Two RecA protein types that mediate different modes of hyperrecombination

RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.     

Hyper-recombinogenic RecA protein from Pseudomonas aeruginosa with enhanced activity of its primary DNA binding site

According to one prominent model, each protomer in the activated nucleoprotein filament of homologous recombinase RecA possesses two DNA-binding sites. The primary site binds (1) single-stranded DNA (ssDNA) to form presynaptic complex and (2) the newly formed double-stranded (ds) DNA whereas the secondary site binds (1) dsDNA of a partner to initiate strand exchange and (2) the displaced ssDNA following the strand exchange. RecA protein from Pseudomonas aeruginosa (RecAPa) promotes in Escherichia coli hyper-recombination in an SOS-independent manner. Earlier we revealed that RecAPa rapidly displaces E.coli SSB protein (SSB-Ec) from ssDNA to form presynaptic complex. Here we show that this property (1) is based on increased affinity of ssDNA for the RecAPa primary DNA binding site while the affinity for the secondary site remains similar to that for E.coli RecA, (2) is not specific for SSB-Ec but is also observed for SSB protein from P.aeruginosa that, in turn, predicts a possibility of enhanced recombination repair in this pathogenic bacterium.     

Phe217 regulates the transfer of allosteric information across the subunit interface of the RecA protein filament

BACKGROUND: ATP-mediated cooperative assembly of a RecA nucleoprotein filament activates the protein for catalysis of DNA strand exchange. RecA is a classic allosterically regulated enzyme in that ATP binding results in a dramatic increase in ssDNA binding affinity. This increase in ssDNA binding affinity results almost exclusively from an ATP-mediated increase in cooperative filament assembly rather than an increase in the inherent affinity of monomeric RecA for DNA. Therefore, certain residues at the subunit interface must play an important role in transmitting allosteric information across the filament structure of RecA. RESULTS: Using electron microscopic analysis of RecA polymer formation in the absence of DNA, we show that while wild-type RecA undergoes a slight decrease in filament length in the presence of ATP, a Phe217Tyr substitution results in a dramatic ATP-induced increase in cooperative filament assembly. Biosensor DNA binding measurements reveal that the Phe217Tyr mutation increases ATP-mediated cooperative interaction between RecA subunits by more than 250-fold. CONCLUSIONS: These studies represent the first identification of a subunit interface residue in RecA (Phe217) that plays a critical role in regulating the flow of ATP-mediated information throughout the protein filament structure. We propose a model by which conformational changes that occur upon ATP binding are propagated through the structure of a RecA monomer, resulting in the insertion of the Phe217 side chain into a pocket in the neighboring subunit. This event serves as a key step in intersubunit communication leading to ATP-mediated cooperative filament assembly and high affinity binding to ssDNA.     

Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional RecA protein to yield presynaptic filaments. Here, electron microscopy has been used to further explore the parameters of this assembly process. The optimal extent of presynaptic filament formation required at least one RecA protein monomer per three nucleotides, high concentrations of ATP (greater than 3 mM in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein assembly.     

Complete inhibition of Streptococcus pneumoniae RecA protein-catalyzed ATP hydrolysis by single-stranded DNA-binding protein (SSB protein): implications for the mechanism of SSB protein-stimulated DNA strand exchange

The ATP-dependent three-strand exchange activity of the Streptococcus pneumoniae RecA protein (RecA(Sp)), like that of the Escherichia coli RecA protein (RecA(Ec)), is strongly stimulated by the single-stranded DNA-binding protein (SSB) from either E. coli (SSB(Ec)) or S. pneumoniae (SSB(Sp)). The RecA(Sp) protein differs from the RecA(Ec) protein, however, in that its ssDNA-dependent ATP hydrolysis activity is completely inhibited by SSB(Ec) or SSB(Sp) protein, apparently because these proteins displace RecA(Sp) protein from ssDNA. These results indicate that in contrast to the mechanism that has been established for the RecA(Ec) protein, SSB protein does not stimulate the RecA(Sp) protein-promoted strand exchange reaction by facilitating the formation of a presynaptic complex between the RecA(Sp) protein and the ssDNA substrate. In addition to acting presynaptically, however, it has been proposed that SSB(Ec) protein also stimulates the RecA(Ec) protein strand exchange reaction postsynaptically, by binding to the displaced single strand that is generated when the ssDNA substrate invades the homologous linear dsDNA. In the RecA(Sp) protein-promoted reaction, the stimulatory effect of SSB protein may be due entirely to this postsynaptic mechanism. The competing displacement of RecA(Sp) protein from the ssDNA substrate by SSB protein, however, appears to limit the efficiency of the strand exchange reaction (especially at high SSB protein concentrations or when SSB protein is added to the ssDNA before RecA(Sp) protein) relative to that observed under the same conditions with the RecA(Ec) protein.     

RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.     

RecA protein inhibits in vitro replication of single-stranded DNA with DNA polymerase III holoenzyme of Escherichia coli

Purified RecA protein from Escherichia coli inhibited 5-10-fold the rate of in vitro replication of both unirradiated and UV-irradiated single-stranded DNA (ssDNA) with DNA polymerase III holoenzyme. Maximal inhibition occurred at a ratio of 1 molecule of RecA per 2-4 nucleotides of DNA, and it affected primarily the initiation of elongation on primed ssDNA. Adding single-strand DNA-binding protein (SSB) caused a relief of inhibition. Under conditions when there was enough SSB to cover the ssDNA completely, RecA protein had no effect on initiation, elongation or dissociation steps of replication. These observations together with data from in vivo studies suggest a role for RecA protein in the arrest of DNA replication observed in cells exposed to UV-radiation and a variety of chemical carcinogens.     

The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing

In homologous pairing, the RecA protein sequentially binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), aligning the two DNA molecules within the helical nucleoprotein filament. To identify the DNA binding region, which stretches from the outside to the inside of the filament, we constructed two mutant RecA proteins, RecAR243Q and RecAK245N, with the amino acid substitutions of Arg243 to Gln and Lys245 to Asn, respectively. These amino acids are exposed to the solvent in the crystal structure of the RecA protein and are located in the central domain, which is believed to be the catalytic center of the homologous pairing activity. The mutations of Arg243 to Gln (RecAR243Q) and Lys245 to Asn (RecAK245N) impair the repair of UV-damaged DNA in vivo and cause defective homologous pairing of ssDNA and dsDNA in vitro. Although RecAR243Q is only slightly defective and RecAK245N is completely proficient in ssDNA binding to form the presynaptic filament, both mutant RecA proteins are defective in the formation of the three-component complex including ssDNA, dsDNA, and RecA protein. The ability to form dsDNA from complementary single strands is also defective in both RecAR243Q and RecAK245N. These results suggest that the region including Arg243 and Lys245 may be involved in the path of secondary DNA binding to the presynaptic filament.     

A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments.

Summary The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein. If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction. In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction. Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein. The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step. In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype. Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities. RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody. However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI. RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis. These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments.Abbreviations RecAwt RecA wind-type protein - ssDNA singlestranded DNA - dsDNA dmble-stranded DNA     

ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange.

When the recA protein (RecA) of Escherichia coli promotes strand exchange between single-stranded DNA (ssDNA) circles and linear double-stranded DNAs (dsDNA) with complementary 5' or 3' ends a polarity is observed. This property of RecA depends on ATP hydrolysis and the ssDNA that is displaced in the reaction since no polarity is observed in the presence of the non-hydrolyzable ATP analog, ATP gamma S, or in the presence of single-strand specific exonucleases. Based on these results a model is presented in which both the 5' and 3' complementary ends of the linear dsDNA initiate pairing with the ssDNA circle but only one end remains stably paired. According to this model, the association/dissociation of RecA in the 5' to 3' direction on the displaced strand determines the polarity of strand exchange by favoring or blocking its reinvasion into the newly formed dsDNA. Reinvasion is favored when the displaced strand is coated with RecA whereas it is blocked when it lacks RecA, remains covered by single-stranded DNA binding protein or is removed by a single-strand specific exonuclease. The requirement for ATP hydrolysis is explained if the binding of RecA to the displaced strand occurs via the dissociation and/or transfer of RecA, two functions that depend on ATP hydrolysis. The energy for strand exchange derives from the higher binding constant of RecA for the newly formed dsDNA as compared with that for ssDNA and not from ATP hydrolysis.     

Changes in the tension in dsDNA alter the conformation of RecA bound to dsDNA�CRecA filaments

The RecA protein is an ATPase that mediates recombination via strand exchange. In strand exchange a single-stranded DNA (ssDNA) bound to RecA binding site I in a RecA/ssDNA filament pairs with one strand of a double-stranded DNA (dsDNA) and forms heteroduplex dsDNA in site I if homology is encountered. Long sequences are exchanged in a dynamic process in which initially unbound dsDNA binds to the leading end of a RecA/ssDNA filament, while heteroduplex dsDNA unbinds from the lagging end via ATP hydrolysis. ATP hydrolysis is required to convert the active RecA conformation, which cannot unbind, to the inactive conformation, which can unbind. If dsDNA extension due to RecA binding increases the dsDNA tension, then RecA unbinding must decrease tension. We show that in the presence of ATP hydrolysis decreases in tension induce decreases in length whereas in the absence of hydrolysis, changes in tension have no systematic effect. These results suggest that decreases in force enhance dissociation by promoting transitions from the active to the inactive RecA conformation. In contrast, increases in tension reduce dissociation. Thus, the changes in tension inherent to strand exchange may couple with ATP hydrolysis to increase the directionality and stringency of strand exchange.     

The N-terminal domain of Escherichia coli RecA have multiple functions in promoting homologous recombination

Escherichia coli RecA mediates homologous recombination, a process essential to maintaining genome integrity. In the presence of ATP, RecA proteins bind a single-stranded DNA (ssDNA) to form a RecA-ssDNA presynaptic nucleoprotein filament that captures donor double-stranded DNA (dsDNA), searches for homology, and then catalyzes the strand exchange between ssDNA and dsDNA to produce a new heteroduplex DNA. Based upon a recently reported crystal structure of the RecA-ssDNA nucleoprotein filament, we carried out structural and functional studies of the N-terminal domain (NTD) of the RecA protein. The RecA NTD was thought to be required for monomer-monomer interaction. Here we report that it has two other distinct roles in promoting homologous recombination. It first facilitates the formation of a RecA-ssDNA presynaptic nucleoprotein filament by converting ATP to an ADP-Pi intermediate. Then, once the RecA-ssDNA presynaptic nucleoprotein filament is stably assembled in the presence of ATPγS, the NTD is required to capture donor dsDNA. Our results also suggest that the second function of NTD may be similar to that of Arg243 and Lys245, which were implicated earlier as binding sites of donor dsDNA. A two-step model is proposed to explain how a RecA-ssDNA presynaptic nucleoprotein filament interacts with donor dsDNA.     

Dynamic light scattering investigations of RecA self-assembly and interactions with single strand DNA

Dynamic light scattering (DLS) measurements were performed on self-assembled solutions of RecA as a function of assembly time under strand exchange ionic strength conditions (10 mM MgCl2, 65 mM NaCl, 10 mM Tris-HCl, pH = 7.5, 1 mM DTT, 3-4 microM RecA) in the absence of ATP. These measurements yield distributions of the translational diffusion coefficients of the changing populations of assembling protein species. Interpretations of results of DLS measurements are made in terms of model hydrodynamic calculations that indicate, under the solution conditions employed, the smallest fundamental quaternary subunit of RecA is a hexamer in a toroidal or lock-washer configuration. Interactions of M13mp19 circular single strand DNA (ssDNA) with RecA assembled to different stages were also investigated. Additions of ssDNA to self-assembled solutions of RecA acts to dissociate the associated structures into hexamer subunits. However, the effect of ssDNA on assembled RecA is highly dependent on the RecA self-assembly state. The longer the assembly time, the less reversible the self-assembled structures of RecA become. Binding isotherms of titrated mixtures of ssDNA with RecA self-assembled to various stages were also determined. Evaluated dissociation constants of RecA/ssDNA complexes were found to increase with increases of the associated state of RecA. These results strongly suggest that, under the solvent conditions employed, the active ssDNA binding form of RecA is a hexamer.     

Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1

The double substitution of Glu156 with Leu and Gly157 with Val in the Escherichia coli RecA protein results in a severely reduced level of recombination and constitutive coprotease behavior. Here we present our examination of the biochemical properties of this mutant protein, RecA N99, in an effort to understand its phenotype and the role of loop 1 (L1) in RecA function. We find that RecA N99 protein has reduced single-stranded DNA (ssDNA)-dependent ATP hydrolysis activity, which is not as sensitive to the presence of SSB protein as wild-type RecA protein. RecA N99 protein is also nearly unable to utilize duplex DNA as a polynucleotide cofactor for ATP hydrolysis, and it shows both a decreased rate of association with ssDNA and a diminished capacity to bind DNA in the secondary binding site. The mutant protein has a corresponding reduction in DNA strand exchange activity, which probably results in the decrease in recombination activity in vivo. The constitutive induction of the SOS response may be a consequence of the impaired ability to repair damaged DNA, resulting in unrepaired ssDNA which can act as a cofactor for the cleavage of LexA repressor. These findings point to an involvement of L1 in both the primary and secondary DNA binding sites of the RecA protein.     

Structure of RecX protein complex with the presynaptic RecA filament: Molecular dynamics simulations and small angle neutron scattering

Using molecular modeling techniques we have built the full atomic structure and performed molecular dynamics simulations for the complexes formed by Escherichia coli RecX protein with a single-stranded oligonucleotide and with RecA presynaptic filament. Based on the modeling and SANS experimental data a sandwich-like filament structure formed two chains of RecX monomers bound to the opposite sides of the single stranded DNA is proposed for RecX::ssDNA complex. The model for RecX::RecA::ssDNA include RecX binding into the grove of RecA::ssDNA filament that occurs mainly via Coulomb interactions between RecX and ssDNA. Formation of RecX::RecA::ssDNA filaments in solution was confirmed by SANS measurements which were in agreement with the spectra computed from the molecular dynamics simulations.     

The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein

The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E. coli ssDNA-binding protein (SSB). The capacity of RecA protein to displace SSB is dramatically enhanced in RecA proteins with C-terminal deletions. The displacement of SSB by RecA protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA protein relative to the full-length protein. The C-terminal deletion mutants also more readily displace yeast replication protein A than does the full-length protein. Thus, the RecA protein has an inherent and robust capacity to displace SSB from ssDNA. However, the displacement function is suppressed by the RecA C terminus, providing another example of a RecA activity with C-terminal modulation. RecADeltaC17 also has an enhanced capacity relative to wild-type RecA protein to bind ssDNA containing secondary structure. Added Mg(2+) enhances the ability of wild-type RecA and the RecA C-terminal deletion mutants to compete with SSB and replication protein A. The overall binding of RecADeltaC17 mutant protein to linear ssDNA is increased further by the mutation E38K, previously shown to enhance SSB displacement from ssDNA. The double mutant RecADeltaC17/E38K displaces SSB somewhat better than either individual mutant protein under some conditions and exhibits a higher steady-state level of binding to linear ssDNA under all conditions.     

Biochemical basis of the temperature-inducible constitutive protease activity of the RecA441 protein of Escherichia coli

We compared the biochemical properties of the RecA441 protein to those of the wild-type RecA protein in an effort to account for the constitutive protease activity observed in recA441 strains. The two RecA proteins have similar properties in the absence of single-stranded DNA binding protein (SSB protein), and the differences that do exist shed little light on the temperature-inducible phenotype observed in recA441 strains. In contrast, several biochemical differences are apparent when the two proteins are compared in the presence of SSB protein, and these are conducive to a hypothesis that explains the temperature-sensitive behavior observed in these strains. We find that both the single-stranded DNA (ssDNA)-dependent ATPase and LexA-protease activities of RecA441 protein are more resistant to inhibition by SSB protein than are the activities of the wild-type protein. Additionally, the RecA441 protein is more capable of using ssDNA that has been precoated with SSB protein as a substrate for ATPase and protease activities, implying that RecA441 protein is more proficient at displacing SSB protein from ssDNA. The enhanced SSB protein displacement ability of the RecA441 protein is dependent on elevated temperature. These observations are consistent with the hypothesis that the RecA441 protein competes more efficiently with SSB protein for limited ssDNA sites and can be activated to cleave repressors at elevated temperature by displacing SSB protein from the limited ssDNA that occurs naturally in Escherichia coli. Neither the ssDNA binding characteristics of the RecA441 protein nor the rate at which it transfers from one DNA molecule to another provides an explanation for its enhanced activities, leading us to conclude that kinetics of RecA441 protein association with DNA may be responsible for the properties of the RecA441 protein.     

The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange

The RecA protein of Escherichia coli optimally promotes DNA strand exchange reactions in the presence of the single strand DNA-binding protein of E. coli (SSB protein). Under these conditions, assembly of RecA protein onto single-stranded DNA (ssDNA) occurs in three steps. First, the ssDNA is rapidly covered by SSB protein. The binding of RecA protein is then initiated by nucleation of a short tract of RecA protein onto the ssDNA. Finally, cooperative polymerization of additional RecA protein accompanied by displacement of SSB protein results in a ssDNA-RecA protein filament (Griffith, J. D., Harris, L. D., and Register, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559). We report here that RecA protein assembly onto circular ssDNA yields RecA protein-covered circles in which greater than 85% are completely covered by RecA protein with no remaining SSB protein-covered segments (as detected by electron microscopy). However, when linear ssDNA is used, 90% of the filaments contain a short segment at one end complexed with SSB protein. This suggests that RecA protein assembly is unidirectional. Visualization of the assembly of RecA protein onto either long ssDNA tails (containing either 5' or 3' termini) or ssDNA gaps generated in double strand DNA allowed us to determine that the RecA protein polymerizes in the 5' to 3' direction on ssDNA and preferentially nucleates at ssDNA-double strand DNA junctions containing 5' termini.     

Effect of RecF protein on reactions catalyzed by RecA protein.

RecF protein is one of at least three single strand DNA (ssDNA) binding proteins which act in recombination and repair in Escherichia coli. In this paper we show that our RecF protein preparation complexes with ssDNA so as to retard its electrophoretic movement in an agarose gel. The apparent stoichiometry of RecF-ssDNA-binding measured in this way is one RecF molecule for every 15 nucleotides and the binding appears to be cooperative. Interaction of the other two ssDNA-binding proteins, RecA and Ssb proteins, has been studied extensively; so in this paper we begin the study of the interaction of RecF and RecA proteins. We found that the RecF protein preparation inhibits the activity of RecA protein in the formation of joint molecules whether added before or after addition of RecA protein to ssDNA. It, therefore, differs from Ssb protein which stimulates joint molecule formation when added to ssDNA after RecA protein. We found that our RecF protein preparation inhibits two steps prior to joint molecule formation: RecA protein binding to ssDNA and coaggregate formation between ssDNA-RecA complexes and dsDNA. We found that it required a much higher ratio of RecF to RecA protein than normally occurs in vivo to inhibit joint molecule formation. The insight that these data give to the normal functioning of RecF protein is discussed.     

Changing of filamentation dynamics of RecA protein, induced by D112R amino acid substitution or ATP to dATP replacement, results in filament steadiness TO THE RecX protein action

It is known that RecX is a negative regulator of RecA protein. We found that the mutant RecA D112R protein exhibits increased resistance to RecX protein comparatively to wild-type RecA protein in vitro and in vivo. Using molecular modeling we showed, that amino acid located in position 112 can not approach RecX closer than 25-28 angstroms. Thus, direct contact between amino acid and RecX is impossible. RecA D112R protein more actively competes with SSB protein for the binding sites on ssDNA and, therefore, differs from the wild-type RecA protein by dynamics of filamentation on ssDNA. On the other hand, after the replacement of ATP by dATP, the wild-type RecA protein, changing the dynamics of filamentation on ssDNA, also becomes more resistant to RecX. Based on these data it is concluded that the dynamics of filamentation has a great, if not dominant role in the stability of RecA filament to RecX relative to the role of RecA-RecX protein-protein interactions discussed earlier. We also propose an improved model of regulation of RecA by RecX protein, where RecA filament elongation along ssDNA is blocked by RecX protein on the ssDNA region, located outside the filament.