Mycobacterium smegmatis RecA protein is structurally similar to but functionally distinct from Mycobacterium tuberculosis RecA

In eubacteria, RecA proteins belong to a large superfamily of evolutionarily conserved, filament-forming, functional homologs of DNA strand exchange proteins. Here, we report the functional characterization of Mycobacterium smegmatis (Ms) and Mycobacterium tuberculosis (Mt) RecA proteins. Although in some respects Ms and Mt RecA proteins are structural and functional homologs of Escherichia coli (Ec) RecA, there are significant differences as well. The single-stranded DNA-binding property of RecA proteins was analyzed by electrophoretic mobility shift assays. We observed that Ms or Mt RecA proteins bound single-stranded DNA in a manner distinct from that of Ec RecA: The former two were able to form protein-DNA complexes in the presence of high salt. Further experiments indicated that Ms or Mt RecA proteins catalyzed adenosine triphosphate hydrolysis at approximately comparable rates across a wide range of pHs. Significantly, DNA strand invasion promoted by Ms or Mt RecA proteins displayed similar kinetics but distinctly different pH profiles. In contrast to MtRecA, MsRecA by itself was unable to form joint molecules across a wide range of pHs. However, regardless of the order in which SSB was added, it was able to stimulate MsRecA to form joint molecules within a narrow pH range, indicating that SSB is a required accessory factor. Together, these results provide a source of sharp contrast between EcRecA and mycobacterial RecAs on the one hand and Mt and Ms RecA proteins on the other.     

Allosteric movements in eubacterial RecA

The action of RecA, an important eubacterial protein involved in recombination and repair, involves the transition from an inactive filament in the absence of DNA to an active filament formed in association with DNA and ATP. The structure of the inactive filament was first established in Escherichia coli RecA (EcRecA). The interaction of RecA with non-hydrolysable ATP analogues and ADP has been thoroughly characterized and the DNA binding loops visualized based on the crystal structures of the RecA proteins from Mycobacterium tuberculosis (MtRecA) and Mycobacterium smegmatis (MsRecA). A switch residue, which triggers the transformation of the information on ATP binding to the DNA binding regions, has been identified. The 20-residue C-terminal stretch of RecA, which is disordered in all other relevant crystal structures, has been defined in an MsRecA-dATP complex. The ordering of the stretch is accompanied by the generation of a new nucleotide binding site which can communicate with the original nucleotide binding site of an adjacent molecule in the filament. The plasticity of MsRecA and its mutants involving the switch residue has been explored by studying crystals grown under different conditions at two different temperatures and, in one instance, at low humidity. The structures of these crystals and those of EcRecA and Deinococcus radiodurans RecA (DrRecA) provide information on correlated movements involving different regions of the molecule. These correlated movements appear to be important in the allosteric transitions of RecA during its action.     

Snapshots of RecA protein involving movement of the C-domain and different conformations of the DNA-binding loops: crystallographic and comparative analysis of 11 structures of Mycobacterium smegmatis RecA

Mycobacterium smegmatis RecA and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals reported here and five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P6(1). They include one obtained by reducing relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 6(1) screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported for Escherichia coli RecA, the variation in the pitch among the three forms correlates well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue, which is believed to provide the trigger for transmitting the effect of nucleotide binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue similar to that caused by nucleotide binding. The ordering of the DNA-binding loops, which present ensembles of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide binding, presumably on account of the movement of the switch residue that forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications for intermolecular communications within the RecA filament. The structures resulting from different orientations of the C domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA at different phases of activity, and provide insights into the mechanism of action of RecA.     

Site-directed mutagenesis of the RecA protein of Escherichia coli. Tyrosine 264 is required for efficient ATP hydrolysis and strand exchange but not for LexA repressor inactivation

The role of Tyr264 in nucleotide binding and hydrolysis catalyzed by the RecA protein of Escherichia coli was investigated by constructing Gly, Ser, and Phe substitution mutations using oligonucleotide-directed mutagenesis. The corresponding mutant recA genes neither restored resistance to killing by ultraviolet irradiation nor increased homologous recombination in a recA strain. The purified RecA(Gly264) protein was unable to bind nucleotide, hydrolyze ATP, or form stable ternary complexes with adenosine 5'-O-thiotriphosphate and DNA although the mutant protein bound DNA normally in the absence of nucleotide. The RecA (Phe264) and RecA(Ser264) proteins hydrolyzed ATP poorly and the rates were reduced approximately 8- and 18-fold, respectively. Although capable of low levels of ATP hydrolysis, neither the RecA(Phe264) nor the RecA(Ser264) protein promoted DNA pairing or strand exchange reactions in vitro. Furthermore, these mutant RecA proteins were impaired in their ability to form salt-resistant ternary complexes with adenosine 5'-O-thiotriphosphate) and DNA as judged by filter binding. Nevertheless, nucleoprotein complexes formed with either RecA(Phe264) or RecA(Ser264) protein directed efficient cleavage of LexA repressor in vitro. These results demonstrate that Tyr264 is required for efficient ATP hydrolysis and for homologous pairing of DNA but does not participate in activating RecA protein for LexA repressor autodigestion.     

Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.     

DEAD-box RNA helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and RNA duplex recognition

DEAD-box helicases catalyze the non-processive unwinding of double-stranded RNA (dsRNA) at the expense of adenosine triphosphate (ATP) hydrolysis. Nucleotide and RNA binding and unwinding are mediated by the RecA domains of the helicase core, but their cooperation in these processes remains poorly understood. We therefore investigated dsRNA and nucleotide binding by the helicase cores and the isolated N- and C-terminal RecA domains (RecA_N, RecA_C) of the DEAD-box proteins Hera and YxiN by steady-state and time-resolved fluorescence methods. Both helicases bind nucleotides predominantly via RecA_N, in agreement with previous studies on Mss116, and with a universal, modular function of RecA_N in nucleotide recognition. In contrast, dsRNA recognition is different: Hera interacts with dsRNA in the absence of nucleotide, involving both RecA domains, whereas for YxiN neither RecA_N nor RecA_C binds dsRNA, and the complete core only interacts with dsRNA after nucleotide has been bound. DEAD-box proteins thus cover a continuum from complete functional independence of their domains, exemplified by Mss116, to various degrees of inter-domain cooperation in dsRNA binding. The different degrees of domain communication and of thermodynamic linkage between dsRNA and nucleotide binding have important implications on the mechanism of dsRNA unwinding, and may help direct RNA helicases to their respective cellular processes.     

Structure and interactions of RecA: plasticity revealed by molecular dynamics simulations

Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from Mycobacterium tuberculosis, Mycobacterium smegmatis and Escherichia coli and their Adenosine triphosphate (ATP) complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural framework within which RecA molecules from different eubacteria function. Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins.     

In vivo splicing and functional characterization of Mycobacterium leprae RecA

The RecA proteins from Mycobacterium tuberculosis and Mycobacterium leprae contain inteins. In contrast to the M. tuberculosis RecA, the M. leprae RecA is not spliced in Escherichia coli. We demonstrate here that M. leprae RecA is functionally spliced in Mycobacterium smegmatis and produces resistance toward DNA-damaging agents and homologous recombination.     

The stimulatory effect of CaCl(2), NaCl and NH(4)NO(3) salts on the ssDNA-binding activity of RecA depends on nucleotide cofactor and buffer pH

The single-stranded DNA binding activity of the Escherichia coli RecA protein is crucial for homologous recombination to occur. This and other biochemical activities of ssDNA binding proteins may be affected by various factors. In this study, we analyzed the effect of CaCl(2), NaCl and NH(4)NO(3) salts in combination with the pH and nucleotide cofactor effect on the ssDNA-binding activity of RecA. The studies revealed that, in addition to the inhibitory effect, these salts exert also a stimulatory effect on RecA. These effects occur only under very strict conditions, and the presence or absence and the type of nucleotide cofactor play here a major role. It was observed that in contrast to ATP, ATPγS prevented the inhibitory effect of NaCl and NH(4)NO(3), even at very high salt concentration. These results indicate that ATPγS most likely stabilizes the structure of RecA required for DNA binding, making it resistant to high salt concentrations.     

Conformationally selective binding of nucleotide analogues to Escherichia coli RecA: a ligand-based analysis of the RecA ATP binding site

The roles of the RecA protein in the survival of bacteria and the evolution of resistance to antibiotics make it an attractive target for inhibition by small molecules. The activity of RecA is dependent on the formation of a nucleoprotein filament on single-stranded DNA that hydrolyzes ATP. We probed the nucleotide binding site of the active RecA protein using modified nucleotide triphosphates to discern key structural elements of the nucleotide and of the binding site that result in the activation of RecA for NTP hydrolysis. Our results show that the RecA-catalyzed hydrolysis of a given nucleotide triphosphate or analogue thereof is exquisitely sensitive to certain structural elements of both the base and ribose moieties. Furthermore, our ligand-based approach to probing the RecA ATP binding site indicated that the binding of nucleotides by RecA was found to be conformationally selective. Using a binding screen that can be readily adapted to high-throughput techniques, we were able to segregate nucleotides that interact with RecA into two classes: (1) NTPs that preferentially bind the active nucleoprotein filament conformation and either serve as substrates for or competitively inhibit hydrolysis and (2) nonsubstrate NTPs that preferentially bind the inactive RecA conformation and facilitate dissociation of the RecA-DNA species. These results are discussed in the context of a recent structural model for the active RecA nucleoprotein filament and provide us with important information for the design of potent, conformationally selective modulators of RecA activities.     

Investigating structural changes induced by nucleotide binding to RecA using difference FTIR

Nucleotide binding to RecA results in either the high-DNA affinity form (Adenosine 5'-triphosphate (ATP)-bound) or the more inactive protein conformation associated with a lower affinity for DNA (Adenosine 5'-diphosphate (ADP)-bound). Many of the key structural differences between the RecA-ATP and RecA-ADP bound forms have yet to be elucidated. We have used caged-nucleotides and difference FTIR in efforts to obtain a comprehensive understanding of the molecular changes induced by nucleotide binding to RecA. The photochemical release of nucleotides (ADP and ATP) from biologically inactive precursors was used to initiate nucleotide binding to RecA. Here we present ATP hydrolysis assays and fluorescence studies suggesting that the caged nucleotides do not interact with RecA before photochemical release. Furthermore, we now compare difference spectra obtained in H2O and D2O as our first attempt at identifying the origin of the vibrations influenced by nucleotide binding. The infrared data suggest that unique alpha-helical, beta structures, and side chain rearrangements are associated with the high- and low-DNA affinity forms of RecA. Difference spectra obtained over time isolate contributions arising from perturbations in the nucleotide phosphates and have provided further information about the protein structural changes involved in nucleotide binding and the allosteric regulation of RecA.     

Structural studies on MtRecA-nucleotide complexes: insights into DNA and nucleotide binding and the structural signature of NTP recognition

RecA protein plays a crucial role in homologous recombination and repair of DNA. Central to all activities of RecA is its binding to Mg(+2)-ATP. The active form of the protein is a helical nucleoprotein filament containing the nucleotide cofactor and single-stranded DNA. The stability and structure of the helical nucleoprotein filament formed by RecA are modulated by nucleotide cofactors. Here we report crystal structures of a MtRecA-ADP complex, complexes with ATPgammaS in the presence and absence of magnesium as well as a complex with dATP and Mg+2. Comparison with the recently solved crystal structures of the apo form as well as a complex with ADP-AlF4 confirms an expansion of the P-loop region in MtRecA, compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP. The ligand bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve in maintaining the network of interactions crucial for nucleotide binding. The nucleotide binding site itself, however, remains relatively unchanged. The analysis also reveals that ATPgammaS rather than ADP-AlF4 is structurally a better mimic of ATP. From among the complexed structures, a definition for the two DNA-binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA. The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide binding pocket, at the molecular level. Modeling studies on the mutants again point to the relative rigidity of the nucleotide binding site. Comparison with other NTP binding proteins reveals many commonalties in modes of binding by diverse members in the structural family, contributing to our understanding of the structural signature of NTP recognition.     

The hRad51 and RecA proteins show significant differences in cooperative binding to single-stranded DNA.

The human Rad51 protein (hRad51), like its bacterial homologue RecA, catalyzes genetic recombination between homologous single and double-stranded DNA substrates. Using IAsys biosensor technology, we have examined the critical first step in this process, the binding of hRad51 and RecA to ssDNA. We show that hRad51 binds cooperatively and with high affinity to an oligonucleotide substrate in both the absence and presence of nucleotide cofactors. In fact, both ATP and ATPgammaS have a slight inhibitory effect on hRad51 binding affinity. We show that this results from a decrease in the intrinsic affinity of a given monomer for ssDNA, which is counterbalanced by an increase in the cooperative assembly of protein onto DNA. In contrast, we show that the dramatic NTP-induced increase in ssDNA binding affinity of RecA is accounted for by a significant increase in cooperative filament assembly and not by an increase in the intrinsic DNA binding affinity of monomeric RecA. These results demonstrate that although the hRad51 and RecA proteins display many structural and functional similarities, they show profound inherent mechanistic differences.     

The extended N-terminus of Mycobacterium smegmatis RecX potentiates its ability to antagonize RecA functions

The members of the RecX family of proteins have a unique capacity to regulate the catalytic activities of RecA/Rad51 proteins in both prokaryotic and eukaryotic organisms. However, our understanding of the functional roles of RecX in pathogenic and non-pathogenic mycobacteria has been limited by insufficient knowledge of the molecular mechanisms of its activity and regulation. Moreover, the significance of a unique 14 amino acid N-terminal extension in Mycobacterium smegmatis RecX (MsRecX) to its function remains unknown. Here, we advance our understanding of the antagonistic roles of mycobacterial RecX proteins and the functional significance of the extended N-terminus of MsRecX. The full-length MsRecX acts as an antagonist of RecA, negatively regulating RecA promoted functions, including DNA strand exchange, LexA cleavage and ATP hydrolysis, but not binding of ATP. The N-terminally truncated MsRecX variants retain the RecA inhibitory activity, albeit with lower efficiencies compared to the full-length protein. Perhaps most importantly, direct visualization of RecA nucleoprotein filaments, which had been incubated with RecX proteins, showed that they promote disassembly of nucleoprotein filaments primarily within the filaments. In addition, interaction of RecX proteins with the RecA nucleoprotein filaments results in the formation of stiff and irregularly shaped nucleoprotein filaments. Thus, these findings add an additional mechanism by which RecX disassembles RecA nucleoprotein filaments. Overall, this study provides strong evidence for the notion that the N-terminal 14 amino acid region of MsRecX plays an important role in the negative regulation of RecA functions and new insights into the molecular mechanism underlying RecX function.     

Crystallographic identification of an ordered C-terminal domain and a second nucleotide-binding site in RecA: new insights into allostery

RecA protein is a crucial and central component of the homologous recombination and DNA repair machinery. Despite numerous studies on the protein, several issues concerning its action, including the allosteric regulation mechanism have remained unclear. Here we report, for the first time, a crystal structure of a complex of Mycobacterium smegmatis RecA (MsRecA) with dATP, which exhibits a fully ordered C-terminal domain, with a second dATP molecule bound to it. ATP binding is an essential step for all activities of RecA, since it triggers the formation of active nucleoprotein filaments. In the crystal filament, dATP at the first site communicates with a dATP of the second site of an adjacent subunit, through conserved residues, suggesting a new route for allosteric regulation. In addition, subtle but definite changes observed in the orientation of the nucleotide at the first site and in the positions of the segment preceding loop L2 as well as in the segment 102–105 situated between the 2 nt, all appear to be concerted and suggestive of a biological role for the second bound nucleotide.     

Altered nucleotide cofactor-dependent properties of the mutant [S240K]RecA protein

Two mutant Escherichia coli RecA proteins were prepared in which the ATP active site residue, Ser240, was replaced with asparagine and lysine (these amino acids are found in the corresponding positions in other bacterial RecA proteins). The S240N mutation had no discernible effect on the ATP-dependent activities of the RecA protein, indicating that serine and asparagine are functionally interchangeable at position 240. The S240K mutation, in contrast, essentially eliminated the ability of the RecA protein to utilize ATP as a nucleotide cofactor. The [S240K]RecA protein was able to catalyze the hydrolysis of dATP, however, suggesting that the absence of the 2'-hydroxyl group reduced an inhibitory interaction with the Lys240 side chain. Interestingly, the [S240K]RecA protein was able to promote an efficient LexA cleavage reaction but exhibited no strand exchange activity when dATP was provided as the nucleotide cofactor. This apparent separation of function may be attributable to the elevated S(0.5) value for dATP for the [S240K]RecA protein (490 μM, compared to 20-30 μM for the wild type and [S240N]RecA proteins), and may reflect a differential dependence of the LexA co-protease and DNA strand exchange activities on the nucleotide cofactor-mediated stabilization of the functionally-active state of the RecA-ssDNA complex.     

Characterization of DNA strand transfer promoted by Mycobacterium smegmatis RecA reveals functional diversity with Mycobacterium tuberculosis RecA

The RecA-like proteins constitute a group of DNA strand transfer proteins ubiquitous in eubacteria, eukarya, and archaea. However, the functional relationship among RecA proteins is poorly understood. For instance, Mycobacterium tuberculosis RecA is synthesized as a large precursor, which undergoes an unusual protein-splicing reaction to generate an active form. Whereas the precursor was inactive, the active form promoted DNA strand transfer less efficiently compared to EcRecA. Furthermore, gene disruption studies have indicated that the frequencies of allele exchange are relatively lower in Mycobacterium tuberculosis compared to Mycobacterium smegmatis. The mechanistic basis and the factors that contribute to differences in allele exchange remain to be understood. Here, we show that the extent of DNA strand transfer promoted by the M. smegmatis RecA in vitro differs significantly from that of M. tuberculosis RecA. Importantly, M. smegmatis RecA by itself was unable to promote strand transfer, but cognate or noncognate SSBs rendered it efficient even when added prior to RecA. In the presence of SSB, MsRecA or MtRecA catalyzed strand transfer between ssDNA and varying lengths of linear duplex DNA with distinctly different pH profiles. The factors that were able to suppress the formation of DNA networks greatly stimulated strand transfer reactions promoted by MsRecA or MtRecA. Although the rate and pH profiles of dATP hydrolysis catalyzed by MtRecA and MsRecA were similar, only MsRecA was able to couple dATP hydrolysis to DNA strand transfer. Together, these results provide insights into the functional diversity in DNA strand transfer promoted by RecA proteins of pathogenic and nonpathogenic species of mycobacteria.     

Splicing before import - an intein in a mitochondrially targeted preprotein folds and is catalytically active in the cytoplasm in vivo

Nuclear-encoded mitochondrial proteins are cytoplasmically synthesized and imported into the organelle. The intein-containing RecA protein of Mycobacterium tuberculosis, with or without the CoxIVp mitochondrial targeting signal (MTS), was used to determine where a protein targeted to mitochondria folds and becomes catalytically active. Analysis of fractions from Saccharomyces cerevisiae cells expressing RecA without the MTS revealed that RecA and intein proteins remained cytoplasmic. With the MTS, most of RecA was directed to mitochondria, while most of the intein remained in the cytoplasm. The intein therefore folds into a catalytically active state in the cytoplasm prior to RecA import into mitochondria.     

Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein.     

Analysis of double stranded DNA-dependent activities of Deinococcus radiodurans RecA protein

In this study, the double-stranded DNA-dependent activities of Deinococcus radiodurans RecA protein (Dr RecA) were characterized. The interactions of the Dr RecA protein with double-stranded DNA were determined, especially dsDNA-dependent ATP hydrolysis by the Dr RecA protein and the DNA strand exchange reaction, in which multiple branch points exist on a single RecA protein-DNA complex. A nucleotide cofactor (ATP or dATP ) was required for the Dr RecA protein binding to duplex DNA. In the presence of dATP, the nucleation step in the binding process occurred more rapidly than in the presence of ATP. Salts inhibited the binding of the Dr RecA protein to double-stranded DNA. Double-stranded DNA-dependent ATPase activities showed a different sensitivity to anion species. Glutamate had only a minimal effect on the double-stranded DNA-dependent ATPase activities, up to a concentration of 0.7 M. In the competition experiment for Dr RecA protein binding, the Dr RecA protein manifested a higher affinity to double-stranded DNA than was observed for single-stranded DNA.