientation for the reaction. LD measurements detect DNA bending by the CRP transcription activator protein, as well as by the UvrB DNA repair protein. LD can thus provide information about the structures of protein-DNA complexes under various conditions and in real time.
Assuntos
DNA , Recombinases Rec A , Recombinases Rec A/metabolismo , DNA/química , DNA de Cadeia Simples , Análise Espectral/métodos , Rad51 Recombinase/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Enzimas de Restrição do DNA , Recombinases Rec A , Arabinose/metabolismo , Proteínas de Bactérias/metabolismo , Dano ao DNA , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/metabolismo , Recombinação Homóloga , Viabilidade Microbiana/efeitos dos fármacos , Mitomicina/farmacologia , Recombinases Rec A/metabolismoRESUMO
Homologous recombination (HR) is a fundamental process common to all species. HR aims to faithfully repair DNA double strand breaks. HR involves the formation of nucleoprotein filaments on DNA single strands (ssDNA) resected from the break. The nucleoprotein filaments search for homologous regions in the genome and promote strand exchange with the ssDNA homologous region in an unbroken copy of the genome. HR has been the object of intensive studies for decades. Because multi-scale dynamics is a fundamental aspect of this process, studying HR is highly challenging, both experimentally and using computational approaches. Nevertheless, knowledge has built up over the years and has recently progressed at an accelerated pace, borne by increasingly focused investigations using new techniques such as single molecule approaches. Linking this knowledge to the atomic structure of the nucleoprotein filament systems and the succession of unstable, transient intermediate steps that takes place during the HR process remains a challenge; modeling retains a very strong role in bridging the gap between structures that are stable enough to be observed and in exploring transition paths between these structures. However, working on ever-changing long filament systems submitted to kinetic processes is full of pitfalls. This review presents the modeling tools that are used in such studies, their possibilities and limitations, and reviews the advances in the knowledge of the HR process that have been obtained through modeling. Notably, we will emphasize how cooperative behavior in the HR nucleoprotein filament enables modeling to produce reliable information.
Assuntos
Recombinação Homóloga , Recombinases Rec A , Recombinases Rec A/metabolismo , DNA de Cadeia Simples/genética , Nucleoproteínas/genética , Quebras de DNA de Cadeia DuplaRESUMO
In E. coli, double strand breaks (DSBs) are resected and loaded with RecA protein. The genome is then rapidly searched for a sequence that is homologous to the DNA flanking the DSB. Mismatches in homologous partners are rare, suggesting that RecA should rapidly reject mismatched recombination products; however, this is not the case. Decades of work have shown that long lasting recombination products can include many mismatches. In this work, we show that in vitro RecA forms readily observable recombination products when 16% of the bases in the product are mismatched. We also consider various theoretical models of mismatch-tolerant homology testing. The models test homology by comparing the sequences of Ltest bases in two single-stranded DNAs (ssDNA) from the same genome. If the two sequences pass the homology test, the pairing between the two ssDNA becomes permanent. Stringency is the fraction of permanent pairings that join ssDNA from the same positions in the genome. We applied the models to both randomly generated genomes and bacterial genomes. For both randomly generated genomes and bacterial genomes, the models show that if no mismatches are accepted stringency is â¼ 99% when Ltest = 14 bp. For randomly generated genomes, stringency decreases with increasing mismatch tolerance, and stringency improves with increasing Ltest. In contrast, in bacterial genomes when Ltest â¼ 75 bp, stringency is â¼ 99% for both mismatch-intolerant and mismatch-tolerant homology testing. Furthermore, increasing Ltest does not improve stringency because most incorrect pairings join different copies of repeats. In sum, for bacterial genomes highly mismatch tolerant homology testing of 75 bp provides the same stringency as homology testing that rejects all mismatches and testing more than â¼75 base pairs is not useful. Interestingly, in vivo commitment to recombination typically requires homology testing of â¼ 75 bp, consistent with highly mismatch intolerant testing.
Assuntos
DNA , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Pareamento de Bases , DNA de Cadeia Simples/genética , Recombinação GenéticaRESUMO
Meiotic crossover (CO) formation between homologous chromosomes ensures their subsequent proper segregation and generates genetic diversity among offspring. In maize, however, the mechanisms that modulate CO formation remain poorly characterized. Here, we found that both maize BREAST CANCER SUSCEPTIBILITY PROTEIN 2 (BRCA2) and AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) act as positive factors of CO formation by controlling the assembly or/and stability of two conserved DNA recombinases RAD51 and DMC1 filaments. Our results revealed that ZmBRCA2 is not only involved in the repair of DNA double-stranded breaks (DSBs), but also regulates CO formation in a dosage-dependent manner. In addition, ZmFIGL1 interacts with RAD51 and DMC1, and Zmfigl1 mutants had a significantly reduced number of RAD51/DMC1 foci and COs. Further, simultaneous loss of ZmFIGL1 and ZmBRCA2 abolished RAD51/DMC1 foci and exacerbated meiotic defects compared with the single mutant Zmbrca2 or Zmfigl1. Together, our data demonstrate that ZmBRCA2 and ZmFIGL1 act coordinately to regulate the dynamics of RAD51/DMC1-dependent DSB repair to promote CO formation in maize. This conclusion is surprisingly different from the antagonistic roles of BRCA2 and FIGL1 in Arabidopsis, implying that, although key factors that control CO formation are evolutionarily conserved, specific characteristics have been adopted in diverse plant species.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Zea mays/genética , Zea mays/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Reparo do DNA , Arabidopsis/genética , Arabidopsis/metabolismo , DNA/metabolismo , Meiose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
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.