Influence of DNA sequence on the positioning of RecA monomers in RecA-DNA cofilaments.

We show that certain DNA sequences have the ability to influence the positioning of RecA monomers in RecA-DNA complexes. A tendency for RecA monomers to be phased was observed in RecA protein complexes with several oligonucleotides containing a recombinational hotspot sequence, the chi-site from Escherichia coli. This influence was observed in both the 5' to 3' and 3' to 5' directions with respect to chi. A 5'-end phosphate group and probably some other features in DNA also influence the phasing of RecA monomers. We conclude that natural DNAs contain a number of features that influence the positioning of RecA monomers. The ability of specific DNA sequences to influence the positioning of RecA monomers demonstrates some specificity in the binding of individual bases at different sites within a RecA monomer and, most likely, reflects the stereochemical non-equivalence of these sites. The possible biological implications of the phasing of RecA monomers in presynaptic DNA-protein cofilaments are discussed.     

Use of ion exchange chromatography for the study of RecA-DNA interaction

In vitro binding of RecA protein to double-stranded DNA (dsDNA) was studied using ion-exchange liquid chromatography. The method allowed quantification of both free DNA and free protein. The results unambiguously showed a binding stoichiometry of 3 base pairs per RecA monomer. The binding exhibited cooperativity, and the stoichiometry suggested that RecA does not form complexes with two molecules of dsDNA. More than 90% of RecA molecules in the sample were active for DNA binding.     

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.     

Atomic force microscopy of RecA--DNA complexes using a carbon nanotube tip

We report high resolution images of RecA-double stranded (ds) DNA complexes obtained by atomic force microscopy (AFM). When a carbon nanotube (CNT) tip was used, AFM images visualized the 10-nm pitch of RecA-dsDNA complexes and RecA filaments as three-dimensional surface topography without reconstruction analysis. The depth of the notch between two pitches was less than 1 nm. When adsorbed on a soft surface covered with proteins, naked DNA, RecA monomers, RecA hexamers, and short RecA filaments were all clearly resolved in one image. The high resolution images with a CNT tip provided valuable information on the initiation process of RecA-dsDNA complex formation.     

Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction

The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E. coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments. The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of RecA protein from one single-stranded DNA molecule to another, were studied. The binding of SSB protein and RecA protein to single-stranded phage M13 DNA is found to be competitive and, therefore, mutually exclusive. In the absence of a nucleotide cofactor, SSB protein binds more tightly to single-stranded DNA than does RecA protein, whereas in the presence of ATP-gamma-S, RecA protein binds more tightly than SSB protein. In the presence of ATP, an intermediate result is obtained that depends on the type of DNA used, the temperature, and the magnesium ion concentration. When complexes of RecA protein, SSB protein and single-stranded M13 DNA are formed under conditions of slight molar excess of single-stranded DNA, no effect of RecA protein on the equilibrium stability of the SSB protein-single-stranded DNA complex is observed. Under similar conditions, SSB protein has no observed effect on the stability of the RecA protein-etheno M13 DNA complex. Finally, measurements of the rate of RecA protein transfer from RecA protein-single-stranded DNA complexes to competing single-stranded DNA show that there is no kinetic stabilization of the RecA protein-etheno M13 DNA complex by SSB protein, but that a tenfold stabilization is observed when single-stranded M13 DNA is used to form the complex. However, this apparent stabilizing effect of SSB protein can be mimicked by pre-incubation of the RecA protein-single-stranded M13 DNA complex in low magnesium ion concentration, suggesting that this effect of SSB protein is indirect and is mediated through changes in the secondary structure of the DNA. Since no direct effect of SSB protein is observed on either the equilibrium or dissociation properties of the RecA protein-single-stranded DNA complex, it is concluded that the likely effect of SSB protein in the strand assimilation reaction is on a slow step in the association of RecA protein with single-stranded DNA. Direct evidence for this conclusion is presented in the accompanying paper.     

RecA interacts with Klenow and enhances fidelity of DNA synthesis in vitro

To understand the molecular basis of RecA-mediated DNA-repair, we tested the replicative fidelity of the large fragment of Pol I (Klenow) in RecA-DNA complexes in vitro. Klenow synthesis was error-prone in naked DNA substrates but essentially error-free in RecA coated complexes. Escherichia coli SSB, causes no such improvement in Klenow fidelity. RecA filaments promote better exonucleolytic proofreading by Klenow than on naked DNA substrates at select sites when replication is "stalled" due to a missing dNTP. Addition of RecA to pyrene sulfonylchloride-labeled Klenow resulted in a specific increase in steady-state fluorescence anisotropy and a concomitant decrease in fluorescence lifetime. These observations suggest the possibility of a direct interaction between RecA and Klenow even in the absence of DNA which may mediate the observed improvement in Klenow fidelity.     

Binding stoichiometry and structure of RecA-DNA complexes studied by flow linear dichroism and fluorescence spectroscopy. Evidence for multiple heterogeneous DNA co-ordination

The interaction between RecA and DNA (in the form of unmodified single-stranded DNA, fluorescent single-stranded DNA and double-stranded DNA) is studied with linear dichroism and fluorescence spectroscopy. RecA is found to form a complex with single-stranded DNA with a binding stoichiometry of about four nucleotides per RecA monomer, in which the DNA bases appear to have a random orientation. Addition of ATP gamma S (a non-hydrolyzable analog of ATP) reduces the stoichiometry to about three nucleotides per RecA and causes the DNA bases to adopt an orientation preferentially perpendicular to the fiber axis. This complex can incorporate an additional strand of single-stranded DNA or double-stranded DNA, yielding a total stoichiometry of six nucleotides or three nucleotides and three base-pairs, respectively, per RecA. RecA, in the presence of ATP gamma S, is also found to interact with double-stranded DNA, with a stoichiometry of about three base-pairs per RecA. In all studied complexes, the tryptophan residues in the RecA protein are oriented with their planes preferentially parallel to the fiber axis, whereas in complexes involving ATP gamma S the planes of the DNA bases are oriented preferentially perpendicular to the fiber. This virtually excludes the possibility that the tryptophan residues are intercalated in the DNA helix. On the basis of these results, a model for the research of homology in the RecA-mediated, strand-exchange reaction in the genetic recombination process is proposed.     

Characterization of the DNA binding activity of stable RecA-DNA complexes. Interaction between the two DNA binding sites within RecA helical filaments

The DNA-binding, annealing and recombinational activities of purified RecA-DNA complexes stabilized by ATP gamma S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is taken into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and this helps to explain how topological problems are avoided during RecA-mediated homologous recombination.     

Energetics of RecA-mediated recombination reactions. Without ATP hydrolysis RecA can mediate polar strand exchange but is unable to recycle

We demonstrate that the step of DNA strand exchange during RecA-mediated recombination reaction can occur equally efficiently in the presence or absence of ATP hydrolysis. The polarity of strand exchange is the same when instead of ATP its non-hydrolyzable analog adenosine-5'-O-(3-thiotriphosphate) is used. We show that the ATP dependence of recombination reaction is limited to the post-exchange stages of the reactions. The low DNA affinity state of RecA protomers, induced after ATP hydrolysis, is necessary for the dissociation of RecA-DNA complexes at the end of the reaction. This dissociation of RecA from DNA is necessary for the release of recombinant DNA molecules from the complexes formed with RecA and for the recycling of RecA protomers for another round of the recombination reaction.     

Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange

The RecA protein of Escherichia coli will drive the pairing and exchange of strands between homologous DNA molecules in a reaction stimulated by single-stranded binding protein. Here, reactions utilizing three homologous DNA pairs which can undergo both paranemic and plectonemic joining were examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally indistinguishable from paranemic joints; (ii) complexes which appeared to be joined both paranemically and plectonemically were present in these reactions in roughly equal numbers; and (iii) in complexes undergoing strand exchange, both DNA partners were often enveloped within a RecA protein filament consisting of hundreds of RecA protein monomers and several kilobases of DNA. These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products.     

Properties of RecA Complexes with Homopolymeric DNA Strands Depend on Sequence Complementarity. Implications for the Mechanism of Strand Exchange

Abstract

We have characterised complexes between RecA and single-stranded homopolynucleotides by linear dichroism spectroscopy and small angle neutron scattering to investigate base pairing possibilities among DNA strands bound in a RecA filament. We find that in the presence of the non-hydrolysable cofactor ATPγS, and very likely also in the presence of ATP, a RecA fiber has three distinct DNA binding sites, each of which can bind one strand of DNA at a stoichiometry of three nucleotides per RecA monomer. The structural and hydrodynamic properties of the complexes are found to depend on the number of strands bound and on sequence complementarity among the strands. For example, RecA-[homopolymer]₃-ATPγS complexes aggregate when either of the strands bound in sites I and II is complementary to the strand bound in site III. We have also studied the RecA catalysed annealing of complementary homopolymers and find it to be most efficient when two strands of one homopolymer are bound per RecA filament prior to the addition of the complementary homopolymer. These results suggest that a DNA strand bound in site III can base-pair with either of the strands in sites I and II, whereas the latter strands are unable to base-pair with each other.     

Competitive Binding Between Unmodified and Etheno DNA Provides Information About Structure and Stoichiometry of RECA-DNA Complexes

Abstract

RecA is found to form three different complexes with single-stranded DNA with stoichiometries of 3, 6, and  9 nucleotides per RecA monomer. In the first two complexes the DNA bases are oriented preferentially perpendicular to the fiber axis of the complex. The second complex is shown to involve two different DNA strands.     

The regulation mechanism of the C-terminus of RecA proteins during DNA strand-exchange process

The C-terminus of Escherichia coli RecA protein can affect the DNA binding affinity, interact with accessory proteins, and regulate the RecA activity. A substantial upward shift in the pH-reaction profile of RecA-mediated DNA strand-exchange reactions was observed for C-terminal-truncated E. coli ΔC17 RecA, Deinococcus radiodurans RecA, and Deinococcus ficus RecA. Here, the process of RecA-mediated strand exchange from the beginning to the end was investigated with florescence resonance energy transfer and tethered particle motion experiments to determine the detailed regulation mechanism. RecA proteins with a shorter C-terminus possess more stable nuclei, higher DNA binding affinities, and lower protonation requirements for the formation of nucleoprotein filaments. Moreover, more stable synaptic complexes in the homologous sequence searching process were also observed for RecA proteins with a shorter C-terminus. Our results suggest that the C-terminus of RecA proteins regulates not only the formation of RecA nucleoprotein filaments but also the entrance of secondary DNA into RecA nucleoprotein filaments.     

Dynamic structures of Bacillus subtilis RecN-DNA complexes

Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end-processing of DNA double-strand ends via homologous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN–ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN–ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the overhangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA–DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3′-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search.     

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).     

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.     

Co-ordination of multiple DNA molecules in RecA fiber evidenced by linear dichroism spectroscopy

Polarized light spectroscopy has been used to study the interaction of RecA protein with DNA. Several different DNA complexes have been identified and characterized with respect to stoichiometries, base orientation and nuclease accessibility. By using spectroscopically distinguishable DNAs, we determined the number of DNA molecules co-ordinated by the RecA fiber in each of these complexes, and established their base pairing abilities. Based on these observations, we discuss the molecular mechanism of the RecA-mediated strand exchange reaction.     

Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS

The filament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPgammaS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA::ATPgammaS::ssDNA were compared by small angle neutron scattering. A model was developed that allowed for an analytical solution for small angle scattering on a long helical filament, making it possible to obtain the helical pitch and the mean diameter of the protein filament from the scattering curves. The results suggest that the structure of the filaments formed by these two RecA proteins, and particularly their complexes with ATPgammaS, is conservative.     

Investigation of RecA--polynucleotide interactions from the measurement of LexA repressor cleavage kinetics. Presence of different types of complex

The proteolysis of the LexA repressor in the presence of RecA and various polynucleotides was studied by measuring the fluorescence decrease of LexA upon cleavage. The results were compared with the DNA binding of RecA to investigate the presence of multiple DNA-RecA complexes. All single-stranded polydeoxyribonucleotides (DNA) efficiently stimulated the proteolysis and the maximum activation was reached in the presence of three or four nucleotides of polynucleotide per monomer of RecA. The stimulative effect was decreased in the presence of larger amounts of poly(dA), poly(dT) or heat-denatured DNA, whereas the excess of single-stranded DNAs chemically modified with chloroacetaldehyde did not present such an inhibitory effect, despite the fact that a second DNA molecule is likely to interact with RecA as monitored by the intrinsic fluorescence of these DNA species. The complicated cleavage promotion and inhibition pattern is tentatively explained by a three-state model assuming that RecA may interact with three single-stranded DNA molecules. According to this model, occupation of the first site would be necessary and sufficient for cleavage promotion, the second site would be neutral with respect to cleavage and the occupation of the third site would inhibit LexA cleavage at least partially. Double-stranded natural DNA did not stimulate cleavage, even under conditions where RecA binds quantitatively to the DNA. No polyribonucleotides (RNA) examined showed a significant stimulative effect either, nor did they appear to interact with RecA.     

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.