recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange.

The recA protein of Escherichia coli promotes the complete exchange of strands between full-length linear duplex and single-stranded circular DNA molecules of bacteriophage phi X-174, converting more than 50% of the single-stranded DNA into heteroduplex replicative form II-like structures. Kinetically, the reaction can be divided into two phases, formation of short heteroduplex regions (D loops) and extension of the D loops via branch migration. recA protein participates directly in both phases. D loops are formed efficiently in the presence of ATP or the nonhydrolyzable ATP analog adenosine 5'-[gamma-thio]triphosphate, whereas D-loop extension requires continuous ATP hydrolysis. Complete strand exchange requires a stoichiometric amount of recA protein and is strongly stimulated by the single-stranded-DNA-binding protein of E. coli.     

Ionic inhibition of formation of RecA nucleoprotein networks blocks homologous pairing.

Conditions that favor the complete coating of single-stranded DNA by RecA protein promote the association of these presynaptic filaments with naked double-stranded DNA to form large nucleoprotein networks before homologous pairing occurs. These RecA nucleoprotein networks sequester virtually all of the DNA in the reaction mixture. Conditions that are suboptimal for the formation of the RecA presynaptic filament rendered both the formation of RecA-DNA networks and the subsequent formation of joint molecules sensitive to inhibition by excess ATP or by pyrophosphate when these were added during synapsis. The rate of homologous pairing was directly related to the degree of inhibition of network formation. Various multivalent cations added during synapsis restored both the formation of networks and the pairing of homologous molecules. These observations support the view that the nucleoprotein network is a synaptic intermediate by means of which RecA protein facilitates the conjunction of DNA molecules and the subsequent processive search for homology. Inhibition by multivalent anions and restoration by multivalent cations suggests in addition, that negative charge repulsion inhibits the binding of naked duplex DNA to presynaptic filaments.     

RecX protein abrogates ATP hydrolysis and strand exchange promoted by RecA: insights into negative regulation of homologous recombination

In many eubacteria, coexpression of recX with recA is essential for attenuation of the deleterious effects of recA overexpression; however, the molecular mechanism has remained enigmatic. Here, we show that Mycobacterium tuberculosis RecX binds directly to M. tuberculosis RecA as well as M. smegmatis and E. coli RecA proteins in vivo and in vitro, but not single-stranded DNA binding protein. The direct association of RecX with RecA failed to regulate the specificity or extent of binding of RecA either to DNA or ATP, ligands that are central to activation of its functions. Significantly, RecX severely impeded ATP hydrolysis and the generation of heteroduplex DNA promoted by homologous, as well as heterologous, RecA proteins. These findings reveal a mode of negative regulation of RecA, and imply that RecX might act as an anti-recombinase to quell inappropriate recombinational repair during normal DNA metabolism.     

RecA.oligonucleotide filaments bind in the minor groove of double-stranded DNA.

Escherichia coli RecA protein, in the presence of ATP or its analog adenosine 5'-[gamma-thio]triphosphate, polymerizes on single-stranded DNA to form nucleoprotein filaments that can then bind to homologous sequences on duplex DNA. The three-stranded joint molecule formed as a result of this binding event is a key intermediate in general recombination. We have used affinity cleavage to examine this three-stranded joint by incorporating a single thymidine-EDTA.Fe (T*) into the oligonucleotide part of the filament. Our analysis of the cleavage patterns from the joint molecule reveals that the nucleoprotein filament binds in the minor groove of an extended Watson-Crick duplex.     

The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA.

A key step in homologous recombination is the alignment and pairing of homologous DNAs. The Escherichia coli RecA protein initiates pairing by binding to single-strand DNA, forming a helical nucleoprotein filament. We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-[gamma-thio]triphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA. RecA can pair as few as eight bases of homology to form such synaptic complexes. The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs. Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search.     

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2' methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2' methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.     

RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins.

We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB). RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA. RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule. At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion. The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo.     

DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.     

Purification and characterization of an activity from Saccharomyces cerevisiae that catalyzes homologous pairing and strand exchange.

An activity that catalyzes the formation of joint molecules from linear M13mp19 replicative form DNA and circular M13mp19 viral DNA was purified 1000- to 2000-fold from mitotic Saccharomyces cerevisiae cells. The activity appeared to reside in a Mr 132,000 polypeptide. The reaction required that the substrates be homologous and also required Mg2+. There was no requirement for ATP. The reaction required stoichiometric amounts of protein and showed a cooperative dependence on protein concentration. Electron microscopic analysis of the joint molecules indicated they were formed by displacement of one strand of the linear duplex by the single-stranded circular molecule. This analysis also showed that heteroduplex formation started at the 3'-homologous end of the linear duplex strand followed by extension of the hybrid region toward the 5'-homologous end of the linear duplex strand (3'-to-5' direction).     

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA

The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.     

Homologous pairing in genetic recombination: complexes of recA protein and DNA.

recA protein, which is essential for general genetic recombination in Escherichia coli, promotes the homologous pairing of single-stranded DNA with double-stranded DNA to form a D loop. The amount of recA protein required for the reaction was directly proportional to the amount of single stranded DNA and was unaffected by similar variations in the amount of double-stranded DNA. The ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), which was not rapidly hydrolyzed by recA protein, blocked the formation of D loops but promoted the formation of stable complexes of recA protein and single-stranded DNA. These complexes, in turn, bound homologous or heterologous double-stranded DNA and partially unwound it. Because ATP gamma S competitively inhibited the ATPase activity of recA protein (Km/Ki approximately 300), we infer that ATP gamma S binds at a site that overlaps the site for ATP and that the functional complexes formed in the presence of the analog probably represent partial steps in the overall reaction. If the complexes formed in the presence of ATP gamma S reflect natural intermediates in the formation of D loops, recA protein must promote homologous pairing either by moving juxtaposed single-stranded and double-stranded DNA relative to one another or by forming and dissociating complexes reiteratively until a homologous match occurs.     

A recombinase from Drosophila melanogaster embryos.

We have partially purified a DNA strand-exchange activity (recombinase) from nuclear extracts of Drosophila melanogaster embryos. The protein fraction forms a joint molecule between a circular single-strand DNA and a homologous linear duplex DNA that is resolved from the substrates by agarose gel electrophoresis. A strand-exchange activity can be obtained from nuclear extracts from embryos as old as 24 hr. The activity is similar to that partially purified from human cells [Hsieh, P., Meyn, S.M. & Camerini-Otero, R.D. (1986) Cell 44, 885-894]. It is homology-dependent, requires Mg2+, appears to be directional in that it prefers to displace the 3' end of the noncomplementary strand, and does not require exogenous ATP. Forty nanograms of protein in the partially purified DNA strand-exchange fraction from D. melanogaster embryos can completely convert 50 ng of substrate single-strand DNA into joint molecules in 10 min. In the electron microscope, joint molecules are seen to consist of a circular single-strand DNA molecule attached to only one end of a linear duplex DNA molecule; a displaced strand is also seen. The region of heteroduplex formation can be as long as 600 base pairs. The demonstration of a strand-exchange activity from wild-type D. melanogaster embryos invites analysis of recombination-defective mutants to explore the role of DNA strand exchange in homologous recombination.     

Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli.

Homogeneous recA protein catalyzes the hybridization of single-stranded DNA to homologous regions in duplex DNA. The products are D-loops, which are formed with equal efficiency in linear and supercoiled molecules. This assimilation reaction can be separated into two partial reactions. In the first, recA protein binds to duplex DNA and produces a reA protein-DNA complex. The binding shows a sigmoidal dependence on recA protein concentration, requires ATP, GTP or the gamma-thio analog of ATP, and Mg2+, but does not require hydrolysis of the nucleoside triphosphate. In the second reaction, single-stranded regions of the recA protein-ATP-duplex DNA intermediate hybridize with free complementary single strands to produce D-loop structures. This reaction is coupled to ATP hydrolysis and is analogous to the renaturation of single-stranded DNA catalyzed by the recA protein [Weinstrock, G.M., McEntee, K. & Lehman, I.R. (1979) Proc. Natl. Acad. Sci. USA 76, 126-130]. Hydrolysis of ATP appears to be required in these reactions for dissociation of recA protein from the DNA.     

Stable three-stranded DNA made by RecA protein.

When RecA protein, in the form of a nucleoprotein filament containing circular single-stranded DNA (plus strand only), reacts with homologous linear duplex DNA, a directional transfer ensues of a strand from the duplex DNA to the nucleoprotein filament, resulting in the displacement of the linear plus strand in the 5' to 3' direction. The initial homologous synapsis, however, can occur at either end of the duplex DNA, or anywhere in between, and when homology is restricted to different regions of the duplex DNA, the joint molecules that form in each region show striking differences in stability upon deproteinization: distal joints greater than proximal joints much greater than medial joints. In the deproteinized distal joints, which are thermostable, 2000 nucleotide residues of the circular plus strand are resistant to P1 nuclease; both strands of the original duplex DNA remain resistant to P1 nuclease, and the potentially displaceable linear plus strand, which has a 3' homologous end, remains resistant to Escherichia coli exonuclease I. These observations suggest that RecA protein promotes homologous pairing and strand exchange via long three-stranded DNA intermediates and, moreover, that, once formed, such triplex structures in natural DNA are stable even when RecA protein has been removed.     

Polar branch migration promoted by recA protein: effect of mismatched base pairs.

Escherichia coli recA protein makes joint molecules from single-stranded circular phage DNA (viral or plus strand) and homologous linear duplex DNA by a polar reaction that displaces the 5' end of the plus strand from the duplex molecule [Kahn, R., Cunningham, R. P., DasGupta, C. & Radding, C. M. (1981) Proc. Natl. Acad. Sci. USA 78, 4786-4790]. Growth of the heteroduplex joint, which results from strand exchange or branch migration, stopped at the borders of regions of nonhomologous DNA that were variously located 145, 630, or 1202 nucleotides from the end. Accumulation of migrating branches at heterologous borders demonstrates that their migration is not the result of random diffusion but is actively driven by recA protein. Growth of the heteroduplex joint was blocked even when a heterologous insertion was located in the single-stranded DNA, a case in which the flexible single-stranded region might conceivably fold out of the way under some condition. The recA protein did not make joint molecules from phage phi X174 and G4DNAs, which are 70% homologous, but did join phage fd and M13DNAs, which are 97% homologous. In the latter case, heteroduplex joints extended through regions containing isolated mismatched base pairs but stopped in a region where the fd and M13 sequences differ by an average of 1 base pair in 10. These results suggest that in genetic recombination the discrimination of perfect or near-perfect homology from a high degree of relatedness may be attributable in part to the mechanism by which recA protein promotes strand transfer.     

Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein.

RecT protein of Escherichia coli promotes the formation of joint molecules between homologous linear double-stranded M13mp19 replicative-form bacteriophage DNA and circular single-stranded M13mp19 DNA in the presence of exonuclease VIII, the recE gene product. The joint molecules were formed by a mechanism involving the pairing of the complementary strand of the linear double-stranded DNA substrate with the circular single-stranded DNA substrate coupled with the displacement of the noncomplementary strand. When the homologous linear double-stranded DNA substrate had homologous 3' or 5' single-stranded tails, then RecT promoted homologous pairing and strand exchange in the absence of exonuclease VIII. Histone H1 could substitute for RecT protein; however, joint molecules formed in the presence of histone H1 did not undergo strand exchange. These results indicate that under the reaction conditions used, the observed strand exchange reaction is promoted by RecT and is not the result of spontaneous branch migration. These results are consistent with the observation that expression of RecE (exonuclease VIII) and RecT substitutes for RecA in some recombination reactions in E. coli.     

RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis.

The polymerization of RecA on individual double-stranded DNA molecules is studied. A linear DNA (lambda DNA, 48.5 Kb), anchored at one end to a cover glass and at the other end to an optically trapped 3-micrometers diameter polystyrene bead, serves as a template. The elongation caused by RecA assembly is measured in the presence of ATP and ATP[gammaS]. By using force extension and hydrodynamic recoil, a value of the persistence length of the RecA-DNA complex is obtained. In the presence of ATP, the polymer length is unstable, first growing to saturation and then decreasing. This suggests a transient dynamics of association and dissociation for RecA on a double-stranded DNA, the process being controlled by ATP hydrolysis. Part of this dynamics is suppressed in the presence of ATP[gammaS], leading to a stabilized RecA-DNA complex. A one-dimensional nucleation and growth model is presented that may account for the protein assembly.     

Torsional stress generated by RecA protein during DNA strand exchange separates strands of a heterologous insert.

Previous studies have shown that the helical RecA nucleoprotein filament formed on a circular single strand of DNA causes the progressive, directional transfer of a complementary strand from naked linear duplex DNA to the nucleoprotein filament, even when the duplex contains a sizable heterologous insertion. Since RecA protein lacks demonstrable helicase activity, the mechanism by which it pushes strand exchange through long heterologous inserts has been a quandary. In the present study, a linear duplex substrate with an insertion of 110 base pairs in its middle yielded the expected products, whereas much less of the heteroduplex product was seen when the insertion was located at either end of the duplex substrate or 160 base pairs from the far end of the duplex substrate. In an ongoing reaction of the substrate with an insertion in its middle, P1 nuclease cleaved intermediates from the point of the insertion to various distal sites. Acting on a duplex substrate that contained a single nick located in the complementary strand just beyond the insertion, RecA protein formed joint molecules but failed to complete strand exchange. These data show that negative torsional stress is generated by distant homologous interactions that occur beyond the heterologous insertion and that such stress is essential for unwinding a heterologous insertion that otherwise halts strand exchange.     

Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein.

The Escherichia coli RecF, RecO, and RecR proteins were analyzed for their effect on RecA-mediated pairing of single-stranded circular DNA and homologous linear duplex DNA substrates. As shown by other workers, joint molecule formation by RecA was inhibited by E. coli single-stranded DNA binding protein (SSB) when it was added to single-stranded DNA before RecA. This inhibitory effect was overcome by the addition of RecO and RecR or RecF, RecO, and RecR. Both the rate and extent of joint molecule formation were restored to the maximal level observed when SSB was added after RecA. RecF, RecO, and RecR proteins had no effect on the conversion of joint molecules to final products and only appeared to stimulate an early step in the pairing reaction. The stimulatory effect of RecF, RecO, and RecR was not seen without SSB or when SSB was added after RecA. RecF protein by itself inhibited reactions in mixtures containing RecA and SSB, and this inhibition was overcome by the addition of RecO and RecR. These data suggest that RecO and RecR, and possibly RecF, help RecA overcome inhibition by SSB and utilize SSB-single-stranded-DNA complexes as substrates.     

Genetic recombination in Escherichia coli: Holliday junctions made by RecA protein are resolved by fractionated cell-free extracts.

Escherichia coli RecA protein catalyzes reciprocal strand-exchange reactions between duplex DNA molecules, provided that one contains a single-stranded gap or tail, to form recombination intermediates containing Holliday junctions. Recombination reactions are thought to occur within helical RecA-nucleoprotein filaments in which DNA molecules are interwound. Structures generated in vitro by RecA protein have been used to detect an activity from fractionated E. coli extracts that resolves the intermediates into heteroduplex recombinant products. Resolution occurs by specific endonucleolytic cleavage at the Holliday junction. The products of cleavage are characteristic of patch and splice recombinants.