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 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.     

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.     

Defective homologous pairing and proficient processive unwinding by the recA430 mutant protein and intermediates of homologous pairing by recA protein

The recA protein promotes the formation and processing of joint molecules of homologous double- and single-stranded DNAs in vitro. Under a set of specified conditions, we found that the substitution of a single amino acid in the recA protein (recA430 mutation) depresses its activity for the homologous pairing to about 1/100 of that by the wild type protein when compared by the rate for the first 2-3 min of the reaction, but that the mutation only slightly, if at all, affects its ability to bind progressively to double-stranded DNA to unwind the double helix ("processive unwinding"). This is in striking contrast to an anti-recA protein monoclonal IgG, ARM193, which severely inhibits the processive unwinding but not the homologous pairing, providing further support for our conclusion that the homologous pairing and processive unwinding are functionally independent of each other. Antibody ARM193 caused the breakdown of spontaneously formed filaments of the recA protein, but the recA430 mutation did not affect the self-polymerization of the protein. The recA430 protein was apparently proficient in the functional binding to a single-stranded DNA and in the hydrolysis of ATP. However, we found that under the above conditions the mutant protein was defective as to homology-independent conjunction of DNA molecules to form a "ternary complex" (of macromolecules). These results suggest that (i) only one DNA-binding site is sufficient for the recA protein to promote the processive unwinding (the ability of the protein to form spontaneous filaments is closely related to this process) and that (ii) two DNA-binding sites on each of the recA polypeptides or those composed of a dimer (or oligomer) of the polypeptide are required for the recA protein to promote both the conjunction of parental DNA molecules and the homologous pairing (the ability to form the spontaneous filaments is not essential to this process). (iii) The simultaneous inactivation of the activity to promote the homologous pairing and that to form the ternary complex by the single substitution of the amino acid provides a physical support for the conclusion that the ternary complex is an indispensable intermediate in the homologous pairing.     

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.     

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 mechanism of the search for homology promoted by recA protein. Facilitated diffusion within nucleoprotein networks

recA protein promotes the homologous pairing of single strands with duplex DNA by polymerizing on the single strands to make presynaptic nucleoprotein filaments which are polyvalent with respect to duplex DNA and which consequently form large networks or coaggregates when duplex DNA is added. Previous work has shown that efficient homologous pairing occurs within these networks. In the experiments described here, we observed that the length of the duplex DNA determined the stability of coaggregates, their steady state level, and the yield of joint molecules. Correspondingly, heterologous duplex DNA when preincubated with presynaptic filaments excluded subsequently added homologous duplex DNA from coaggregates and inhibited homologous pairing; the extents of exclusion and inhibition were determined by the length of the heterologous duplex DNA. On the other hand, long heterologous duplex DNA when added together with short homologous duplex DNA was capable of stimulating the absorption of the homologous molecules into coaggregates and increasing the rate of homologous pairing. In reactions involving short duplex molecules, polyamines exerted comparable effects on coaggregation and homologous pairing. We conclude that coaggregates are instrumental in homologous pairing, that they constitute distinct domains that are responsible for the processive or first order character of the pairing reaction, and that they act by concentrating DNA and facilitating diffusion.     

Can recA protein promote homologous pairing between duplex regions of DNA?

RecA protein has been shown to promote the formation of joint molecules between intact duplex DNA and homologous gapped DNA. When examined by electron microscopy, such joint molecules display a junction that is, in most cases, distant from the site of the gap. This led us to test whether the observed location of the joint was due to pairing at the gap followed by branch migration, or whether recA-promoted pairing could also take place between duplex homologous regions away from the gap. To test the latter possibility, intact duplex DNA was incubated with DNA which contained a gap in a region of non-homology. Joint molecules were detected by filter binding assay and by electron microscopy at about one-third of the yield observed for fully homologous molecules. These results indicate that initial homologous pairing promoted by recA protein is not restricted to the single-stranded region in the gap but can also take place in regions where both molecules are duplex.     

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.     

rec-A protein-promoted recombination reaction consists of two independent processes, homologous matching and processive unwinding. A study involving an anti-rec-A protein-monoclonal IgG

recA protein promotes the formation and processing of joint molecules of homologous double-stranded DNA and single-stranded DNA. We studied the effects of an anti-recA protein monoclonal IgG (ARM193) on two processes carried out by the recA protein. The homologous matching, i.e. pairing of double-stranded DNA and single-stranded DNA by forming intermolecular base-pairing at homologous regions was found to occur even in the presence of an excess amount of antibody ARM193. On the other hand, processive unwinding, i.e. the propagation of the unwinding of double-stranded DNA through a processive reaction of recA protein, which occurs even in the absence of single-stranded DNA, was found to be very sensitive to the inhibition by antibody ARM193. Therefore, we conclude that homologous matching and processive unwinding are independent of each other. Analysis of the effect of antibody ARM193 on the various activities of recA protein suggests that the entire reaction of the formation of joint molecules and their processing can be rationalized in terms of these two underlying processes, homologous matching and processive unwinding. This analysis also suggests that homologous matching seems to require only the binding itself of active units of recA protein to single-stranded DNA but not necessarily either the cooperativity of the protein or unwinding.     

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.     

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.     

Basis for avid homologous DNA strand exchange by human Rad51 and RPA

Human Rad51 (hRad51), a member of a conserved family of general recombinases, is shown here to have an avid capability to make DNA joints between homologous DNA molecules and promote highly efficient DNA strand exchange of the paired molecules over at least 5.4 kilobase pairs. Furthermore, maximal efficiency of homologous DNA pairing and strand exchange is strongly dependent on the heterotrimeric single-stranded DNA binding factor hRPA and requires conditions that lessen interactions of the homologous duplex with the hRad51-single-stranded DNA nucleoprotein filament. The homologous DNA pairing and strand exchange system described should be valuable for dissecting the action mechanism of hRad51 and for deciphering its functional interactions with other recombination factors.     

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.     

Use of structure-directed DNA ligands to probe the binding of recA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing.

We have used circular dichroism and structure-directed drugs to identify the role of structural features, wide and narrow grooves in particular, required for the cooperative polymerization, recognition of homologous sequences, and the formation of joint molecules promoted by recA protein. The path of cooperative polymerization of recA protein was deduced by its ability to cause quantitative displacement of distamycin from the narrow groove of duplex DNA. By contrast, methyl green bound to the wide groove was retained by the nucleoprotein filaments comprised of recA protein-DNA. Further, the mode of binding of these ligands and recA protein to DNA was confirmed by DNaseI digestion. More importantly, the formation of joint molecules was prevented by distamycin in the narrow groove while methyl green in the wide groove had no adverse effect. Intriguingly, distamycin interfered with the production of coaggregates between nucleoprotein filaments of recA protein-M13 ssDNA and naked linear M13 duplex DNA, but not with linear phi X174 duplex DNA. Thus, these data, in conjunction with molecular modeling, suggest that the narrow grooves of duplex DNA provide the fundamental framework required for the cooperative polymerization of recA protein and alignment of homologous sequences. These findings and their significance are discussed in relation to models of homologous pairing between two intertwined DNA molecules.     

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.     

Homologous pairing in genetic recombination: recA protein makes joint molecules of gapped circular DNA and closed circular DNA

The recA protein, which is essential for genetic recombination in E. coli, promotes the homologous pairing of double-stranded DNA and linear single-stranded DNA, thereby forming a three-stranded joint molecule called a D loop. Single-stranded DNA stimulates recA protein to unwind double-stranded DNA. By a presumably related mechanism, recA protein promoted the homologous pairing of two circular double-stranded molecules when one of them had a gap in one strand. The two molecules were joined at homologous sites by noncovalent bonds. The covalently closed molecule remained intact and was not topologically linked to the intact circular strand of the gapped substrate. Electron microscopy showed that molecules were usually linked at two or more nearby points. The junctions in most molecules were shorter than 300 nucleotides. Sometimes the region between two extreme points was separated into two arms, producing an ellipsoidal loop (called an eye loop). The junctions in these biparental joint molecules were frequently remote from the site of the gap. We infer that a free end of the interrupted strand crossed over to form a structure like a D loop which moved away from the gap by branch migration.     

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.