Interdependence of the kinetics of NTP hydrolysis and the stability of the RecA-ssDNA complex

The ssDNA-dependent NTP hydrolysis activity of the RecA protein was examined using a series of dTn oligomers ranging in size from dT10 to dT2000 as the ssDNA effector. There were three distinct manifestations of the dTn-dependent NTP hydrolysis reaction, depending on the length of the dTn effector that was used. With longer dTn oligomers, NTP hydrolysis occurred with a turnover number of 20-25 min(-1) and the observed S0.5 value for the NTP was independent of the concentration of the dTn oligomer (DNA concentration-independent hydrolysis). With dTn oligomers of intermediate length, NTP hydrolysis still occurred with a turnover number of 20-25 min(-1), but the observed S0.5 for the NTP decreased with increasing dTn concentration until reaching a value similar to that obtained with the longer dTn oligomers (DNA concentration-dependent hydrolysis). With shorter dTn oligomers, the NTP hydrolysis activity was effectively eliminated. Although this general progression of kinetic behavior was observed for the three structurally related NTPs (dATP, ATP, and GTP), the dTn oligomer length at which DNA concentration-independent, DNA concentration-dependent, and no NTP hydrolysis was observed depended on the NTP being considered. For example, dATP (S0.5 = 35 microM) was hydrolyzed in the presence of dT20, whereas ATP (S0.5 = 70 microM) and GTP (S0.5 = 1200 microM) required at least dT50 and dT200 for hydrolysis, respectively. These results are discussed in terms of a kinetic model in which the stability of the RecA-ssDNA-NTP complex is dependent on the intrinsic S0.5 value of the NTP being hydrolyzed.     

Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein: Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA

The effect that Escherichia coli single-stranded DNA binding (SSB) protein has on the single-stranded DNA-dependent ATPase activity of RecA protein is shown to depend upon a number of variables such as order of addition, magnesium concentration, temperature and the type of single-stranded DNA substrate used. When SSB protein is added to the DNA solution prior to the addition of RecA protein, a significant inhibition of ATPase activity is observed. Also, when SSB protein is added after the formation of a RecA protein-single-stranded DNA complex using either etheno M13 DNA, poly(dA) or poly(dT), or using single-stranded phage M13 DNA at lower temperature (25 °C) and magnesium chloride concentrations of 1 mm or 4 mm, a time-dependent inhibition of activity is observed. These results are consistent with the conclusion that SSB protein displaces the RecA protein from these DNA substrates, as described in the accompanying paper. However, if SSB protein is added last to complexes of RecA protein and single-stranded M13 DNA at elevated temperature (37 °C) and magnesium chloride concentrations of 4 mm or 10 mm, or to poly(dA) and poly(dT) that was renatured in the presence of RecA protein, no inhibition of ATPase activity is observed; in fact, a marked stimulation is observed for single-stranded M13 DNA. A similar effect is observed if the bacteriophage T4-coded gene 32 protein is substituted for SSB protein. The apparent stoichiometry of DNA (nucleotides) to RecA protein at the optimal ATPase activity for etheno M13 DNA, poly(dA) and poly(dT) is 6(±1) nucleotides per RecA protein monomer at 4 mm-MgCl₂ and 37 °C. Under the same conditions, the apparent stoichiometry obtained using single-stranded M13 DNA is 12 nucleotides per RecA protein monomer; however, the stoichiometry changes to 4.5 nucleotides per RecA protein monomer when SSB protein is added last. In addition, a stoichiometry of four nucleotides per RecA protein can be obtained with single-stranded M13 DNA in the absence of SSB protein if the reactions are carried out in 1 mm-MgCl₂. These data are consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecA protein to these regions. The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA. In addition, our results have disclosed an additional property of the RecA protein-single-stranded DNA complex: namely, in the presence of complementary base-pairing and at elevated temperatures and magnesium concentrations, a unique RecA protein-DNA complex forms that is resistant to inhibition by SSB protein.     

An NMR study on the interaction of Escherichia coli DinI with RecA-ssDNA complexes

The SOS response, a set of cellular phenomena exhibited by eubacteria, is initiated by various causes that include DNA damage-induced replication arrest, and is positively regulated by the co- protease activity of RecA. Escherichia coli DinI, a LexA-regulated SOS gene product, shuts off the initiation of the SOS response when overexpressed in vivo. Biochemical and genetic studies indicated that DinI physically interacts with RecA to inhibit its co-protease activity. Using nuclear magnetic resonance (NMR) spectroscopy, we show that DinI tightly binds to the central region of RecA (between the N- and C-terminal domains) and that this interaction is enhanced upon the oligomerisation of RecA. On the other hand, DinI did not inhibit the interaction between 4mer single-stranded (ss)DNA and RecA– ATPγS, but had a slight effect on the structure of ssDNA–RecA–ATPγS complexes involving 8mer and 12mer ssDNA. We hypothesise that prevention of repressor binding to the intermolecular cleft region of RecA protomers by DinI, with the possibility of a slight conformational change induced in the DinI-bound ssDNA–RecA–ATPγS complex, together function to inhibit the co-protease activity of RecA.     

DNA sequence dependence of ATP hydrolysis by RecA protein

The DNA sequence dependence of the ATPase activity of RecA protein has been investigated for a variety of single strand octamer and hexadecamer homopolymers and alternating copolymers. Under assay conditions where the single strand DNA concentration exceeds the RecA protein concentration, significant differences in the rates of ATP hydrolysis for the various single strand DNA oligomer cofactors are observed. Under the conditions examined, the order of efficiency of the DNA cofactors in inducing RecA mediated ATPase activity is found to be: dA16 greater than dT16 greater than d(TC)16 greater than dT8 greater than dC16 greater than dA8 = dG8 greater than dG16 greater than dC8 greater than d(AG)16. These results demonstrate not only a dependence of RecA ATPase activity on the sequence composition of short single strand DNA they further reveal ATPase activity can be affected by the nearest neighbor nucleotide sequence of short DNA cofactors.     

RecA dimers serve as a functional unit for assembly of active nucleoprotein filaments

All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments and for their coordinated activity during the catalysis of homologous recombination.     

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.     

RecA protein-promoted cleavage of LexA repressor in the presence of ADP and structural analogues of inorganic phosphate, the fluoride complexes of aluminum and beryllium

Complexes formed from A13+ or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein. In contrast, poly(dT)-RecA-ADP complexes, which are inactive for cleavage of LexA protein, become fully active in the presence of AlF4- or BeF3- ions. These data suggest that fluoride complexes of aluminum and beryllium (called herein X) convert RecA-ADP complexes, which bind weakly to single-stranded DNA, into RecA-ADP-X complexes, which bind tightly to single-stranded DNA, the ADP-X moiety behaving as a nonhydrolyzable analogue of ATP. We propose that AlF4- and BeF3- ions act as analogues of inorganic phosphate by binding to the site of the gamma-phosphate of ATP on RecA-ADP complexes, hence mimicking the single-stranded DNA-RecA-ADP-Pi transition state. We conclude that the elementary reaction that switches RecA protein from a high affinity single-stranded DNA binding state to a low affinity single-stranded DNA binding state is not ATP hydrolysis per se but Pi release.     

Biochemical basis of hyper-recombinogenic activity of Pseudomonas aeruginosa RecA protein in Escherichia coli cells

The replacement of Escherichia coli recA gene (recA_Ec) with the Pseudomonas aeruginosa recA_Pa gene in Escherichia coli cells results in constitutive hyper-recombination (high frequency of recombination exchanges per unit length of DNA) in the absence of constitutive SOS response. To understand the biochemical basis of this unusual in vivo phenotype, we compared in vitro the recombination properties of RecA_Pa protein with those of RecA_Ec protein. Consistent with hyper-recombination activity, RecA_Pa protein appeared to be more proficient both in joint molecule formation, producing extensive DNA networks in strand exchange reaction, and in competition with single-stranded DNA binding (SSB) protein for single-stranded DNA (ssDNA) binding sites. The RecA_Pa protein showed in vitro a normal ability for cleavage of the E. coli LexA repressor (a basic step in SOS regulon derepression) both in the absence and in the presence (i.e. even under suboptimal conditions for RecA_Ec protein) of SSB protein. However, unlike other hyper-recombinogenic proteins, such as RecA441 and RecA730, RecA_Pa protein displaced insufficient SSB protein from ssDNA at low magnesium concentration to induce the SOS response constitutively. In searching for particular characteristics of RecA_Pa in comparison with RecA_Ec, RecA441 and RecA803 proteins, RecA_Pa showed unusually high abilities: to be resistant to the displacement by SSB protein from poly(dT); to stabilize a ternary complex RecA::ATP::ssDNA to high salt concentrations; and to be much more rapid in both the nucleation of double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-dependent ATP hydrolysis at pH 7.5. We hypothesized that the high affinity of RecA_Pa protein for ssDNA, and especially dsDNA, is the factor that directs the ternary complex to bind secondary DNA to initiate additional acts of recombination instead of to bind LexA repressor to induce constitutive SOS response.     

The DinI protein stabilizes RecA protein filaments

When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI protein was present at very high concentrations, generally superstoichiometric, relative to the RecA protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.     

Organized unidirectional waves of ATP hydrolysis within a RecA filament

The RecA protein forms nucleoprotein filaments on DNA, and individual monomers within the filaments hydrolyze ATP. Assembly and disassembly of filaments are both unidirectional, occurring on opposite filament ends, with disassembly requiring ATP hydrolysis. When filaments form on duplex DNA, RecA protein exhibits a functional state comparable to the state observed during active DNA strand exchange. RecA filament state was monitored with a coupled spectrophotometric assay for ATP hydrolysis, with changes fit to a mathematical model for filament disassembly. At 37 °C, monomers within the RecA-double-stranded DNA (dsDNA) filaments hydrolyze ATP with an observed k_cat of 20.8 ± 1.5 min⁻¹. Under the same conditions, the rate of end-dependent filament disassembly (k_off) is 123 ± 16 monomers per minute per filament end. This rate of disassembly requires a tight coupling of the ATP hydrolytic cycles of adjacent RecA monomers. The relationship of k_cat to k_off infers a filament state in which waves of ATP hydrolysis move unidirectionally through RecA filaments on dsDNA, with successive waves occurring at intervals of approximately six monomers. The waves move nearly synchronously, each one transiting from one monomer to the next every 0.5 s. The results reflect an organization of the ATPase activity that is unique in filamentous systems, and could be linked to a RecA motor function.     

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.     

Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant

Rad51 protein promotes homologous recombination in eukaryotes. Recombination activities are activated by Rad51 filament assembly on ssDNA. Previous studies of yeast Rad51 showed that His352 occupies an important position at the filament interface, where it could relay signals between subunits and active sites. To investigate, we characterized yeast Rad51 H352A and H352Y mutants, and solved the structure of H352Y. H352A forms catalytically competent but salt-labile complexes on ssDNA. In contrast, H352Y forms salt-resistant complexes on ssDNA, but is defective in nucleotide exchange, RPA displacement and strand exchange with full-length DNA substrates. The 2.5 Å crystal structure of H352Y reveals a right-handed helical filament in a high-pitch (130 Å) conformation with P6₁ symmetry. The catalytic core and dimer interface regions of H352Y closely resemble those of DNA-bound Escherichia coli RecA protein. The H352Y mutation stabilizes Phe187 from the adjacent subunit in a position that interferes with the γ-phosphate-binding site of the Walker A motif/P-loop, potentially explaining the limited catalysis observed. Comparison of Rad51 H352Y, RecA–DNA and related structures reveals that the presence of bound DNA correlates with the isomerization of a conserved cis peptide near Walker B to the trans configuration, which appears to prime the catalytic glutamate residue for ATP hydrolysis.     

Characterization of RecA424 and RecA670 proteins from Deinococcus radiodurans.

RecA protein is considered to be the most important participant in the radiation resistance of Deinococcus radiodurans. However, it is still unclear how RecA contributes to the resistance. In this study, we identified a new recA mutation (recA424) in the DNA-repair deficient mutant strain KI696, the phenotype of which is remarkably different from mutant strain rec30 carrying recA670. The properties of the gene products from the recA mutants were compared. recA424 could not complement the deficiency in Escherichia coli RecA, as found for recA670. In vitro, neither RecA424 nor RecA670 could promote DNA strand exchange under conditions in which wild-type RecA promoted the reaction, indicating that both RecA424 and Rec670 are defective in recombination activity. RecA424 promoted the autocleavage reaction of LexA in vitro, whereas RecA670 did not. The intracellular LexA level in KI696 was decreased following gamma-irradiation. However, the LexA level in strain rec30 was constant irrespective of irradiation. These results indicate that RecA424 retains co-protease activity, whereas RecA670 does not. While strain rec30 is extremely radiation sensitive, strain KI696 is only slightly sensitive. Together, these observations suggest that the co-protease activity rather than the recombination activity of RecA contributes to radiation resistance in D. radiodurans.     

Bacillus subtilis RecN binds and protects 3'-single-stranded DNA extensions in the presence of ATP

Bacillus subtilis RecN appears to be an early detector of breaks in double-stranded DNA. In vivo, RecN forms discrete nucleoid-associated structures and in vitro exhibits Mg²⁺-dependent single-stranded (ss) DNA binding and ssDNA-dependent ATPase activities. In the presence of ATP or ADP, RecN assembles to form large networks with ssDNA molecules (designated complexes CII and CIII) that involve ATP binding and requires a 3′-OH at the end of ssDNA molecule. Addition of dATP–RecA complexes dissociates RecN from these networks, but this is not observed following addition of an ssDNA binding protein. Apparently, ATP modulates the RecN–ssDNA complex for binding to ssDNA extensions and, in vivo, RecN–ATP bound to 3′-ssDNA might sequester ssDNA ends within complexes that protect the ssDNA while the RecA accessory proteins recruit RecA. With the association of RecA to ssDNA, RecN would dissociate from the DNA end facilitating the subsequent steps in DNA repair.     

Enzymatic properties of the RecA803 protein, a partial suppressor of recF mutations.

The RecA803 protein suppresses the recombinational repair defect of recF mutations and displays enhanced joint molecule formation in vitro (Madiraju et al., 1988). To understand the physical basis for these phenomena, the biochemical properties of RecA803 protein were compared with those of the wild-type protein. The RecA803 protein shows greater DNA-dependent ATPase activity than the wild-type protein with either M13 single-stranded (ss) DNA, which contains secondary structure, or double-stranded DNA. This increased activity reflects an enhanced ability of the mutant protein to form active complexes with these DNA molecules rather than an enhanced catalytic turnover activity, because identical kcat values for ATP hydrolysis are obtained when DNA substrates lacking secondary structure are examined. In addition, the ssDNA-dependent ATPase activity of RecA803 protein displays greater resistance to inhibition by SSB (single-stranded DNA binding) protein. These properties of the RecA803 protein are not due to either an increased binding affinity for ssDNA or an increased kinetic lifetime of RecA803 protein-ssDNA complexes, demonstrating that altered protein-DNA stability is not the basis for the enhanced properties of RecA803 protein. However, the nucleation-limited rate of association with ssDNA is more rapid for the RecA803 protein than for wild-type RecA protein. Consequently, we suggest that altered protein-protein interactions may account for the differences between these two proteins. The implications of these results with regard to the partial suppression of recF mutations by recA803 are discussed (Madiraju et al., 1988).     

Probing the DNA sequence specificity of Escherichia coli RECA protein

Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5′-GCTGGTGG-3′). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a λ repressor fragment in the presence of RecA, ADP-AlF₄ and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed.     

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.     

DNA pairing and strand exchange by the Escherichia coli RecA and yeast Rad51 proteins without ATP hydrolysis: on the importance of not getting stuck

The bacterial RecA protein and the homologous Rad51 protein in eukaryotes both bind to single-stranded DNA (ssDNA), align it with a homologous duplex, and promote an extensive strand exchange between them. Both reactions have properties, including a tolerance of base analog substitutions that tend to eliminate major groove hydrogen bonding potential, that suggest a common molecular process underlies the DNA strand exchange promoted by RecA and Rad51. However, optimal conditions for the DNA pairing and DNA strand exchange reactions promoted by the RecA and Rad51 proteins in vitro are substantially different. When conditions are optimized independently for both proteins, RecA promotes DNA pairing reactions with short oligonucleotides at a faster rate than Rad51. For both proteins, conditions that improve DNA pairing can inhibit extensive DNA strand exchange reactions in the absence of ATP hydrolysis. Extensive strand exchange requires a spooling of duplex DNA into a recombinase-ssDNA complex, a process that can be halted by any interaction elsewhere on the same duplex that restricts free rotation of the duplex and/or complex, I.e. the reaction can get stuck. Optimization of an extensive DNA strand exchange without ATP hydrolysis requires conditions that decrease nonproductive interactions of recombinase-ssDNA complexes with the duplex DNA substrate.     

Effect of ions and nucleotides on the interactions of yeast Rad51 protein with single-stranded oligonucleotides

Rad51 protein is a eukaryotic homologue of RecA protein that is essential for homologous recombination. We developed a simple procedure for purifying yeast Rad51 protein, characterized its interaction with DNA, and compared it with those of RecA from Escherichia coli and Rad51 from higher eukaryotes. Fractionation of crude extract with 0.2% polyethylenimine eliminated contaminant proteins and nucleic acids, which can perturb the subsequent purification steps. Binding of Rad51 to single-stranded DNA was detected in solution by measuring the fluorescence anisotropy of a fluorescein probe attached to the 5' end of the oligonucleotides. The interaction was stabilized by ATP, as is that of RecA, but was neither stabilized by a non-hydrolysable analog of ATP, nor destabilized by ADP, unlike the interaction of RecA. This character was very similar to that of Xenopus XRad51.1, although the binding of yeast Rad51 to DNA was more sensitive to Mg(2+) ion in both the presence and absence of ATP, and was optimal at 5--10 mM Mg(2+). The dissociation of Rad51 protein from DNA is not, therefore, favored by the hydrolysis of ATP to ADP, in contrast to that of RecA. On the other hand, the high DNA-binding state of the Rad51-DNA complex promoted by ATP appeared to be short-lived. These features may be linked to the lower activity of Rad51 and the fact that Rad51 activity does not require the hydrolysis of ATP.