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.     

ATP-dependent unwinding of the double helix and extensive supercoiling by Escherichia coli recA protein in the presence of topoisomerase

recA protein, which is essential for genetic recombination in Escherichia coli, causes extensive unwinding of the double helix by an ATP-dependent reaction and accumulation of positive supercoiling in closed circular double-stranded DNA. Initiation of the extensive unwinding was largely dependent on homologous single-stranded DNA. Therefore, it is likely that the extensive unwinding is initiated mainly at the site of D-loops. "Nascent D-loops" in which the two DNA molecules did not interwind were also good initiation sites of extensive unwinding. When the concentration of Mg2+ was decreased from the standard conditions for D-loop formation (13 mM MgCl2; the higher Mg2+ condition) to the lower Mg2+ condition (1 to 2 mM MgCl2), extensive unwinding by recA protein was initiated very quickly in the absence of single-stranded DNA. Results showed that this single-stranded DNA-independent initiation of extensive unwinding (i) requires negative superhelicity of the double-stranded DNA and (ii) is a first order reaction with respect to the DNA. These observations suggest that, under the lower Mg2+ condition, the extensive unwinding starts at a transiently denatured site in the negative superhelical DNA. Once initiated, the unwinding by recA protein is propagated extensively, even under conditions that do not allow its initiation. Therefore, the propagation of unwinding is a processive reaction ("processive unwinding"). Previous studies indicated that recA protein promotes "distributive unwinding" of double helix which depends on single-stranded DNA. Therefore, recA protein promotes unwinding of the double helix by either of two distinct pathways. Stress caused by the processive unwinding could explain the dissociation of D-loops and reversible inactivation of the double-stranded DNA in a D-loop cycle.     

Specific defects in double-stranded DNA unwinding and homologous pairing of a mutant RecA protein

The DNA molecules bound to RecA filaments are extended 1.5-fold relative to B-form DNA. This extended DNA structure may be important in the recognition of homology between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). In this study, we show that the K286N mutation specifically impaired the dsDNA unwinding and homologous pairing activities of RecA, without an apparent effect on dsDNA binding itself. In contrast, the R243Q mutation caused defective dsDNA unwinding, due to the defective dsDNA binding of the C-terminal domain of RecA. These results provide new evidence that dsDNA unwinding is essential to homology recognition between ssDNA and dsDNA during homologous pairing.     

Intermediates in homologous pairing promoted by recA protein. Isolation and characterization of active presynaptic complexes

recA protein promotes homologous pairing and strand exchange by an ordered reaction in which the protein first polymerizes on single-stranded DNA. This presynaptic intermediate, which can be formed either in the presence or absence of Escherichia coli single-stranded binding protein (SSB), has been isolated by gel filtration and characterized. At saturation, purified complexes contained one molecule of recA protein per 3.6 nucleotide residues of single-stranded DNA. Complexes that had been formed in the presence of SSB contained up to one molecule of SSB per 15 nucleotide residues, but the content of SSB in different preparations of isolated complexes appeared to be inversely related to the content of recA protein. Even when they have lost as much as a third of their recA protein, presynaptic complexes can retain activity, because the formation of stable joint molecules depends principally on the binding of recA protein to the single-stranded DNA in the localized region that corresponds to the end of the duplex substrate.     

Triple-helical DNA pairing intermediates formed by recA protein

RecA protein aligns homologous single- and double-stranded DNA molecules in three-stranded joints that can extend over thousands of base pairs. When cross-linked by 4'-amino-4,5',8-trimethyl-psoralen the joint structure observed in nonuniform and divided into multiple substructures each a few hundred base pairs long. Two paired substructures are observed; at least one, and possibly both, are right-handed triple helices. Sites of homologous contact are interspersed with regions where the DNA molecules are arranged side-by-side without contact. These substructures alternate in all combinations. The length and frequency of joints is much greater when one of the DNA substrates is linear, and interwinding is unrestricted, than when there are topological restrictions between the pairing partners. The results are consistent with the idea that recA protein facilitates the formation of a right-handed triple-helical DNA pairing intermediate during strand exchange. The results further suggest that recA filaments do not promote the formation of structures that provide efficient topological compensation for right-handed interwinding of two paired DNA molecules.     

Networks of DNA and RecA protein are intermediates in homologous pairing

Partial coating of single-stranded DNA by recA protein causes its aggregation, but conditions that promote complete coating inhibit independent aggregation of single strands and, instead, cause the mutually dependent conjunction of single- and double-stranded DNA in complexes that sediment at more than 10 000 S. This coaggregation is independent of homology but otherwise shares key properties of homologous pairing of single strands with duplex DNA: both processes require ATP, MgCl2, and stoichiometric amounts of recA protein; both are very sensitive to inhibition by salt and ADP. Coaggregates are closed domains that are intermediates in homologous pairing: they form faster than joint molecules, they include virtually all of the DNA in the reaction mixture, and they yield joint molecules nearly an order of magnitude faster than they exchange DNA molecules with the surrounding solution. The independent aggregation of single-stranded DNA differs in all respects except the requirement for Mg2+, and its properties correlate instead with those associated with the renaturation of complementary single strands by recA protein.     

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.     

Genetic recombination: recA protein promotes homologous pairing between duplex DNA molecules without strand unwinding.

The recA protein of Escherichia coli promotes pairing in vitro between covalent circular duplex DNA and homologous circular duplex DNA containing a single stranded region. We have used a filter binding assay to investigate the frequency of homologous pairing between gapped and intact duplex DNA when unwinding of the free 3' and 5' ends of the gapped molecules was blocked. In order to obtain DNA without free ends, the gapped DNA was treated with trimethylpsoralen and 360 nm light so as to introduce about 6 crosslinks per DNA molecule and the double stranded regions on either side of the gaps were then digested up to the first crosslinks with exonuclease III and lambda exonuclease. This treatment did not diminish the frequency of homologous pairing, an observation which is difficult to reconcile with models for recombination requiring strand unwinding before pairing.     

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.     

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.     

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.     

Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins

We have established an in vitro reaction in which heteroduplex DNA formation is dependent on the concerted actions of recA and recBCD proteins, the major components of the recBCD pathway of genetic recombination in vivo. We find that heteroduplex DNA formation requires three distinct enzymatic functions: first, the helicase activity of recBCD enzyme initiates heteroduplex DNA formation by unwinding the linear double-stranded DNA molecule to transiently form single-stranded DNA (ssDNA); second, recA protein traps this ssDNA before it reanneals; third, recA protein catalyzes the pairing of this ssDNA molecule with another homologous ssDNA molecule, followed by the renaturation of these molecules to form heteroduplex DNA. The first two functions should be important to all in vitro reactions involving recA and recBCD proteins, whereas the third will be specific to the DNA substrates used. The rate-limiting step of heteroduplex DNA formation is the trapping by recA protein of the ssDNA produced by recBCD enzyme. A model for this reaction is described.     

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

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

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.     

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.     

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.     

Inhibition of protein-mediated homologous pairing by a DNA helicase.

Protein-mediated exchange of homologous DNA strands is a central reaction in general genetic recombination and the mechanism by which proteins mediate this process in vivo is a topic of keen interest. The dda protein of the bacteriophage T4 is a DNA helicase that has been shown to accelerate branch migration catalyzed by the phage uvsX and gene 32 proteins in vitro (Kodadek, T., and Alberts, B.M. (1987) Nature 326, 312-314). This study did not address the potential role of the helicase in protein-mediated homologous pairing, the first phase of the overall strand-exchange reaction. It is shown here that the dda protein inhibits uvsX protein-mediated pairing between homologous single and double-stranded DNAs. Experiments using deproteinized heteroduplex joints demonstrate that the dda helicase is capable of unwinding these structures to some extent and suggests that this activity may be responsible for the observed inhibition of pairing. It is found that the helicase also reduces the level of uvsX protein-mediated, single-stranded DNA-dependent ATP hydrolysis in the strand-exchange reactions, suggesting that the helicase may also act to destabilize the uvsX protein-DNA filaments that are important intermediates in the pairing reaction. Three other helicases are found to have no effect on the uvsX protein-mediated pairing reaction. A model rationalizing the ability of the dda protein to both inhibit homologous pairing and stimulate branch migration is presented and possible in vivo roles for this interesting activity are discussed.     

Homologous pairing of single-stranded circular DNAs catalyzed by recA protein.

RecA protein catalyzes annealing between pairs of circular single-stranded DNA molecules containing complementary sequences varying in length from 3550 nucleotides to 181 nucleotides. The reaction requires ATP and catalytic amounts of recA protein. Molecules containing large complementary inserts are annealed by recA protein to form large multimeric aggregates that migrate slowly in agarose gels. In contrast the products formed from circular molecules containing short complementary regions are principally dimeric structures. We have used electron microscopy, thermal denaturation and kinetic studies to analyze these reaction products. Our results indicate that recA protein catalyzes multiple nucleation events between complementary DNA sequences in the absence of a free end and when these sequences are flanked by extensive noncomplementary regions.     

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.     

Kinetics of homologous pairing promoted by RecA protein: effects of ends and internal sites in DNA

When recA protein was preincubated with single-stranded DNA in the presence of an ATP-regenerating system prior to the addition of homologous duplex DNA, a slow presynaptic step was eliminated, and the subsequent homologous pairing was revealed as a reaction whose rate exceeds by 1 or 2 orders of magnitude the calculated rate of spontaneous renaturation in 0.15 M NaCl at Tm -25 degrees C. The pairing reaction displayed saturation kinetics with respect to both single-stranded and double-stranded DNA, indicating the existence of a rate-limiting enzyme-substrate complex. The signal observed in the assay of the pairing reaction was due to pairing at free homologous ends of the duplex DNA, as well as pairing in the middle of the duplex molecule, away from a free end. The apparent rate of pairing of circular single strands with linear duplex DNA was equal to the sum of the rates of pairing at sites located at either end of the duplex DNA or at interior sites, but the apparent rates attributable to ends were greater, and nicks also stimulated the apparent rate.