Homology-dependent changes in adenosine 5'-triphosphate hydrolysis during recA protein promoted DNA strand exchange: evidence for long paranemic complexes

As a first step in DNA strand exchange, recA protein forms a filamentous complex on single-stranded DNA (ssDNA), which contains stoichiometric (one recA monomer per four nucleotides) amounts of recA protein. recA protein monomers within this complex hydrolyze ATP with a turnover number of 25 min-1. Upon introduction of linear homologous duplex DNA to initiate strand exchange, this rate of ATP hydrolysis drops by 33%. The decrease in rate is complete in less than 2 min, and the rate of ATP hydrolysis then remains constant during and subsequent to the strand exchange reaction. This drop is completely dependent upon homology in the duplex DNA. In addition, the magnitude of the drop is linearly dependent upon the length of the homologous region in the linear duplex DNA. Linear DNA substrates in which pairing is topologically restricted to a paranemic joint also follow this relationship. Taken together, these properties imply that all of the available homology in the incoming duplex DNA is detected very early in the DNA strand exchange reaction, with the linear duplex DNA paired paranemically with the homologous ssDNA in the complex throughout its length. The results indicate that paranemic joints can extend over thousands of base pairs. We note elsewhere [Pugh, B. F., & Cox, M. M. (1987b) J. Biol. Chem. 262, 1337-1343] that this duplex acquires resistance to digestion by DNase with a much slower time course (30 min), which parallels the progress of strand exchange. Together these results imply that the duplex DNA is paired with the ssDNA but remains outside the nucleoprotein filament. Finally, the results also support the notion that ATP hydrolysis occurs throughout the recA nucleoprotein filament.     

RecA protein promoted homologous pairing in vitro. Pairing between linear duplex DNA bound to HU Protein (nucleosome cores) and nucleoprotein filaments of recA protein-single-stranded DNA

RecA protein promotes two distinct types of synaptic structures between circular single strands and duplex DNA; paranemic joints, where true intertwining of paired strands is prohibited and the classically intertwined plectonemic form of heteroduplex DNA. Paranemic joints are less stable than plectonemic joints and are believed to be the precursors for the formation of plectonemic joints. We present evidence that under strand exchange conditions the binding of HU protein, from Escherichia coli, to duplex DNA differentially affects homologous pairing in vitro. This conclusion is based on the observation that the formation of paranemic joint molecules was not affected, whereas the formation of plectonemic joint molecules was inhibited from the start of the reaction. Furthermore, introduction of HU protein into an ongoing reaction stalls further increase in the rate of the reaction. By contrast, binding of HU protein to circular single strands has neither stimulatory nor inhibitory effect. Since the formation of paranemic joint molecules is believed to generate positive supercoiling in the duplex DNA, we have examined the ability of positive superhelical DNA to serve as a template in the formation of paranemic joint molecules. The inert positively supercoiled DNA could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. Taken collectively, these results indicate that the structural features of the bacterial chromosome which include DNA supercoiling and organization of DNA into nucleosome-like structures by HU protein modulate homologous pairing promoted by the nucleoprotein filaments of recA protein single-stranded DNA.     

Patterns of nuclease protection during strand exchange. recA protein forms heteroduplex DNA by binding to strands of the same polarity

recA protein, in the presence of ATP, polymerizes on single-stranded DNA (plus strand) to form a presynaptic nucleoprotein filament that pairs with linear duplex DNA and actively displaces the plus strand from the recipient molecule in a polarized fashion to form a new heteroduplex molecule. The interaction between recA protein and DNA during strand exchange was studied by labeling different strands and probing the intermediate with pancreatic deoxyribonuclease I (DNase I) or restriction endonuclease. The incoming single strand was resistant to DNase I in the original nucleoprotein filament and remained resistant even after extensive strand exchange had occurred. Both strands of the parental duplex molecule were sensitive to DNase I in the absence of joint molecule formation; but as strand exchange progressed following homologous pairing, increasing stretches of the parental plus strand became resistant, whereas the complementary parental minus strand remained sensitive to DNase I throughout the reaction. Except for a region of 50-100 base pairs at the end of the newly formed heteroduplex DNA where strand exchange was initiated, the rest of the heteroduplex region was resistant to cleavage by restriction endonucleases. The data suggest that recA protein promotes strand exchange by binding both the incoming and outgoing strands of the same polarity, whereas the complementary strand, which must switch pairing partners, is unhindered by direct contact with the protein.     

Synapsis and the formation of paranemic joints by E. coli RecA protein

E. coli RecA protein promotes the homologous pairing of a single strand with duplex DNA even when certain features of the substrates, such as circularity, prohibit the true intertwining of the newly paired strands. The formation of such nonintertwined or paranemic joints does not require superhelicity, and indeed can occur with relaxed closed circular DNA. E. coli topoisomerase I can intertwine the incoming single strand in the paranemic joint with its complement, thereby topologically linking single-stranded DNA to all of the duplex molecules in the reaction mixture. The efficiency of formation of paranemic joints, the time course, and estimates of their length, all suggest that they represent true synaptic intermediates in the pairing reaction promoted by RecA protein.     

Putative three-stranded DNA pairing intermediate in recA protein-mediated DNA strand exchange: no role for guanine N-7.

As an early step in DNA strand exchange reactions, the recA protein aligns homologous sequences within two DNA molecules to form a putative triple-stranded intermediate. In virtually all models for three-stranded DNA proposed to date, hydrogen bonds involving the N-7 position of guanine have played a prominent structural role. To determine whether the N-7 position of guanine is required for triple helix and heteroduplex formation in the recA protein-mediated DNA pairing reaction, guanine was completely replaced by the base analog 7-deazaguanine in both strands of the duplex DNA substrate using polymerase chain reaction. This modified double-strand DNA was reacted with unmodified single-strand DNA in vitro. The 7-deazaguanine-substituted DNA functioned as well as the unsubstituted DNA in recA protein-mediated DNA three-strand exchange reactions. Strand exchange reactions involving four strands also proceeded normally when three of the four strands contained 7-deazaguanine rather than guanine. In fact, the rate of strand exchange improved somewhat when the modified DNA substrates were used. This indicates either that the N-7 position of guanine is not essential for the formation of the putative triple-stranded DNA pairing intermediate, or that a three-stranded (or four-stranded) structure is not an obligate intermediate in recA protein-mediated DNA strand exchange.     

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.     

On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. II. Four-strand exchanges.

RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski et al., 1990), calling into question the role of ATP hydrolysis in this reaction. We demonstrate here that the ATP gamma S-mediated process is restricted to homologous strand exchange reactions involving three strands. In four-strand exchanges between a gapped duplex circle and a second linear duplex, joint molecules are formed in the gap but are not extended into the four-strand region when ATP gamma S is present. This result provides evidence that one function of ATP hydrolysis in the recA system is to facilitate reciprocal DNA strand exchange involving four strands. Implications with respect to the role of four-stranded pairing intermediates and the mechanistic relationship between three- and four-strand exchange reactions are discussed.     

DNase protection by recA protein during strand exchange. Asymmetric protection of the Holliday structure

When recA protein pairs linear duplex DNA with a homologous duplex molecule that has a single-stranded tail, it produces a recombination intermediate called the Holliday structure and causes reciprocal or symmetric strand exchange, whereas the pairing of a linear duplex molecule with fully single-stranded DNA leads to an asymmetric exchange. To study the location of recA protein on DNA molecules undergoing symmetric exchange, we labeled individually each end of the four strands involved and looked for protection against DNase I or restriction endonucleases. As expected, because of its preferred binding to single-stranded DNA, recA protein protected the single-stranded tails of either substrates, or products. In addition however, strong protection extended into the newly formed heteroduplex DNA along the strand to which recA protein was initially bound. Experiments with uniformly labeled DNA showed a corresponding homology-dependent asymmetry in the protection of the tailed substrate versus its fully duplex partner. Restriction experiments showed that protection extended 50-75 base pairs beyond the point where strand exchange was blocked by a long region of heterology. When compared with earlier observations (Chow, S. A., Honigberg, S. M., Bainton, R. J., and Radding, C. M. (1986) J. Biol. Chem. 261, 6961-6971), the present experiments reveal a pattern of association of recA protein with DNA that suggests a common mechanism of asymmetric and symmetric strand exchange.     

Reversibility of strand invasion promoted by recA protein and its inhibition by Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein

When recA protein promotes homologous pairing and strand exchange involving circular single strands and linear duplex DNA, the protein first polymerizes on the single-stranded DNA to form a nucleoprotein filament which then binds naked duplex DNA to form nucleoprotein networks, the existence of which is independent of homology, but requires the continued presence of recA protein (Tsang, S. S., Chow, S. A., and Radding, C. M. (1985) Biochemistry 24, 3226-3232). Further experiments revealed that within a few minutes after the beginning of homologous pairing and strand exchange, these networks began to be replaced by a distinct set of networks with inverse properties: their formation depended upon homology, but they survived removal of recA protein by a variety of treatments. Formation of this second kind of network required that homology be present specifically at the end of the linear duplex molecule from which strand exchange begins. Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein largely suppressed the formation of this second population of networks by inactivating the newly formed heteroduplex DNA, which, however, could be reactivated when recA protein was dissociated by incubation at 0 degrees C. We interpret these observations as evidence of reinitiation of strand invasion when recA protein acts in the absence of auxiliary helix-destabilizing proteins. These observations indicate that the nature of the nucleoprotein products of strand exchange determines whether pairing and strand exchange are reversible or not, and they further suggest a new explanation for the way in which E. coli single-stranded DNA-binding protein and gene 32 protein accelerate the apparent forward rate of strand exchange promoted by recA protein, namely by suppressing initiation of the reverse reaction.     

Separation of the presynaptic and synaptic phases of homologous pairing promoted by recA protein

Homologous pairing of single strands with duplex DNA promoted by recA protein occurred without a lag only when the protein was preincubated with ATP and single-stranded DNA. The rate-limiting presynaptic interaction of recA protein and single strands showed a high temperature coefficient: it proceeded 30 times more slowly at 30 degrees C than at 37 degrees C, whereas synapsis showed a normal temperature coefficient. Thus, the presynaptic phase could be separated experimentally from the rest of the reaction by preincubation of single strands with recA protein and ATP at 37 degrees C, followed by a shift to 30 degrees C before double-stranded DNA was added. The presynaptic phase was an order of magnitude more sensitive to inhibition by ADP than was subsequent strand exchange. Presynaptic complexes that were formed at 37 degrees C decayed only slowly at 30 degrees C, but Escherichia coli single strand binding protein caused complexes to form rapidly at 30 degrees C which indicates that single strand binding protein accelerated the rate of formation of complexes. Preincubation synchronized the initial pairing reaction, and further revealed the rapid formation of nascent heteroduplex DNA 250-300 base pairs in length.     

The mechanics of winding and unwinding helices in recombination: torsional stress associated with strand transfer promoted by RecA protein

Homologous recombination usually involves the production of heteroduplex DNA, DNA containing strands contributed from two different duplexes. RecA protein of E. coli can promote the formation of heteroduplex DNA in vitro by the exchange of DNA strands between two helical structures, duplex DNA and a helical recA nucleoprotein filament containing a single strand of DNA. Complete unwinding of the parental duplex and the rewinding of one strand with a new complement requires rotation of the helical structures about one another, or about their respective longitudinal axes. The observations described here demonstrate an association of torsional stress with strand exchange, and suggest that exchange is accomplished principally by concomitant rotation of duplex DNA and the recA nucleoprotein filament, each about its longitudinal axis.     

Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA.

The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected. The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction. The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA. We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination.     

Concerted strand exchange and formation of Holliday structures by E. coli RecA protein

RecA protein makes stable joint molecules from fully duplex DNA and molecules that are partially single-stranded; the latter may be either duplex molecules with an internal gap in one strand or molecules with single-stranded ends. Stable joint molecules form only when the end of at least one strand is in a homologous region. When RecA protein pairs linear duplex molecules and tailed molecules that share the same sequence end to end, the joints, which are located away from the single-stranded tails in most instances, have the electron microscopic appearance associated with the Holliday structure resulting from the reciprocal exchange of strands. The reaction leading to reciprocal strand exchange involves the concerted displacement of a strand from the end of the duplex molecule. These observations support the view that RecA protein makes stable joint molecules only by transferring strands and not by the side-by-side pairing of duplex regions.     

An unpaired 3' terminus stimulates recA protein-promoted DNA strand exchange

In the presence of 100 mM NaCl, the efficient exchange of strands between a circular single strand and an homologous DNA duplex promoted by the recA and single-stranded DNA binding proteins of Escherichia coli requires an unpaired 3' terminus. Of the duplex DNAs tested, only those with 4 unpaired bases at the 3' termini are effective. Without added NaCl, strand exchange proceeds efficiently with all duplex DNA termini examined including a nicked circular duplex. Thus, at approximately physiological salt concentrations, factors in addition to the recA and single-stranded DNA binding proteins are needed to promote efficient strand exchange. One such factor may be a DNA helicase(s).     

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.     

Homopurine and homopyrimidine strands complementary in parallel orientation form an antiparallel duplex at neutral pH with A-C,G-T,and T-C mismatched base pairs

DNA sequences d-TGAGGAAAGAAGGT (a 14-mer) and d-CTCCTTTCTTCC (a 12-mer) are complementary in parallel orientation forming either Donahue (reverse Watson-Crick) base pairing at neutral pH or Hoogsteen base pairing at slightly acidic pH. The structure of the complex formed by dissolving the two strands in equimolar ratio in water has been investigated by nmr. At neutral pH, the system forms an ordered antiparallel duplex with five A : T and four G : C Watson-Crick base pairs and three mismatches, namely G-T, A-C, and T-C. The nuclear Overhauser effect cross-peak pattern suggests an overall B-DNA conformation with major structural perturbations near the mismatches. The duplex has a low melting point and dissociates directly into single strands with a broad melting profile. The hydrogen-bonding schemes in the mismatched base pairs have been investigated. It has been shown earlier that in acidic pH, the system prefers a triple-stranded structure with two pyrimidine strands and one purine strand. One of the pyrimidine strands has protonated cytosines, forms Hoogsteen base pairing, and is aligned parallel to the purine strand; the other has nonprotonated cytosines and has base-pairing scheme similar to the one discussed in this paper. The parallel duplex is therefore less stable than either the antiparallel duplex or the triplex, in spite of its perfect complementarity. © 1997 John Wiley & Sons, Inc. Biopoly 41: 773–784, 1997     

The formation of plectonemic joints by the recA protein of Escherichia coli. Requirement for ATP hydrolysis

Formation of D-loops during the exchange of strands between a circular single-stranded DNA and a completely homologous linear duplex proceeds optimally when the duplex DNA is added to the complex of recA protein and single-stranded DNA formed in the presence of single-stranded DNA-binding protein and ATP. D-loops are undetectable when 200 microM adenosine 5'-O-(thiotriphosphate) is substituted for ATP. D-loops can be formed in the presence of adenosine 5'-O-(thiotriphosphate) if recA protein is the last component added to the reaction. However, these D-loops, which depend upon homologous sequences, are unstable upon deproteinization and are formed to a more limited extent than the structures formed with ATP. This finding indicates that D-loops formed under these conditions may be largely nonintertwined paranemic structures rather than plectonemic structures in which two of the strands are interwoven. When adenosine 5'-O-(thiotriphosphate) is added to an ongoing reaction containing ATP, formation of plectonemic structures and ATP hydrolysis is inhibited to an equivalent extent. We, therefore, conclude that ATP hydrolysis is required for the formation of plectonemic structures.     

Homologous pairing in duplex DNA regions and the formation of four-stranded paranemic joints promoted by RecA protein. Effects of gap length and negative superhelicity

RecA protein catalyzes homologous pairing of partially single-stranded duplex DNA and fully duplex DNA to form stable joint molecules. We constructed circular duplex DNA with various defined gap lengths and studied the pairing reaction between the gapped substrate with fully double-stranded DNA. The reaction required a stoichiometric amount of RecA protein, and the optimal reaction was achieved at a ratio of 1 RecA monomer per 4 base pairs. The length of the gap, ranging from 141 to 1158 nucleotides, had little effect on the efficiency of homologous pairing. By using a circular gapped duplex DNA prepared from the chimeric phage M13Gori1, we were able to show the formation of nonintertwined or paranemic joints in duplex regions between the gapped and fully duplex molecules. The formation of such paranemic joints occurred efficiently and included nearly all of the DNA in the reaction mixture. The reaction required negative superhelicity, and pairing was greatly reduced with linear or nicked circular DNA. We conclude that one functional role of the single-stranded gap is for facilitating the binding of RecA protein to the duplex region of the gapped DNA. Once the nucleoprotein filament is formed, homologous pairing between the gapped and fully duplex DNA can take place anywhere along the length of the nucleoprotein complex.     

Production of triple-stranded recombination intermediates by RecA protein, in vitro

During the directional strand exchange that is promoted by RecA protein between linear duplex DNA and circular single-stranded DNA, a triple-stranded DNA intermediate was formed and persisted even after the completion of strand transfer followed by deproteinization. In the deproteinized three-stranded DNA complexes, the sequestered linear third strand resisted digestion by E coli exonuclease I. In relation to polarity of strand exchange which defines the proximal and distal ends of the duplex DNA, when homology was restricted to the distal region of duplex substrate, the joints formed efficiently and were stable even upon complete deproteinization. Enzymatic probing of deproteinized distal joints with nuclease P1 revealed that the joints consist of long three-stranded structures that at neutral pH lack significant single-stranded character in any of the three strands. Instead of circular single-stranded DNA, when a linear single strand is recombined with partially homologous duplex DNA, in the presence of SSB, the formation of homologous joints by RecA protein, is significantly more efficient at distal end than at the proximal. Taken together, these observations suggest that with any single-stranded DNA (circular or linear), RecA protein efficiently promotes the formation of distal joints, from which, however, authentic strand exchange may not occur. Moreover, these joints might represent an intermediate which is trapped into a stable triple stranded state.     

Three-stranded paranemic joints: architecture, topological constraints and movement

The RecA and SSB proteins will catalyze the joining of two DNA molecules containing homologous sequences but lacking homologous ends in a reaction termed paranemic joining. The absence of homologous ends can be achieved by (1) pairing two circular DNAs or (2) using linear DNA(s) with ends lacking homology to the pairing partner. Here we have used electron microscopy (EM) to examine such pairings. Circular M13 single-stranded (ss) DNA enveloped by RecA protein into a presynaptic filament was paired with linear M13mp7 double-stranded (ds) DNA containing non-M13 sequences at its ends. Joint complexes were frequently seen in which the dsDNA was joined with the presynaptic filament over several kilobase (10(3) bases) lengths of the dsDNA. In this region, the presynaptic filament appeared disorganized as contrasted to the customary helical structure of the filament containing only a single strand of DNA. The same ultrastructure, but with greater detail, was observed when the samples were prepared for EM without fixation using a new method of fast-freezing and freeze-drying. EM immunogold staining demonstrated the presence of SSB protein in the disorganized region containing all three strands, but not in the regular helically arranged region. Psoralen photo-crosslinking of the DNA in the joint complexes revealed that the three DNA strands were in close proximity only over a single short (200 to 300 base-pairs) region. The joining of nicked circular M13 dsDNA and presynaptic filaments containing circular M13 ssDNA resulted in the intertwining of the dsDNA about the circular presynaptic filament. The joints produced in this case were short, as was the single region of psoralen photo-crosslinking of the three DNA strands. A model of how these long three-stranded joints form is presented involving the movement of a short "true" paranemic joint along the presynaptic filament.