ATPase Activity Of RecA Research Articles

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.

The ATPase activity of E. coli RecA prevents accumulation of toxic complexes formed by erroneous binding to undamaged double stranded DNA.

The Escherichia coli RecA protein catalyzes the central step of homologous recombination using its homology search and strand exchange activity. RecA is a DNA-dependent ATPase, but its homology search and strand exchange activities are largely independent of its ATPase activity. ATP hydrolysis converts a high affinity DNA binding form, RecA-ATP, to a low affinity form RecA-ADP, thereby supporting an ATP hydrolysis-dependent dynamic cycle of DNA binding and dissociation. We provide evidence for a novel function of RecA’s dynamic behavior; RecA’s ATPase activity prevents accumulation of toxic complexes caused by direct binding of RecA to undamaged regions of dsDNA. We show that a mutant form of RecA, RecA-K250N, previously shown to be toxic to E. coli, is a loss-of-function ATPase-defective mutant. We use a new method for detecting RecA complexes involving nucleoid surface spreading and immunostaining. The method allows detection of damage-induced RecA foci; STED microscopy revealed these to typically be between 50 and 200 nm in length. RecA-K250N, and other toxic variants of RecA, form spontaneous DNA-bound complexes that are independent of replication and of accessory proteins required to load RecA onto tracts of ssDNA in vivo, supporting the hypothesis that RecA’s expenditure of ATP serves an error correction function.

The Influence of pH and Buffers on the Thermal Stability and Activity of RecA

RecA is a DNA repair protein found in Escherichia coli, with homologues found in mammalian species. RecA is a DNA dependent ATPase that hydrolyzes ATP in the presence of single or double-stranded DNA. RecA has pH dependent affinities for ssDNA and dsDNA binding and ATP hydrolysis. Previous studies in our laboratory have shown that a variety of salts, substrates and pH conditions alter RecA structure and stability. In this study, three buffers were used to study the thermal unfolding and ATPase activity of RecA. RecA unfolding in HEPES, MES, and potassium phosphate buffers is compared to unfolding in Tris buffer at a variety of pH levels (6.5, 7.0, 7.5, 8.0, 8.5). Circular Dichroism was used to follow the unfolding transitions and to determine the melting temperature of RecA at each given pH in each of the various buffers. Activity assays were conducted for each of the solution conditions used for the CD studies in order to study how buffer composition and pH influences RecA activity. Differences in melting temperatures between buffers at a given pH suggest that the buffer itself may alter the aggregation state and stability of RecA.

High-Throughput Screening for RecA Inhibitors Using a Transcreener Adenosine 5′-O-Diphosphate Assay

The activities of the bacterial RecA protein are involved in the de novo development and transmission of antibiotic resistance genes, thus allowing bacteria to overcome the metabolic stress induced by antibacterial agents. RecA is ubiquitous and highly conserved among bacteria, but has only distant homologs in human cells. Together, this evidence points to RecA as a novel and attractive antibacterial drug target. All known RecA functions require the formation of a complex formed by multiple adenosine 5'-O-triphosphate (ATP)-bound RecA monomers on single-stranded DNA. In this complex, RecA hydrolyzes ATP. Although several methods for assessing RecA's ATPase activity have been reported, these assay conditions included relatively high concentrations of enzyme and ATP and thereby restricted the RecA conformational state. Herein, we describe the validation of commercial reagents (Transcreener(®) adenosine 5'-O-diphosphate [ADP](2) fluorescence polarization assay) for the high-throughput measurement of RecA's ATPase activity with lower concentrations of ATP and RecA. Under optimized conditions, ADP detection by the Transcreener reagent provided robust and reproducible activity data (Z'=0.92). Using the Transcreener assay, we screened 113,477 small molecules against purified RecA protein. In total, 177 small molecules were identified as confirmed hits, of which 79 were characterized by IC(50) values ≤ 10 μM and 35 were active in bioassays with live bacteria. This set of compounds comprises previously unidentified scaffolds for RecA inhibition and represents tractable hit structures for efforts aimed at tuning RecA inhibitory activity in both biochemical and bacteriological assays.

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.

Dynamics of RecA filaments on single-stranded DNA

RecA, the key protein in homologous recombination, performs its actions as a helical filament on single-stranded DNA (ssDNA). ATP hydrolysis makes the RecA–ssDNA filament dynamic and is essential for successful recombination. RecA has been studied extensively by single-molecule techniques on double-stranded DNA (dsDNA). Here we directly probe the structure and kinetics of RecA interaction with its biologically most relevant substrate, long ssDNA molecules. We find that RecA ATPase activity is required for the formation of long continuous filaments on ssDNA. These filaments both nucleate and extend with a multimeric unit as indicated by the Hill coefficient of 5.4 for filament nucleation. Disassembly rates of RecA from ssDNA decrease with applied stretching force, corresponding to a mechanism where protein-induced stretching of the ssDNA aids in the disassembly. Finally, we show that RecA–ssDNA filaments can reversibly interconvert between an extended, ATP-bound, and a compressed, ADP-bound state. Taken together, our results demonstrate that ATP hydrolysis has a major influence on the structure and state of RecA filaments on ssDNA.

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.

Complexes of RecA with LexA and RecX Differentiate Between Active and Inactive RecA Nucleoprotein Filaments

The bacterial RecA protein has been the dominant model system for understanding homologous genetic recombination. Although a crystal structure of RecA was solved ten years ago, we still do not have a detailed understanding of how the helical filament formed by RecA on DNA catalyzes the recognition of homology and the exchange of strands between two DNA molecules. Recent structural and spectroscopic studies have suggested that subunits in the helical filament formed in the RecA crystal are rotated when compared to the active RecA-ATP-DNA filament. We examine RecA-DNA-ATP filaments complexed with LexA and RecX to shed more light on the active RecA filament. The LexA repressor and RecX, an inhibitor of RecA, both bind within the deep helical groove of the RecA filament. Residues on RecA that interact with LexA cannot be explained by the crystal filament, but can be properly positioned in an existing model for the active filament. We show that the strand exchange activity of RecA, which can be inhibited when RecX is present at very low stoichiometry, is due to RecX forming a block across the deep helical groove of the RecA filament, where strand exchange occurs. It has previously been shown that changes in the nucleotide bound to RecA are associated with large motions of RecA's C-terminal domain. Since RecX binds from the C-terminal domain of one subunit to the nucleotide-binding core of another subunit, a stabilization of RecA's C-terminal domain by RecX can likely explain the inhibition of RecA's ATPase activity by RecX.

Escherichia coli RecX Inhibits RecA Recombinase and Coprotease Activities in Vitro and in Vivo

In Escherichia coli the RecA protein plays a pivotal role in homologous recombination, DNA repair, and SOS repair and mutagenesis. A gene designated recX (or oraA) is present directly downstream of recA in E. coli; however, the function of RecX is unknown. In this work we demonstrated interaction of RecX and RecA in a yeast two-hybrid assay. In vitro, substoichiometric amounts of RecX strongly inhibited both RecA-mediated DNA strand exchange and RecA ATPase activity. In vivo, we showed that recX is under control of the LexA repressor and is up-regulated in response to DNA damage. A loss-of-function mutation in recX resulted in decreased resistance to UV irradiation; however, overexpression of RecX in trans resulted in a greater decrease in UV resistance. Overexpression of RecX inhibited induction of two din (damage-inducible) genes and cleavage of the UmuD and LexA repressor proteins; however, recX inactivation had no effect on any of these processes. Cells overexpressing RecX showed decreased levels of P1 transduction, whereas recX mutation had no effect on P1 transduction frequency. Our combined in vitro and in vivo data indicate that RecX can inhibit both RecA recombinase and coprotease activities.

Saturation mutagenesis of the E. coli RecA loop L2 homologous DNA pairing region reveals residues essential for recombination and recombinational repair

The disordered mobile loop L2 of the Escherichia coli RecA protein is known to play a central role in DNA binding and pairing. To investigate the local chemical environment in relation to function we performed saturation mutagenesis of the loop L2 region (amino acid positions 193-212) using a site-directed mutagenesis procedure, and determined the recombinational proficiency of the 380 mutants using genetic assays for homologous recombination and recombinational repair. Residues Asn193, Gln194, Arg196, Glu207, Thr209, Gly211, and Gly212 were identified as stringently required for recombinational events in bacterial cells. In addition, our findings suggest the involvement of loop L2 in the ATPase activity of RecA, and a role for residues Gln194, Arg196, Lys198 and Thr209 in the DNA-dependent hydrolysis of ATP. Finally, since 20 residue peptides that comprise this region can pair homologous DNAs by forming filamentous β-structures, we propose how the information from the mutant analysis might facilitate the use of a simplified amino acid alphabet to design β-structure forming L2 peptides with improved RecA-like activities.

Analysis of two distinct single-stranded DNA binding sites on the recA nucleoprotein filament.

The binding stoichiometry of Escherichia coli recA protein to single-stranded DNA (ssDNA) determined by two separate assays differs by a factor of 2.2-2.4. Using the fluorescence of etheno-DNA (epsilon DNA), a chemically modified ssDNA, the stoichiometry was found to be 7.0 +/- 0.6 bases/recA protein monomer in a nucleo-protein filament. Using a competition assay, a similar stoichiometry, 7.5 bases/recA, is found for unmodified poly(dT). Using the DNA-dependent ATPase of recA, which monitors bound protein rather than bound DNA, we find that each recA monomer needs to bind only 3.1 +/- 0.5 bases to fully activate the ATPase. The difference in site size determined by the two assays indicates that there are two DNA binding sites with differential effects on ATPase activation. When recA protein is mixed with ssDNA at a ratio of 7 bases/recA or greater, the complex that forms contains 7 bases/recA and acts as a kinetic trap for the ssDNA. Upon further addition of recA protein, no additional ATPase activity is observed. If, on the other hand, the ssDNA is initially mixed with excess recA (at a ratio of 3-3.5 bases/recA or less) the ATPase activity is twice as high. Analysis of the binding curves suggests that the first DNA strand binds recA to form a filament with a stoichiometry of 3-3.5 bases/protein monomer. The ATPase activity of recA is completely active in this complex. A second strand of DNA can then be bound to this filament yielding a final stoichiometry of approximately 7 bases/protein monomer. The presence of this second strand neither enhances nor inhibits ATP hydrolysis. This ternary complex may mimic the structures formed by recA in searching for homologous DNA sequences and/or in the strand exchange reaction.

The ATPase activity of RecA is needed to push the DNA strand exchange through heterologous regions.

The role of ATP hydrolysis during the RecA-mediated recombination reaction is addressed in this paper. Recent studies indicated that the RecA-promoted DNA strand exchange between completely homologous double- and single-stranded DNA can be very efficient in the absence of ATP hydrolysis. In this work we demonstrate that the energy derived from the ATP hydrolysis is strictly needed to drive the DNA strand exchange through the regions where the interacting DNA molecules are not in a homologous register. Therefore, in addition to the role of the ATP hydrolysis in promoting the dissociation of RecA from the products of the recombination reaction, as described earlier, ATP hydrolysis also plays a crucial role in the actual process of strand exchange, provided that the lack of homologous register obstructs the process of branch migration.