RecA Activity Research Articles

A Mechanism for Single-stranded DNA-binding Protein (SSB) Displacement from Single-stranded DNA upon SSB-RecO Interaction

Displacement of single-stranded DNA (ssDNA)-binding protein (SSB) from ssDNA is necessary for filament formation of RecA on ssDNA to initiate homologous recombination. The interaction between RecO and SSB is considered to be important for SSB displacement; however, the interaction has not been characterized at the atomic level. In this study, to clarify the mechanism underlying SSB displacement from ssDNA upon RecO binding, we examined the interaction between Thermus thermophilus RecO and cognate SSB by NMR analysis. We found that SSB interacts with the C-terminal positively charged region of RecO. Based on this result, we constructed some RecO mutants. The R127A mutant had considerably decreased binding affinity for SSB and could not anneal SSB-coated ssDNAs. Further, the mutant in the RecOR complex prevented the recovery of ssDNA-dependent ATPase activity of RecA from inhibition by SSB. These results indicated that the region surrounding Arg-127 is the binding site of SSB. We also performed NMR analysis using the C-terminal peptide of SSB and found that the acidic region of SSB is involved in the interaction with RecO, as seen in other protein-SSB interactions. Taken together with the findings of previous studies, we propose a model for SSB displacement from ssDNA where the acidic C-terminal region of SSB weakens the ssDNA binding affinity of SSB when the dynamics of the C-terminal region are suppressed by interactions with other proteins, including RecO.

Contribution of Listeria monocytogenes RecA to acid and bile survival and invasion of human intestinal Caco-2 cells

The food-borne pathogen Listeria monocytogenes is able to colonize the human gastro-intestinal tract and subsequently cross the intestinal barrier. Thus, for L. monocytogenes to become virulent, it must survive the low pH of the stomach, high bile concentrations in the small intestine, and invade the epithelial cells. In this study, we show that RecA, which is an important factor in DNA repair and the activator of the SOS response, contributes to the resistance against acid and bile and to the ability of L. monocytogenes to adhere and invade human intestine epithelial cells. Activation of recA was shown with a promoter reporter after exposure to low pH and high bile concentrations and during adhesion and invasion of Caco-2 intestinal epithelial cells. Furthermore, an in-frame recA deletion mutant showed reduced survival after exposure to low pH and high bile concentrations. This mutant also showed a deficiency in adhesion and invasion of Caco-2 cells. These results suggest that RecA may contribute to the colonization of the human gastro-intestinal tract and crossing of the intestinal barrier.

Use of RecA fusion proteins to induce genomic modifications in zebrafish

The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA–DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB.

Less Is More: Neisseria gonorrhoeae RecX Protein Stimulates Recombination by Inhibiting RecA

Escherichia coli RecX (RecX(Ec)) is a negative regulator of RecA activities both in the bacterial cell and in vitro. In contrast, the Neisseria gonorrhoeae RecX protein (RecX(Ng)) enhances all RecA-related processes in N. gonorrhoeae. Surprisingly, the RecX(Ng) protein is not a RecA protein activator in vitro. Instead, RecX(Ng) is a much more potent inhibitor of all RecA(Ng) and RecA(Ec) activities than is the E. coli RecX ortholog. A series of RecX(Ng) mutant proteins representing a gradient of functional deficiencies provide a direct correlation between RecA(Ng) inhibition in vitro and the enhancement of RecA(Ng) function in N. gonorrhoeae. Unlike RecX(Ec), RecX(Ng) does not simply cap the growing ends of RecA filaments, but it directly facilitates a more rapid RecA filament disassembly. Thus, in N. gonorrhoeae, recombinational processes are facilitated by RecX(Ng) protein-mediated limitations on RecA(Ng) filament presence and/or length to achieve maximal function.

X-ray Crystal Structure of the Bacterial Conjugation Factor PsiB, a Negative Regulator of RecA

During bacterial conjugation, genetic material from one cell is transferred to another as single-stranded DNA. The introduction of single-stranded DNA into the recipient cell would ordinarily trigger a potentially deleterious transcriptional response called SOS, which is initiated by RecA protein filaments formed on the DNA. During F plasmid conjugation, however, the SOS response is suppressed by PsiB, an F-plasmid-encoded protein that binds and sequesters free RecA to prevent filament formation. Among the many characterized RecA modulator proteins, PsiB is unique in using sequestration as an inhibitory mechanism. We describe the crystal structure of PsiB from the Escherichia coli F plasmid. The stucture of PsiB is surprisingly similar to CapZ, a eukaryotic actin filament capping protein. Structure-directed neutralization of electronegative surfaces on PsiB abrogates RecA inhibition whereas neutralization of an electropositive surface element enhances PsiB inhibition of RecA. Together, these studies provide a first molecular view of PsiB and highlight its use as a reagent in studies of RecA activity.

Microarray-based STR genotyping using RecA-mediated ligation

We describe a novel assay capable of accurately determining the length of short tandem repeat (STR) alleles. STR genotyping is achieved utilizing RecA-mediated ligation (RML), which combines the high fidelity of RecA-mediated homology searching with allele-specific ligation. RecA catalyzes the pairing of synthetic oligonucleotides with one strand of a double-stranded DNA target, in this case a PCR amplicon. Ligation occurs only when two adjacent oligonucleotides are base paired to the STR region without any overlap or gap. RecA activity is required to overcome the inherent difficulty of annealing repeated sequences in register. This assay is capable of determining STR genotypes of human samples, is easily adapted to high throughput or automated systems and can have widespread utility in diagnostic and forensic applications.

The RdgC protein employs a novel mechanism involving a finger domain to bind to circular DNA

The DNA-binding protein RdgC has been identified as an inhibitor of RecA-mediated homologous recombination in Escherichia coli. In Neisseria species, RdgC also has a role in virulence-associated antigenic variation. We have previously solved the crystal structure of the E. coli RdgC protein and shown it to form a toroidal dimer. In this study, we have conducted a mutational analysis of residues proposed to mediate interactions at the dimer interfaces. We demonstrate that destabilizing either interface has a serious effect on in vivo function, even though a stable complex with circular DNA was still observed. We conclude that tight binding is required for inhibition of RecA activity. We also investigated the role of the RdgC finger domain, and demonstrate that it plays a crucial role in the binding of circular DNA. Together, these data allow us to propose a model for how RdgC loads onto DNA. We discuss how RdgC might inhibit RecA-mediated strand exchange, and how RdgC might be displaced by other DNA metabolism enzymes such as polymerases and helicases.

Repression of recA Induction by RecX Is Independent of the RecA Protein in Deinococcus radiodurans

Besides inhibiting RecA activity at the protein level, Deinococcus radiodurans RecX can suppress RecA induction at the transcriptional level. The regulation of RecX on recA induction is independent of RecA activity, and its N terminus is involved in this process.

Concentration and Ion Induced Effects on Nucleotide Binding, Aggregation and Thermal Unfolding Transitions of Reca

The Escherichia coli protein, RecA, catalyzes the DNA pairing and strand exchange reactions that are utilized in DNA recombination and repair. Buffer and salt conditions are known to influence the activity, aggregation state and thermal unfolding of RecA. We have used circular dichroism (CD), fluorescence, infrared and dynamic light scattering studies (DLS) to better understand the salt-induced effects on RecA structure, substrate binding and unfolding. CD and infrared studies were performed in order to monitor the thermally induced unfolding of RecA in the presence of a variety of salts and/or nucleotide and DNA substrates. Previous studies in our laboratory have shown that the concentration and identity of the salt ions resulted in unique influences on RecA unfolding transitions and stability. Unfolding studies performed under salt conditions known to activate RecA's ATPase activity in the absence of DNA showed thermally stable RecA structures. Additional characterization of these stable structures using DLS and fluorescence experiments shows unique aggregation states and nucleotide binding for some of the thermally stable RecA structures. A comparison of the influences of different ions on RecA unfolding, aggregation and nucleotide binding will be presented. These studies may help to elucidate how different ions influence RecA activity, structure, aggregation, and stability.

Inhibitors of RecA Activity Discovered by High-Throughput Screening: Cell-Permeable Small Molecules Attenuate the SOS Response in Escherichia coli

The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with nonclassical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by high-throughput screening, for example, RecA, can complement these efforts. Recent evidence suggests that RecA-controlled processes are responsible for tolerance to antibiotic chemotherapy and are involved in pathways that ultimately lead to full-fledged antibiotic resistance. Therefore inhibitors of RecA may serve as therapeutic adjuvants in combination chemotherapy of bacterial infectious diseases. Toward the goal of validating RecA as a novel target in the chemotherapy of bacterial infections, the authors have screened 35,780 small molecules against RecA. In total, 80 small molecules were identified as primary hits and could be clustered in 6 distinct chemotype clades. The most potent class of hits was further examined, and 1 member compound was found to inhibit RecA-mediated strand exchange and prevent ciprofloxacin-induced SOS expression in Escherichia coli. This compound represents the first small molecule demonstrating an ability to inhibit the bacterial SOS response in live bacterial cell cultures.

RecX is Involved In the Switch between DNA Damage Response and Normal Metabolism in D. radiodurans

Apart from inhibiting RecA activity through protein-protein interactions, Deinococcus radiodurans RecX inhibits the expression of RecA and two other anti-oxidant proteins. To identify the repertoire of proteins regulated by RecX, comparative proteomic studies were undertaken on a wild-type strain (R1) and recX null mutant (RecX(-)). Two-dimensional electrophoresis followed by MALDI-TOF identification revealed 35 differentially expressed proteins, including 12 up-regulated and 23 down-regulated proteins in the mutant. The 12 up-regulated proteins are DNA repair proteins, stress response proteins, and metabolism-related proteins. Most of these have been previously characterized as ionizing radiation-induced proteins. The 23 down-regulated proteins are mainly involved in cellular metabolism, and some of these are key enzymes in the metabolic pathway. Thus, RecX is suggested to be involved in the switch between DNA damage response and normal metabolism in D. radiodurans.

Stochastic analysis of the SOS response in Escherichia coli.

BackgroundDNA damage in Escherichia coli evokes a response mechanism called the SOS response. The genetic circuit of this mechanism includes the genes recA and lexA, which regulate each other via a mixed feedback loop involving transcriptional regulation and protein-protein interaction. Under normal conditions, recA is transcriptionally repressed by LexA, which also functions as an auto-repressor. In presence of DNA damage, RecA proteins recognize stalled replication forks and participate in the DNA repair process. Under these conditions, RecA marks LexA for fast degradation. Generally, such mixed feedback loops are known to exhibit either bi-stability or a single steady state. However, when the dynamics of the SOS system following DNA damage was recently studied in single cells, ordered peaks were observed in the promoter activity of both genes (Friedman et al., 2005, PLoS Biol. 3(7):e238). This surprising phenomenon was masked in previous studies of cell populations. Previous attempts to explain these results harnessed additional genes to the system and deployed complex deterministic mathematical models that were only partially successful in explaining the results.Methodology/Principal FindingsHere we apply stochastic methods, which are better suited for dynamic simulations of single cells. We show that a simple model, involving only the basic components of the circuit, is sufficient to explain the peaks in the promoter activities of recA and lexA. Notably, deterministic simulations of the same model do not produce peaks in the promoter activities.Conclusion/SignificanceWe conclude that the double negative mixed feedback loop with auto-repression accounts for the experimentally observed peaks in the promoter activities. In addition to explaining the experimental results, this result shows that including additional regulations in a mixed feedback loop may dramatically change the dynamic functionality of this regulatory module. Furthermore, our results suggests that stochastic fluctuations strongly affect the qualitative behavior of important regulatory modules even under biologically relevant conditions, thus emphasizing the importance of stochastic analysis of regulatory circuits.

The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec + cells, only the full-length RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible N-terminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1–32 (which initiates at Met33 and encodes RecUΔ1–32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB·ATPγS·Mg 2+ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1–32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecA-mediated DNA strand exchange. This study shows that the flexible N-terminus of RecU is essential for protein activity.

Genetic instabilities of (CCTG)·(CAGG) and (ATTCT)·(AGAAT) disease‐associated repeats reveal multiple pathways for repeat deletion

The DNA repeats (CTG).(CAG), (CGG).(CCG), (GAA).(TTC), (ATTCT).(AGAAT), and (CCTG).(CAGG), undergo expansion in humans leading to neurodegenerative disease. A genetic assay for repeat instability has revealed that the activities of RecA and RecB during replication restart are involved in a high rate of deletion of (CTG).(CAG) repeats in E. coli. This assay has been applied to (CCTG).(CAGG) repeats associated with myotonic dystrophy type 2 (DM2) that expand to 11 000 copies and to spinocerebellar ataxia type 10 (SCA10) (ATTCT).(AGAAT) repeats that expand to 4500 copies in affected individuals. DM2 (CCTG).(CAGG) repeats show a moderate rate of instability, less than that observed for the myotonic dystrophy type 1 (CTG).(CAG) repeats, while the SCA10 (ATTCT).(AGAAT) repeats were remarkably stable in E. coli. In contrast to (CTG).(CAG) repeats, deletions of the DM2 and SCA10 repeats were not dependent on RecA and RecB, suggesting that replication restart may not be a predominant mechanism by which these repeats undergo deletion. These results suggest that different molecular mechanisms, or pathways, are responsible for the instability of different disease-associated DNA repeats in E. coli. These pathways involve simple replication slippage and various sister strand exchange events leading to deletions or expansions, often associated with plasmid dimerization. The differences in the mechanisms of repeat deletion may result from the differential propensity of these repeats to form various DNA secondary structures and their differential proclivity for primer-template misalignment during replication.

The Effect of High Concentration Salt on the Structure, Stability, and Aggregation of RecA

The Escherichia coli protein, RecA, is critical for maintaining genetic integrity. RecA catalyzes DNA pairing and strand exchange reactions that are utilized in DNA recombination and DNA repair. Buffer and salt conditions influence the aggregation and activity of RecA. In low salt conditions, RecA is a DNA-dependent ATPase. However, prior research demonstrated that high salt concentrations allow RecA to hydrolyze ATP in the absence of DNA and at levels comparable to those obtained in the presence of DNA [Pugh, B. F. and Cox, M. M. (1988) Journal of Biological Chemistry 263, 76-83]. We have used circular dichroism (CD) and fluorescence spectroscopies to better understand the salt-induced effects on RecA structure and function. CD and fluorescence studies were performed in order to monitor the thermally induced unfolding of RecA in the presence of a variety of salts. We found that different salts had unique effects on RecA unfolding transitions and stability. Unfolding studies performed under salt conditions known to activate RecA's ATPase activity showed unique, thermally stable RecA structures. A comparison of the influences of different ions on RecA unfolding will be presented. These studies may help to elucidate how different ions influence RecA activity, structure, aggregation, and stability.

Inhibitors of RecA Activity Discovered by High-Throughput Screening: Cell-Permeable Small Molecules Attenuate the SOS Response in Escherichia coli

Inhibitors of RecA Activity Discovered by High-Throughput Screening: Cell-Permeable Small Molecules Attenuate the SOS Response in Escherichia coli

UvrD303, a Hyperhelicase Mutant That Antagonizes RecA-Dependent SOS Expression by a Mechanism That Depends on Its C Terminus

Genomic integrity is critical for an organism's survival and ability to reproduce. In Escherichia coli, the UvrD helicase has roles in nucleotide excision repair and methyl-directed mismatch repair and can limit reactions by RecA under certain circumstances. UvrD303 (D403A D404A) is a hyperhelicase mutant, and when expressed from a multicopy plasmid, it results in UV sensitivity (UV(s)), recombination deficiency, and antimutability. In order to understand the molecular mechanism underlying the UV(s) phenotype of uvrD303 cells, this mutation was transferred to the E. coli chromosome and studied in single copy. It is shown here that uvrD303 mutants are UV sensitive, recombination deficient, and antimutable and additionally have a moderate defect in inducing the SOS response after UV treatment. The UV-sensitive phenotype is epistatic with recA and additive with uvrA and is partially suppressed by removing the LexA repressor. Furthermore, uvrD303 is able to inhibit constitutive SOS expression caused by the recA730 mutation. The ability of UvrD303 to antagonize SOS expression was dependent on its 40 C-terminal amino acids. It is proposed that UvrD303, via its C terminus, can decrease the levels of RecA activity in the cell.

Cloning of recA gene of Corynebacterium glutamicum and phenotypic complementation of Escherichia coli recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm2, while non-transformed cells tolerated only up to dose 0.08 J/cm2. It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.

Crystal structure of RecX: A potent regulatory protein of RecA fromXanthomonas campestris

Crystal structure of RecX: A potent regulatory protein of RecA from<i>Xanthomonas campestris</i>

Overexpression of the LexA-regulated tisAB RNA in E. coli inhibits SOS functions; implications for regulation of the SOS response

The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOS-inducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage λ and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene.