Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.

Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.     

RecA protein-promoted homologous pairing between duplex molecules: functional role of duplex regions of gapped duplex DNA

RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped 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.     

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.     

Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation

Saccharomyces cerevisiae Rad51, Rad54, and replication protein A (RPA) proteins work in concert to make heteroduplex DNA joints during homologous recombination. With plasmid length DNA substrates, maximal DNA joint formation is observed with amounts of Rad51 substantially below what is needed to saturate the initiating single-stranded DNA template, and, relative to Rad51, Rad54 is needed in only catalytic quantities. RPA is still indispensable for optimal reaction efficiency, but its role in this instance is to sequester free single-stranded DNA, which otherwise inhibits Rad51 and Rad54 functions. We also demonstrate that Rad54 helps overcome various reaction constraints in DNA joint formation. These results thus shed light on the function of Rad54 in the Rad51-mediated homologous DNA pairing reaction and also reveal a novel role of RPA in the presynaptic stage of this reaction.     

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.     

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.     

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.     

Homologous pairing and the formation of nascent synaptic intermediates between regions of duplex DNA by RecA protein

The RecA protein from E. coli gains access to duplex DNA, by nucleation from a short single-stranded gap, to form a spiral nucleoprotein filament that is capable of interaction with homologous duplex DNA. The observations described here demonstrate that any part of the nucleoprotein filament, whether it contains single- or double-stranded DNA, is capable of pairing with homologous duplex DNA. Homologous contacts between regions of duplex DNA lead to an increase in the initial rate and final extent of joint molecule formation. The experiments indicate that pairing is facilitated by the formation of nascent synaptic intermediates between duplex DNA sequences. Using chimeric form I DNA, which is incapable of forming an inter-wound or plectonemic joint with the gapped DNA due to the presence of flanking heterologous sequences, we show that these duplex-duplex pairing reactions involve extensive underwinding of the double helix.     

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology

The RecA protein ofEscherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.     

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.     

Catalysis of strand exchange by the HSV-1 UL12 and ICP8 proteins: potent ICP8 recombinase activity is revealed upon resection of dsDNA substrate by nuclease

The replication of herpes simplex virus type 1 (HSV-1) is associated with a high degree of homologous recombination, which is likely to be mediated, in part, by HSV-1-encoded proteins. We have previously shown that the HSV-1 encoded ICP8 protein and alkaline nuclease UL12 are capable of catalyzing an in vitro strand-exchange reaction. Here, we show, by electron microscopy, that the products of the strand exchange reaction between linear double-stranded DNA and circular single-stranded DNA consist of the expected joint molecule forms: sigma, alpha, and gapped circles. Other exonucleases, such as lambda Red alpha, which, like UL12, digests 5'-3', as well as Escherichia coli exonuclease III (ExoIII), which digests 3'-5', could substitute for UL12 in the strand exchange reaction by providing a resected DNA end. ICP8 generated the same intermediates and strand exchange products when the double-stranded DNA substrate was preresected by any of the nucleases. Using substrates with large regions of non-homology we found that pairing by ICP8 could be initiated from the middle of a DNA molecule and did not require a homologous end. In this reaction, the resection of a DNA end by the nuclease is required to reveal homologous sequences capable of being paired by ICP8. This study further illustrates the complexity of the multi-functional ICP8 protein.     

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.     

The human Rad51 protein: polarity of strand transfer and stimulation by hRP-A.

The human Rad51 protein is homologous to the RecA protein and catalyses homologous pairing and strand transfer reactions in vitro. Using single-stranded circular and homologous linear duplex DNA, we show that hRad51 forms stable joint molecules by transfer of the 5' end of the complementary strand of the linear duplex to the ssDNA. The polarity of strand transfer is therefore 3' to 5', defined relative to the ssDNA on which hRad51 initiates filament formation. This polarity is opposite to that observed with RecA. Homologous pairing and strand transfer require stoichiometric amounts of hRad51, corresponding to one hRad51 monomer per three nucleotides of ssDNA. Joint molecules are not observed when the protein is present in limiting or excessive amounts. The human ssDNA binding-protein, hRP-A, stimulates hRad51-mediated reactions. Its effect is consistent with a role in the removal of secondary structures from ssDNA, thereby facilitating the formation of continuous Rad51 filaments.     

RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA.

In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament. Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 mM-MgCl2, optimized the binding of RecA protein and increased the formation of joint molecules. The gapped duplex formed stable joints with fully duplex DNA that had a 5' or 3' terminus complementary to the single-stranded region of the gapped molecule. However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5' or 3' end. Pairing between gapped DNA and fully duplex linear DNA with a 3' complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products. By contrast, pairing between gapped and fully duplex DNA with a 5' complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange. The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure. We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5' to 3' direction with respect to the initiating strand. The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism.     

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.     

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.     

Multiple interactions with the Rad51 recombinase govern the homologous recombination function of Rad54

In eukaryotes, Rad51 and Rad54 functionally cooperate to mediate homologous recombination and the repair of damaged chromosomes by recombination. Rad51, the eukaryotic counterpart of the bacterial RecA recombinase, forms filaments on single-stranded DNA that are capable of pairing the bound DNA with a homologous double-stranded donor to yield joint molecules. Rad54 enhances the homologous DNA pairing reaction, and this stimulatory effect involves a physical interaction with Rad51. Correspondingly, the ability of Rad54 to hydrolyze ATP and introduce superhelical tension into covalently closed circular plasmid DNA is stimulated by Rad51. By controlled proteolysis, we show that the amino-terminal region of yeast Rad54 is rather unstructured. Truncation mutations that delete the N-terminal 113 or 129 amino acid residues of Rad54 attenuate or ablate physical and functional interactions with Rad51 under physiological ionic strength, respectively. Surprisingly, under less stringent conditions, the Rad54 Delta129 protein can interact with Rad51 in affinity pull-down and functional assays. These results highlight the functional importance of the N-terminal Rad51 interaction domain of Rad54 and reveal that Rad54 contacts Rad51 through separable epitopes.