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.     

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.     

Left-handed DNA and the synaptic pairing reaction promoted by Ustilago rec1 protein

Left-handed Z-DNA binds tightly to Ustilago rec1 protein. The binding reaction is strongly dependent on ATP, but complexes formed are rapidly dissociated by ADP. The parallel between the kinetics of Z-DNA binding and the synaptic pairing reaction leading to paranemic joint molecules suggests that formation of nascent heteroduplex structures in recombination is coupled with formation of left-handed Z-like DNA on the protein. Equilibrium and kinetic studies show that rec1 protein appears to have a strong Z-DNA binding site that binds Z-DNA 75 times tighter than the B form of the DNA. We propose that DNA with a structure approximated best by a left-handed Z-DNA conformation is a key intermediate in homologous pairing promoted by rec1 protein.     

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.     

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.     

recA protein promoted DNA strand exchange

recA protein and circular single-stranded DNA form a stable complex in the presence of single-stranded DNA binding protein (SSB), in which one recA protein monomer is bound per two nucleotides of DNA. These complexes are kinetically significant intermediates in the exchange of strands between the single-stranded DNA and an homologous linear duplex. After completion of strand exchange, the recA protein remains tightly associated with the circular duplex product of the reaction and the SSB is bound to the displaced linear single strand. Upon addition of ADP, the recA protein-duplex DNA complex dissociates. RecA protein also interacts with single-stranded DNA in the absence of SSB; however, the amount of recA protein bound is substantially reduced. These findings provide direct physical evidence for the participation of SSB in the formation of the recA protein-single-stranded DNA complexes inferred earlier from kinetic analysis. Moreover, they confirm the ability of recA protein to equilibrate between bound and free forms in the absence of SSB.     

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.     

Homologous pairing promoted by the human Rad52 protein.

The Rad52 protein, which is unique to eukaryotes, plays important roles in the Rad51-dependent and the Rad51-independent pathways of DNA recombination. In the present study, we have biochemically characterized the homologous pairing activity of the HsRad52 protein (Homo sapiens Rad52) and found that the presynaptic complex formation with ssDNA is essential in its catalysis of homologous pairing. We have identified an N-terminal fragment (amino acid residues 1-237, HsRad52(1-237)) that is defective in binding to the human Rad51 protein, which catalyzed homologous pairing as efficiently as the wild type HsRad52. Electron microscopic visualization revealed that HsRad52 and HsRad52(1-237) both formed nucleoprotein filaments with single-stranded DNA. These lines of evidence suggest the role of HsRad52 in the homologous pairing step of the Rad51-independent recombination pathway. Our results reveal the striking similarity between HsRad52 and the Escherichia coli RecT protein, which functions in a RecA-independent recombination pathway.     

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.     

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.     

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.     

Inhibition of recA protein promoted ATP hydrolysis. 2. Longitudinal assembly and disassembly of recA protein filaments mediated by ATP and ADP

There are at least two major conformations of recA nucleoprotein filaments formed on poly-(deoxythymidylic acid) [poly(dT)], one stabilized by ATP [or adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S)] and one stabilized by ADP. Assembly of filaments in the ATP conformation is much faster than assembly in the ADP conformation. A third conformation may be present in the absence of nucleotides. The ATP and ADP conformations are mutually exclusive. When a mixture of ATP and ADP is present, recA protein binding is a function of the ADP/ATP ratio. Complete dissociation is observed when the ratio becomes 1.0-1.5. When a mixture of ATP and ADP is present at the beginning of a reaction, a transient phase lasting several minutes is observed in which the system approaches the state characteristic of the new ADP/ATP ratio. This phase is manifested by a lag in ATP hydrolysis when ATP is added to preformed ADP filaments, and by a burst in ATP hydrolysis in all other cases. More than 15 ATPs are hydrolyzed per bound recA monomer during the burst phase. The transient phase reflects an end-dependent disassembly process propagated longitudinally through the filament, rather than a slow conformation change in individual recA monomers or a slow exchange of one nucleotide for the other. The hysteresis exhibited by the system provides a number of insights relevant to the mechanism of recA-mediated DNA strand exchange.     

Biologically active recombinant formed through DNA pairing by purified recA protein in vitro

Summary We have detected in vitro homologous recombination mediated by purified recA protein of Escherichia coli as a recombinant phage produced by using the DNA packaging system of phage . When double-stranded DNA of phage carrying amber mutations is incubated with double-stranded DNA carrying the wild-type genes in the presence of recA protein, Mg⁺⁺ and ATP, and the DNA packaged, amber ⁺ recombinant phage is produced at a high frequency. This reaction depends completely upon the function of the wild-type recA protein. After incubation of ³²P-labeled linear DNA (Form III) with bromouracil-labeled circular DNA (Form I-Form II mixture) in the presence of recA protein, Mg⁺⁺ and ATP, about 10% of the ³²P-counts band at an intermediate density in CsCl equilibrium gradient. This fraction yields a high percentage of the recombinant phage after DNA packaging and shows the -shaped and -shaped joint molecules of linear and circular DNA under the electron microscope. Furthermore, we demonstrate that a non-homologous region inhibits the recombination reaction when it is between the marker concerned and the closer cos end. Our results indicate thatrecA protein acts directly in the initial step of recombination to join the homologous double-stranded DNA and that the resulting molecule can be matured into the recombinant DNA.Abbreviations kb kilobase pairs - PFU plaque forming units - Form I superhelical closed circular DNA - Form II open circular DNA - Form III linear DNA     

Kinetic modeling of the recA protein promoted renaturation of complementary DNA strands

Quantitative agarose gel assays reveal that the recA protein promoted renaturation of complementary DNA strands (phi X DNA) proceeds in two stages. The first stage results in the formation of unit-length duplex DNA as well as a distribution of other products ("initial products"). In the second stage, the initial products are converted to complex multipaired DNA structures ("network DNA"). In the presence of ATP, the initial products are formed within 2 min and are then rapidly converted to network DNA. In the absence of ATP, the initial products are formed nearly as fast as with ATP present, but they are converted to network DNA at a much lower rate. The time-dependent formation of initial products and network DNA from complementary single strands for both the ATP-stimulated and ATP-independent reactions can be modeled by using a simple two-step sequential kinetic scheme. This model indicates that the primary effect of ATP in the recA protein promoted renaturation reaction is not on the initial pairing step (which leads to the formation of initial products) but rather is to increase the rate at which subsequent pairing events can occur.     

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.     

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.     

Homologous pairing in genetic recombination. Purification and characterization of Escherichia coli recA protein

RecA protein, which is essential for genetic recombination in Escherichia coli, was extensively purified from a strain of E. coli which contained the recA gene cloned in a plasmid (Sancar, A., and Rupp, W. D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3144-3148). Using the DNA-dependent ATPase activity of recA protein as an assay, we obtained about 60 mg of purified recA protein from 100 g of cells. Ten micrograms or 1 microgram of the purified protein exhibited only one detectable band with Mr approximately = 40,000 upon sodium dodecyl sulfate-acrylamide gel electrophoresis. More than 99% of the ATPase activity of purified recA protein was dependent on single-stranded DNA. Purified recA protein had no detectable DNase, topoisomerase, or ligase activities. The enzyme was stable for a least a year when stored at 0-4 degrees C. The half-life of the ATPase activity of 25 microM recA protein was 37 min at 51 degrees C. Purified recA protein binds to single-stranded and double-stranded DNA, unwinds duplex DNA by a mechanism that is stimulated by single-stranded DNA or oligonucleotides, and pairs homologous single strands with duplex DNA.     

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.