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.     

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.     

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.     

Dissociation pathway for recA nucleoprotein filaments formed on linear duplex DNA

recA protein forms stable filaments on duplex DNA at low pH. When the pH is shifted above 6.8, recA protein remains stably bound to nicked circular DNA, but not to linear DNA. Dissociation of recA protein from linear duplex DNA proceeds to a non-zero endpoint. The kinetics and final extent of dissociation vary with several experimental parameters. The instability on linear DNA is most readily explained by a progressive unidirectional dissociation of recA protein from one end of the filament. Dissociation of recA protein from random points in the filament is eliminated as a possible mechanism by several observations: (1) the requirement for a free end; (2) the inverse and linear dependence of the rate of dissociation on DNA length (at constant DNA base-pair concentration); and (3) the kinetics of exposure of a restriction endonuclease site in the middle of the DNA. Evidence against another possible mechanism, ATP-mediated translocation of the filament along the DNA, is provided by a novel effect of the non-hydrolyzable ATP analog, ATP gamma S, which generally induces recA protein to bind any DNA tightly and completely inhibits ATP hydrolysis. We find that very low, sub-saturating levels of ATP gamma S completely stabilize the filament, while most of the ATP hydrolysis continues. If these levels of ATP gamma S are introduced after dissociation has commenced, further dissociation is blocked, but re-association does not occur. These observations are inconsistent with movement of recA protein along DNA that is tightly coupled to ATP hydrolysis. The recA nucleoprotein filament is polar and the protein binds the two strands asymmetrically, polymerizing mainly in the 5' to 3' direction on the initiating strand of a single-stranded DNA tailed duplex molecule. A model consistent with these results is presented.     

The pairing activity of stable nucleoprotein filaments made from recA protein, single-stranded DNA, and adenosine 5'-(gamma-thio)triphosphate

Under conditions that diminish secondary structure in single-stranded DNA, stable presynaptic filaments can be formed by recA protein in the presence of the nonhydrolyzable analog ATP gamma S, without the need for Escherichia coli single strand binding protein. Such stable presynaptic filaments resemble those formed in the presence of ATP and pair efficiently with homologous duplex DNA. Since this kind of stable filament does not displace a strand from the duplex molecule, it provides a model substrate to study synapsis independent of the earlier and later stages of the recA reaction. Even though detectable strand displacement did not occur in the presence of ATP gamma S, both single strand and double strand breaks in duplex DNA stimulated homologous pairing. These and related observations support the view that the presynaptic nucleoprotein filament and naked duplex DNA intertwine to form a nascent joint in which the duplex DNA is partially unwound, i.e. in which the pitch of the involved duplex segment is reduced.     

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.     

Assembly and disassembly of RecA protein filaments occur at opposite filament ends. Relationship to DNA strand exchange

RecA protein primarily associates with and dissociates from opposite ends of nucleoprotein filaments formed on linear duplex DNA. RecA nucleoprotein filaments that are hydrolyzing ATP therefore engage in a dynamic process under some conditions that has some of the properties of treadmilling. We have also investigated whether the net polymerization of recA protein at one end of the filament and/or a net depolymerization at the other end drives unidirectional strand exchange. There is no demonstrable correlation between recA protein association/dissociation and the strand exchange reaction. RecA protein-mediated DNA strand exchange is affected minimally by changes in reaction conditions (dilution, pH shift, or addition of small amounts of adenosine-5'-O-(3-thiotriphosphate) that have large and demonstrable effects on recA protein association, dissociation, or both. Rather than driving strand exchange, these assembly and disassembly processes may simply represent the mechanism by which recA nucleoprotein filaments are recycled in the cell.     

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.     

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.     

Topological testing of the mechanism of homology search promoted by RecA protein

To initiate homologous recombination, sequence similarity between two DNA molecules must be searched for and homology recognized. How the search for and recognition of homology occurs remains unproven. We have examined the influences of DNA topology and the polarity of RecA–single-stranded (ss)DNA filaments on the formation of synaptic complexes promoted by RecA. Using two complementary methods and various ssDNA and duplex DNA molecules as substrates, we demonstrate that topological constraints on a small circular RecA–ssDNA filament prevent it from interwinding with its duplex DNA target at the homologous region. We were unable to detect homologous pairing between a circular RecA–ssDNA filament and its relaxed or supercoiled circular duplex DNA targets. However, the formation of synaptic complexes between an invading linear RecA–ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a triplex structure formed by non-Watson–Crick hydrogen bonding is unlikely to be an intermediate in homology searching promoted by RecA. Rather, a model in which RecA-mediated homology searching requires unwinding of the duplex DNA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA–ssDNA does not affect its ability to pair and interwind with its circular target duplex DNA.     

RecA protein-facilitated DNA strand breaks. A mechanism for bypassing DNA structural barriers during strand exchange

RecA protein promotes an unexpectedly efficient DNA strand exchange between circular single-stranded DNA and duplex DNAs containing short (50-400-base pair) heterologous sequences at the 5' (initiating) end. The major mechanism by which this topological barrier is bypassed involves DNA strand breakage. Breakage is both strand and position specific, occurring almost exclusively in the displaced (+) strand of the duplex within a 15-base pair region of the heterology/homology junction. Breakage also requires recA protein, ATP hydrolysis, and homologous sequences 3' to the heterology. Although the location of the breaks and the observed requirements clearly indicate a major role for recA protein in this phenomenon, the molecular mechanism is not yet clear. The breakage may reflect a DNA structure and/or some form of structural stress within the DNA during recA protein-mediated DNA pairing which either exposes the DNA at this precise position to the action of a contaminating nuclease or induces a direct mechanical break. We also find that when heterology is located at the 3' end of the linear duplex, strand exchange is halted (without DNA breakage) about 500 base pairs from the homology/heterology junction.     

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.     

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.     

On RecA protein-mediated homologous alignment of two DNA molecules. Three strands versus four strands

The recA protein from Escherichia coli can homologously align two duplex DNA molecules; however, this interaction is much less efficient than the alignment of a single strand and a duplex. Three strand paranemic joints are readily detected. In contrast, duplex-duplex pairing is detected only when the incoming (second) duplex is negatively supercoiled, and even here the pairing is inefficient. The recA protein-promoted four strand exchange reaction is initiated in a three strand region, with efficiency increasing with the length of potential three strand pairing available for initiation. This indicates that a paranemic joint involving three DNA strands may be an important intermediate in all recA protein-mediated DNA strand exchange reactions and that the presence of three strands rather than four is a fundamental structural parameter of paranemic joints.     

Stable binding of recA protein to duplex DNA. Unraveling a paradox

recA protein binding to duplex DNA is a complicated, multistep process. The final product of this process is a stably bound complex of recA protein and extensively unwound double-stranded DNA. recA monomers within the complex hydrolyze ATP with an apparent kcat of approximately 19-22 min-1. Once the final binding state is achieved, binding and ATP hydrolysis by this complex becomes pH independent. The weak binding of recA protein to duplex DNA reported in previous studies does not, therefore, reflect an intrinsically unfavorable binding equilibrium. Instead, this apparent weak binding reflects a slow step in the association pathway. The rate-limiting step in this process involves the initiation rather than the propagation of DNA binding and unwinding. This step exhibits no dependence on recA protein concentration at pH 7.5. Extension or propagation of the recA filament is fast relative to the overall process. Initiation of binding is pH dependent and represents a prominent kinetic barrier at pH 7.5. ATP hydrolysis occurs only after the duplex DNA is unwound. The binding density of recA protein on double-stranded DNA is approximately one monomer/4 base pairs. A model for this process is presented. These results provide an explanation for several paradoxical observations about recA protein-promoted DNA strand exchange. In particular, they demonstrate that there is no thermodynamic requirement for dissociation of recA protein from the heteroduplex DNA product of strand exchange.     

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.     

A chimeric Rec-A protein that implicates non-Watson-Crick interactions in homologous pairing.

The helical filament formed by RecA protein on single-stranded DNA plays an important role in homologous recombination and pairs with a complementary single strand or homologous duplex DNA. The RecA nucleoprotein filament also recognizes an identical single strand. The chimeric protein, RecAc38, forms a nucleoprotein filament that recognizes a complementary strand but is defective in recognition of duplex DNA, and is associated with phenotypic defects in repair and recombination. As described here, RecAc38 nucleoprotein filament is also defective in recognition of an identical strand, either when the filament has within it a single strand or duplex DNA. A model that postulates three DNA binding sites rationalizes these observations and suggests that the third binding site mediates non-Watson-Crick interactions that are instrumental in recognition of homology in duplex DNA.     

Exchange of recA protein between adjacent recA protein-single-stranded DNA complexes

We have examined the exchange of recA protein between stable complexes formed with single-stranded DNA (ssDNA) and (a) other complexes and (b) a pool of free recA protein. We have also examined the relationship of ATP hydrolysis to these exchange reactions. Exchange was observed between two different recA X ssDNA complexes in the presence of ATP. Complete equilibration between two sets of complexes occurred with a t1/2 of 3-7 min under a set of conditions previously found to be optimal for recA protein-promoted DNA strand exchange. Approximately 200 ATPs were hydrolyzed for every detected migration of a recA monomer from one complex to another. This exchange occurred primarily between adjacent complexes, however. Little or no exchange was observed between recA X ssDNA complexes and the free recA protein pool, even after several hundred molecules of ATP had been hydrolyzed for every recA monomer present. ATP hydrolysis is not coupled to complete dissociation or association of recA protein from or with recA X ssDNA complexes under these conditions.     

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.     

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

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