Homologous pairing and topological linkage of DNA molecules by combined action of E. coli recA protein and topoisomerase I

E. coil RecA protein and topolsomerase I, acting on superhelical DNA and circular single strands in the presence of ATP and Mg²⁺, topologically link single-stranded molecules to one another, and single-stranded molecules to duplex DNA. When super-helical DNA is relaxed by prior incubation with topoisomerase, it is a poor substrate for catenation. Extensive homology stimulates the catenation of circular single-stranded DNA and superhelical DNA, whereas little reaction occurs between these forms of the closely related DNAs of phages φX174 and G4, indicating that, in conjunction with topoisomerase I, RecA protein can discriminate perfect or nearly perfect homology from a high degree of relatedness. Circular single-stranded G4 DNA reacts with superhelical DNA of a chimeric phage, M13Goril, to form catenanes, at least half of which survive heating at 80°C following restriction cleavage in the M13 region, but few of which survive following restriction cleavage in the G4 region. Electron microscopic examination of catenated molecules cleaved in the M13 region reveals that in most cases the single-stranded G4 DNA is joined to the linear duplex M13(G4) DNA in the homologous G4 region. The junction frequently has the appearance of a D loop, with an extent equivalent to 100 or more bp. We conclude that a significant fraction of catenanes were hemicatenanes, in which the single-stranded circle was topologically linked, probably by multiple turns, to its complementary strand in the duplex DNA. These observations support the previous conclusion that RecA protein can pair a single strand with its complementary strand in duplex DNA in a side-by-side fashion without a free end in any of the three strands.     

Synapsis promoted by Ustilago rec1 protein

Ustilago rec 1 protein pairs homologous DNA molecules by promoting both synapsis and strand transfer. Complexes formed with rec 1 protein and a homologous combination of single-stranded and duplex DNA that appear to be synaptic structures can be detected by use of a nitrocellulose filter assay. The nascent heteroduplex formed during synapsis is a paranemic joint in which the single-stranded DNA pairs, but does not interwind, with its complement in the duplex molecule. Formation of the paranemic joint is accompanied by duplex unwinding and genesis of left-handed Z-DNA.     

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.     

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.     

On the mechanism of pairing of single- and double-stranded DNA molecules by the recA and single-stranded DNA-binding proteins of Escherichia coli

The pairing of single- and double-stranded DNA molecules at homologous sequences promoted by recA and single-stranded DNA-binding proteins of Escherichia coli follows apparent first-order kinetics. The initial rate and first-order rate constant for the reaction are maximal at approximately 1 recA protein/3 and 1 single-stranded DNA-binding protein/8 nucleotides of single-stranded DNA. The initial rate increases with the concentration of duplex DNA; however, the rate constant is independent of duplex DNA concentration. Both the rate constant and extent of reaction increase linearly with increasing length of duplex DNA over the range 366 to 8623 base pairs. In contrast, the rate constant is independent of the size of the circular single-stranded DNA between 6,400 and 10,100 nucleotides. No significant effect on reaction rate is observed when a single-stranded DNA is paired with 477 base pairs of homologous duplex DNA joined to increasing lengths of heterologous DNA (627-2,367 base pairs). Similarly, heterologous T7 DNA has no effect on the rate of pairing. These findings support a mechanism in which a recA protein-single-stranded DNA complex interacts with the duplex DNA to produce an intermediate in which the two DNA molecules are aligned at homologous sequences. Conversion of the intermediate to a paranemic joint then occurs in a rate-determining unimolecular process.     

RecA-mediated strand exchange reactions between duplex DNA molecules containing damaged bases, deletions, and insertions

RecA protein from Escherichia coli promotes homologous pairing and strand exchange between duplex DNA molecules if one is partially single-stranded. Using linear duplexes and circles with a single-stranded gap as the substrates, this reaction generates nicked circular heteroduplex DNA and linear molecules with single-stranded ends. The completion of strand exchange can be demonstrated by the production of nicked circular heteroduplex DNA detected by gel electrophoresis and autoradiography using radiolabeled linear molecules. When the effect of ultraviolet damage to the substrate DNA was tested, strand exchange was found to pass 30 or more pyrimidine dimers in each duplex. In contrast, exchanges were blocked or severely slowed by interstrand cross-links and monoadducts produced by psoralen and 360 nm light. Deletions and insertions of from 4 to 38 base pairs in the DNA substrates had little effect on the production of nicked circular heteroduplex DNA. However, those of 120 base pairs, or greater, reduced the product yield to a level below the threshold of detection. These results contrast with those obtained in related three-stranded reactions (Bianchi, M. E., and Radding, C. M. (1984) Cell 35, 511-520), in which stable heteroduplex products with 500 or 1300 unpaired bases were obtained when the insert was located within a single-stranded circular substrate.     

By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology

RecA protein promotes homologous pairing by a reaction in which the protein first binds stoichiometrically to single-stranded DNA in a slow presyn-aptic step, and then conjoins single-stranded and duplex DNA, thereby forming a ternary complex. RecA protein did not pair molecules that shared only 30 bp homology, but, with full efficiency, it paired circular single-stranded and linear duplex molecules in which homology was limited to 151 bp at one end of the duplex DNA. The initial rate of the pairing reaction was directly related to the length of the heterologous part of the duplex DNA, which we varied from 0 to 3060 base pairs. Since interactions involving the heterologous part of a molecule speed the location of a small homologous region, we conclude that RecA protein promotes homologous alignment by a processive mechanism involving relative motion of conjoined molecules within the ternary complex.     

Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA

Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA.     

Underwinding of DNA associated with duplex-duplex pairing by RecA protein

Homologous pairing between gapped circular and partially homologous form I DNA, catalyzed by Escherichia coli RecA protein, leads to the formation of nascent synaptic joints between regions of duplex DNA. These duplex-duplex interactions result in underwinding of the form I DNA, as detected by a topoisomerase assay. Underwound DNA species have been studied with regard to their formation, stability, and topological requirements. The synaptic joints are short-lived and of low frequency compared with those formed between single-stranded and duplex DNA. Measurement of the degree of underwinding indicates joints 300-400 base pairs in length, in which the two DNA molecules are presumed to be interwound within the RecA-nucleoprotein filament. Underwound DNA was not detected in reactions between gapped DNA and partially homologous nicked circular or relaxed covalently closed DNA. We have also investigated the requirements for the initiation of strand exchange. Previous results have shown that strand exchange requires a homologous 3'-terminus complementary to the gapped region. We now show that the minimum length of overlap required for efficient initiation of strand exchange is one to two turns of DNA within the RecA-DNA nucleoprotein filament.     

Hysteretic regulation of Ustilago rec1 protein during synapsis

rec1 protein slowly loses activity in synaptic pairing reactions. The explanation for this result would appear to be that the overall reaction is limited by a slow transition of the protein to an inactive state. When preincubated with single-stranded DNA and ADP, rec1 protein forms a dead-end complex that is unable to promote homologous pairing. Its pairing activity can be restored by lowering the temperature for several minutes or can be maintained if duplex DNA sharing no homology with the single-stranded DNA is also included. These results suggest a mechanism for attenuating pairing activity during recombination. After recognition of homologous sequences, rec1 protein undergoes a slow, ligand-induced conformational change to an inactive form.     

Translocation of Escherichia coli recA protein from a single-stranded tail to contiguous duplex DNA

Duplex DNA with a contiguous single-stranded tail was nearly as effective as single-stranded DNA in acting as a cofactor for the ATPase activity of recA protein at neutral pH and concentrations of MgCl2 that support homologous pairing. The ATP hydrolysis reached a steady state rate that was proportional to the length of the duplex DNA attached to a short 5' single-stranded tail after a lag. Separation of the single-stranded tail from most of the duplex portion of the molecule by restriction enzyme cleavage led to a gradual decline in ATP hydrolysis. Measurement of the rate of hydrolysis as a function of DNA concentration for both tailed duplex DNA and single-stranded DNA cofactors indicated that the binding site size of recA protein on a duplex DNA lattice, about 4 base pairs, is similar to that on a single-stranded DNA lattice, about four nucleotides. The length of the lag phase preceding steady state hydrolysis depended on the DNA concentration, length of the duplex region, and the polarity of the single-stranded tail, but was comparatively independent of tail length for tails over 70 nucleotides in length. The lag was 5-10 times longer for 3' than for 5' single-stranded tailed duplex DNA molecules, whereas the steady state rates of hydrolysis were lower. These observations show that, after nucleation of a recA protein complex on the single-stranded tail, the protein samples the entire duplex region via an interaction that is labile and not strongly polarized.     

Unwinding of heterologous DNA by RecA protein during the search for homologous sequences.

The search for homologous sequences promoted by RecA protein in vitro involves a presynaptic filament and naked duplex DNA, the multiple contacts of which produce nucleoprotein networks or coaggregates. The single-stranded DNA within the presynaptic filaments, however, is extended to an axial spacing 1.5 times that of B-form DNA. To investigate this paradoxical difference between the spacing of bases in the RecA presynaptic filament versus the target duplex DNA, we explored the effect of heterologous contacts on the conformation of DNA, and vice versa. In the presence of wheat germ topoisomerase I, RecA presynaptic filaments induced a rapid, limited reduction in the linking number of heterologous circular duplex DNA. This limited unwinding of heterologous duplex DNA, termed heterologous unwinding, was detected within 30 seconds and reached a steady state within a few minutes. Presynaptic filaments that were formed in the presence of ATP gamma S and separated from free RecA protein by gel filtration also generated a ladder of topoisomers upon incubation with relaxed duplex DNA and topoisomerase. The inhibition of heterologous contacts by 60 mM-NaCl or 5 mM-ADP resulted in a corresponding decrease in heterologous unwinding. In reciprocal fashion, the stability or number of heterologous contacts with presynaptic filaments was inversely related to the linking number of circular duplex DNA. These observations show that heterologous contacts with the presynaptic filament cause a limited unwinding of the duplex DNA, and conversely that the ability of the DNA to unwind stabilizes transient heterologous contacts.     

The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity

The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined. A variety of DNA substrates were used to characterize this ATP-dependent activity. Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction. Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility. ATP and MgCl2 were required for this reaction. With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive. The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates. In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence. This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein. When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound. This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate. The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2). Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound. In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity.     

Visualization of recA protein and its association with DNA: a priming effect of single-strand-binding protein

A stoichiometric interaction of RecA protein with single-stranded DNA promotes homologous pairing of the single strand with duplex DNA and subsequent polar formation of a heteroduplex joint. Escherichia coli single-strand-binding (SSB) protein augments these reactions. Electron microscopic observations suggest structural bases for these interactions. Without triphosphates or DNA, RecA protein forms short linear filaments. With added circular single-stranded DNA, it forms extended circular filaments as well as collapsed and aggregated complexes of protein and DNA. The extended circular filaments are stiff and regular in appearance, contrasting with the convoluted structure formed by SSB protein and single-stranded DNA. Together, these two proteins form mixed filaments, which mostly resemble the extended structures containing RecA protein; moreover, SSB protein accelerates formation of extended filaments more than 50-fold, increasing the yield of these structures at the expense of heterogeneous aggregates. Other observations further define the interactions of RecA protein with partially single-stranded DNA, and the effects of ATP gamma S on the tendency of RecA protein to form polymeric structures even in the absence of DNA.     

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.     

Homologous pairing of DNA molecules promoted by a protein from Ustilago

A protein from mitotic cells of Ustilago maydis was purified on the basis of its ability to reanneal complementary single strands of DNA. The protein catalyzed the uptake of linear single strands by super-helical DNA, but only in reactions with homologous combinations of single-strand fragments and super-helical DNA from phages phi X174 and fd. No reaction occurred with heterologous combinations. The protein also efficiently paired circular single strands and linear duplex DNA molecules. The product was a joint molecule in which the circular single strand displaced one strand of the duplex. Efficient pairing depended upon ATP, and ATPase activity was found associated with the purified protein. ATP-dependent reannealing of complementary single strands was not detectable in the rec1 mutant of Ustilago, which is deranged in meiotic recombination, as complete tetrads are rare, and is defective in radiation-induced mitotic gene conversion.     

Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.     

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.     

The REC2 gene encodes the homologous pairing protein of Ustilago maydis.

Amino acid sequence analysis has established that the homologous pairing protein of Ustilago maydis, known previously in the literature as rec1, is encoded by REC2, a gene essential for recombinational repair and meiosis with regional homology to Escherichia coli RecA. The 70-kDa rec1 protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously during sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product, the REC2 gene was overexpressed from a vector that fused a hexahistidine leader sequence onto the amino terminus, enabling isolation of the REC2 protein on an immobilized metal affinity column. The purified protein exhibits ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from cell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds between identical DNA strands. A size threshold of about 50 bp was found to govern pairing between linear duplex molecules and homologous single-stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the duplex was composed of RNA. Linear duplex molecules with hairpin caps also formed joint molecules when as few as three RNA residues were present.     

The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerases.

The binding of linear and circular forms of DNA to yeast DNA topoisomerase II or its complex with AMPPNP, the nonhydrolyzable beta,gamma-imido analog of ATP, was carried out to probe the ATP analog-induced conformational change of the enzyme. Binding of the ATP analog is shown to convert the enzyme to a circular clamp with an annulet, through which only a linear DNA can pass; subsequent circularization of the bound linear DNA forms a salt-stable catenane between the protein circular clamp and the DNA ring. Analysis of catenane formation between a small DNA ring originally bound to the topoisomerase and a large DNA ring subsequently added, under conditions such that the two do not exchange, supports a model in which a second DNA double-helix can enter the open jaws of a DNA-bound protein clamp, and the closure of the jaws upon ATP-binding traps the second duplex and transports it through an enzyme-operated gate in the first DNA duplex.