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.     

Crystal structures of Escherichia coli RecA in complex with MgADP and MnAMP-PNP

RecA catalyzes the DNA pairing and strand-exchange steps of homologous recombination, an important mechanism for repair of double-stranded DNA breaks. The binding of RecA to DNA is modulated by adenosine nucleotides. ATP increases the affinity of RecA for DNA, while ADP decreases the affinity. Previously, the crystal structures of E. coli RecA and its complex with ADP have been determined to resolutions of 2.3 and 3.0 A, respectively, but the model for the RecA-ADP complex did not include magnesium ion or side chains. Here, we have determined the crystal structures of RecA in complex with MgADP and MnAMP-PNP, a nonhydrolyzable analogue of ATP, at resolutions of 1.9 and 2.1 A, respectively. Both crystals grow in the same conditions and have RecA in a right-handed helical form with a pitch of approximately 82 A. The crystal structures show the detailed interactions of RecA with the nucleotide cofactors, including the metal ion and the gamma phosphate of AMP-PNP. There are very few conformational differences between the structures of RecA bound to ADP and AMP-PNP, which differ from uncomplexed RecA only in a slight opening of the P-loop residues 66-73 upon nucleotide binding. To interpret the functional significance of the structure of the MnAMP-PNP complex, a coprotease assay was used to compare the ability of different nucleotides to promote the active, extended conformation of RecA. Whereas ATPgammaS and ADP-AlF(4) facilitate a robust coprotease activity, ADP and AMP-PNP do not activate RecA at all. We conclude that the crystal structure of the RecA-MnAMP-PNP complex represents a preisomerization state of the RecA protein that exists after ATP has bound but before the conformational transition to the active state.     

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.     

Intersubunit electrostatic complementarity in the RecA nucleoprotein filament regulates nucleotide substrate specificity and conformational activation

The Escherichia coli RecA protein is the prototypical member of a family of molecular motors that transduces ATP binding and hydrolysis for mechanical function. While many general mechanistic features of RecA action are known, specific structural and functional insights into the molecular basis of RecA activation remain elusive. Toward a more complete understanding of the interdependence between ATP and DNA binding by RecA, we report the characterization of a mutant RecA protein wherein the aspartate residue at position 100 within the ATP binding site is replaced by arginine. Physiologically, D100R RecA was characterized by an inducible, albeit reduced, activation of the SOS response and a diminished ability to promote cellular survival after UV irradiation. Biochemically, the D100R substitution caused a surprisingly modest perturbation of RecA-ATP interactions and an unexpected and significant decrease in the affinity of RecA for ssDNA. Moreover, in vitro assays of RecA activities requiring the coordinated processing of ATP and DNA revealed (1) a 2-5-fold decrease in steady-state turnover of ATP; (2) no formation of mixed nucleoprotein filaments when wild-type and D100R RecA compete for limiting ssDNA; and (3) no formation of strand exchange reaction products. Taken together, these results suggest that the D100R mutational effects on isolated RecA activities combine synergistically to perturb its higher-order functions. We conclude that the replacement of Asp100 resulted in a change in the electrostatic complementarity between RecA monomers during active filament assembly that prevents the protein from fully accessing the active multimeric state.     

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay.

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.     

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.     

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.     

Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy

Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former "long pitch" helix is found only when RecA protein is bound to DNA. The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.     

Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

Among strains of Escherichia coli that have evolved to survive extreme exposure to ionizing radiation, mutations in the recA gene are prominent and contribute substantially to the acquired phenotype. Changes at amino acid residue 276, D276A and D276N, occur repeatedly and in separate evolved populations. RecA D276A and RecA D276N exhibit unique adaptations to an environment that can require the repair of hundreds of double strand breaks. These two RecA protein variants (a) exhibit a faster rate of filament nucleation on DNA, as well as a slower extension under at least some conditions, leading potentially to a distribution of the protein among a higher number of shorter filaments, (b) promote DNA strand exchange more efficiently in the context of a shorter filament, and (c) are markedly less inhibited by ADP. These adaptations potentially allow RecA protein to address larger numbers of double strand DNA breaks in an environment where ADP concentrations are higher due to a compromised cellular metabolism.     

Physico-chemical basis of RecA nucleo-protein filament formation on single

The analysis of RecA protein playing a central role in homologous recombination of E. coli with single-stranded DNAs of various structure and length on quantitative level is carried out for the first time. It was shown that weak additive interactions between protein monomers of filament and different structural elements of DNA provide DNA recognition. Orthophosphate and dNMPs (I50 = 12-20 mM) were shown to be the minimal inhibitors of RecA filamentation on d(pN)20. The lengthening of homooligonucleotides from d(pN)2 to d(pN)20 by one unit leads to monotonic increase in the affinity by a factor approximately 2 (factor f) due to weak additive contacts of RecA with every internucleoside phosphate group of DNA (f = 1.56) and specific interactions with each of T and C bases (f = 1.32). RecA filament does not practically interact with bases of d(pA)n, but contacts with internucleoside phosphate groups of the first turn (n < 10; f = 2.1) more effective than with additional turns of d(pA)n (n > 10; f = 1.3). The affinity of RecA protein for d(pN)n, containing typical and a number of different modified bases depends on a type of base, peculiarities of DNA structure and conformation of its sugar-phosphate backbone. The affinity is increased significantly if the bases contain exocyclic proton accepting groups. The possible reasons of preferable complexation of RecA with DNA of definite structure and length are analyzed. The mechanism of single-stranded DNA recognition by RecA and hypothetical mechanism of homological DNA strands exchange are proposed.     

Formation of nascent heteroduplex structures by RecA protein and DNA

E. coli RecA protein promotes homologous pairing in two distinguishable phases: synapsis and strand exchange. With circular single strands (plus strand only) and linear duplex DNA, polarized or unidirectional strand exchange appeared to cause heteroduplex joints to form and grow from a unique end of the duplex DNA. However, a variety of other pairs of substrates appeared to form joint molecules without regard to the polarity of the strands involved. This paradox has been resolved by observations that show that synapsis is fast, nonpolar and sensitive to inhibition by ADP, whereas strand exchange is slow, directional and relatively insensitive to inhibition by ADP. Thus a heteroduplex joint initiated at one end of the duplex DNA grows by continued strand exchange, whereas a joint initiated at the other end dissociates and is unable to start again because accumulating ADP inhibits synapsis. RecA protein appears to form a nascent protein-DNA structure, the RecA synaptic structure, in which at least 100-300 bp in the duplex molecule are held in an unwound configuration and in which the incoming strand is aligned with its complement.     

Regulation of RecA protein binding to DNA by opposing effects of ATP and ADP on inter-domain contacts: analysis by urea-induced unfolding of wild-type and C-terminal truncated RecA

RecA protein requires ATP and its hydrolysis to ADP to complete the DNA strand-exchange reaction. We investigated how the nucleotides activate RecA by examining their effect on urea-induced unfolding, which could reflect domain-domain contact of protein. RecA is folded into three continuous domains: the N-terminal, central and C-terminal domains. The fluorescence of tyrosine residues, which lie mainly in the central domain, was modified in 1-3 M urea, while the red shift of fluorescence peak of the tryptophan residues located in the C-terminal domain occurred only in 3-6 M urea. Thus, the C-terminal domain of RecA is unfolded after the central part unfolds. The change in intensity of tryptophan fluorescence without a large shift in the peak at low concentrations of urea suggests that there are weak interactions between the central and C-terminal domains. This is supported by our observation that RecA protein lacking the C-terminal tail unfolded at lower concentrations of urea than the entire RecA, and with clear transitions, unlike the entire RecA. ATP and its unhydrolyzable analog (ATPgammaS), which enhance the binding of RecA to DNA, facilitated the urea-induced change in RecA tryptophan fluorescence, while ADP, an antagonist of ATP, prevented the change. ATP probably weakens the domain-domain contact and facilitates the DNA binding, while ADP stabilizes the contact and inhibits it. Supporting this conclusion, the binding of RecA lacking the C-terminal tail to DNA was not inhibited by ADP, while that of the intact RecA was.     

Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides.

The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.     

ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome

ATP is bound to dnaA protein with high affinity (KD = 0.03 microM) and hydrolyzed slowly to ADP in the presence of DNA. ADP is also bound tightly to dnaA protein and exchanges with ATP very slowly. The ATP form is active in replication; the ADP form is not. A unique conformation of oriC, formed in an early initiation stage, depends on dnaA protein being in the ATP form. The subsequent entry of dnaB protein to form a prepriming complex also requires ATP binding and is blocked by bound ADP. Inasmuch as hydrolysis of ATP is far slower than these initiation reactions and since the poorly hydrolyzable analogue ATP gamma S can replace ATP, the ATP function appears to be allosteric. The extraordinary affinity of ATP for dnaA protein, its slow hydrolysis to ADP, the profound inhibition of dnaA functions by ADP, and the very slow exchange of ADP all point to a possible regulatory role for these nucleotides in the cell cycle.     

Biochemical characterization of the human RAD51 protein. III. Modulation of DNA binding by adenosine nucleotides

Adenosine nucleotides affect the ability of RecA small middle dotsingle-stranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATPgammaS affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATPgammaS, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51 small middle dotssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA small middle dotssDNA binding form. The available evidence suggests that a low molecular weight hRAD51 small middle dotssDNA binding form (hRAD51 small middle dotssDNA(low)) correlates with active ADP and ATP processing. A high molecular weight hRAD51 small middle dotssDNA aggregate (hRAD51 small middle dotssDNA(high)) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.     

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.     

ADP-dependent DNA strand exchange by the mutant [P67G/E68A] RecA protein

We have prepared a mutant RecA protein in which proline 67 and glutamic acid 68 in the NTP binding site were replaced by a glycine and alanine residue, respectively. The [P67G/E68A]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of ATP and is able to promote the standard ATP-dependent three-strand exchange reaction between a circular bacteriophage phiX174 (phiX) single-stranded DNA molecule and a homologous linear phiX double-stranded (ds) DNA molecule (5.4 kilobase pairs). The strand exchange activity differs from that of the wild type RecA protein, however, in that it is (i) completely inhibited by an ATP regeneration system, and (ii) strongly stimulated by the addition of high concentrations of ADP to the reaction solution. These results indicate that the strand exchange activity of the [P67G/E68A]RecA protein is dependent on the presence of both ATP and ADP. The ADP dependence of the reaction is reduced or eliminated when (i) a shorter linear phiX dsDNA fragment (1.1 kilobase pairs) is substituted for the full-length linear phiX dsDNA substrate, or (ii) the Mg(2+) concentration is reduced to a level just sufficient to complex the ATP present in the reaction solution. These results indicate that it is the branch migration phase (and not the initial pairing step) of the [P67G/E68A]RecA protein-promoted strand exchange reaction that is dependent on ADP. It is likely that the [P67G/E68A]RecA mutation has revealed a requirement for ADP that also exists (but is not as readily apparent) in the strand exchange reaction of the wild type 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.     

Formation of nucleoprotein RecA filament on single-stranded DNA. Analysis by stepwise increase in ligand complexity

RecA protein plays a pivotal role in homologous recombination in Escherichia coli. RecA polymerizes on single-stranded (ss) DNA forming a nucleoprotein filament. Then double-stranded (ds) DNA is bound and searched for segments homologous to the ssDNA. Finally, homologous strands are exchanged, a new DNA duplex is formed, and ssDNA is displaced. We report a quantitative analysis of RecA interactions with ss d(pN)n of various structures and lengths using these oligonucleotides as inhibitors of RecA filamentation on d(pT)20. DNA recognition appears to be mediated by weak interactions between its structural elements and RecA monomers within a filament. Orthophosphate and dNMP are minimal inhibitors of RecA filamentation (I50 = 12-20 mM). An increase in homo-d(pN)2-40 length by one unit improves their affinity for RecA (f factor) approximately twofold through electrostatic contacts of RecA with internucleoside phosphate DNA moieties (f approximately = 1.56) and specific interactions with T or C bases (f approximately = 1.32); interactions with adenine bases are negligible. RecA affinity for d(pN)n containing normal or modified nucleobases depends on the nature of the base, features of the DNA structure. The affinity considerably increases if exocyclic hydrogen bond acceptor moieties are present in the bases. We analyze possible reasons underlying RecA preferences for DNA sequence and length and propose a model for recognition of ssDNA by RecA.     

Affinity chromatography of RecA protein and RecA nucleoprotein complexes on RecA protein-agarose columns

We have analyzed the nature of RecA protein-RecA protein interactions using an affinity column prepared by coupling RecA protein to an agarose support. When radiolabeled soluble proteins from Escherichia coli are applied to this column, only the labeled RecA protein from the extract was selectively retained and bound tightly to the affinity column. Efficient binding of purified 35S-labeled RecA protein required Mg2+, and high salt did not interfere with the binding of RecA protein to the column. Complete removal of the bound enzyme from the affinity column required treatment with guanidine HCl (5 M) or urea (8 M). These and other properties suggest that hydrophobic interactions contribute significantly to RecA protein subunit recognition in solution. Using a series of truncated RecA proteins synthesized in vitro, we have obtained evidence that at least some of the sequences involved in protein recognition are localized within the first 90 amino-terminal residues of the protein. Based on the observation that RecA proteins from three heterologous bacteria are specifically retained on the E. coli RecA affinity column, it is likely that this binding domain is highly conserved and is required for interaction and association of RecA protein monomers. Stable ternary complexes of RecA protein and single-stranded DNA were formed in the presence of the nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) and applied to the affinity columns. Most of the complexes formed with M13 DNA could be eluted in high salt, whereas a substantial fraction of those formed with the oligonucleotide (dT)25-30 remained bound in high salt and were quantitatively eluted with guanidine HCl (5 M). The different binding properties of these RecA protein-DNA complexes likely reflect differences in the availability of a hydrophobic surface on RecA protein when it is bound to long polynucleotides compared to short oligonucleotides.