Basis for Avid Homologous DNA Strand Exchange by Human Rad51 and RPA*

Stefan Sigurdsson, Kelly Trujillo, BinWei Song, Sabrina Stratton, and Patrick SungDagger

From the Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207

Received for publication, November 3, 2000, and in revised form, December 12, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Genetic studies in various eukaryotic organisms have indicated that homologous recombination processes are mediated by a group of evolutionarily conserved genes known as the RAD52 epistasis group. As revealed in studies on meiotic recombination and mating type switching in Saccharomyces cerevisiae, DNA double-strand breaks are formed and then processed exonucleolytically to yield long single-stranded tails with a 3' extremity. Nucleation of various RAD52 group proteins onto these ssDNA1 tails renders them recombinogenic, leading to the search for a homologous DNA target (sister chromatid or homologous chromosome), formation of DNA joints with the target, and an exchange of genetic information with it. The repair by recombination of DNA double-strand breaks induced by ionizing radiation and other DNA damaging agents very likely follows the same mechanistic route, as it too is dependent on genes of the RAD52 epistasis group (reviewed in Refs. 1 and 2).

Among members of the RAD52 group, the RAD51-encoded product is of particular interest because of its structural and functional similarities to the Escherichia coli recombination protein RecA (2-5). RecA promotes the pairing and strand exchange between homologous DNA molecules to form heteroduplex DNA (4, 5), an enzymatic activity believed to be germane for the central role of RecA in recombination and DNA repair processes. Likewise, homologous DNA pairing and strand exchange activities have been shown for S. cerevisiae Rad51 (yRad51) (6). Under optimized conditions, the length of heteroduplex DNA joints formed by yRad51 and RecA can extend over quite a few kilobase pairs (4, 5, 7).

In published studies, human Rad51 (hRad51) was found to have the ability to make DNA joints but the maximal potential for forming only about 1 kilobase pairs of heteroduplex DNA (8-11). Furthermore, while yRad51 and RecA require their cognate single-strand DNA binding factors, SSB and yRPA, for optimal recombinase activity, hRPA has been suggested to stimulate the hRad51-mediated homologous pairing and strand exchange reaction only when the hRad51 concentration is suboptimal (9, 10).

Given the central role of hRad51 in recombination processes and the fact that the activities of hRad51 are apparently subject to modulation by tumor suppressor proteins such as BRCA2 (reviewed in Ref. 12), establishing an efficient hRad51-mediated DNA strand exchange system will be important for dissecting the functional interactions among hRad51, other recombination factors, and tumor suppressors. In this work, a variety of reaction parameters that could influence the recombinase activity of hRad51 were explored. We demonstrate that under certain conditions, hRad51 makes DNA joints avidly and promotes highly efficient strand exchange over at least 5.4 kilobase pairs. Importantly, under the new reaction conditions, the efficiency of the hRad51-mediated DNA strand exchange reaction is strongly dependent on hRPA over a wide range of Rad51 concentrations tested.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA Substrates-- phi X174 viral (+)-strand was purchased from New England Biolabs and phi X174 replicative form I DNA was from Life Technologies, Inc. The replicative form I DNA was linearized with ApaLI. The pBluescript DNA was prepared from E. coli XL-1 Blue (Stratagene), purified by banding in cesium chloride gradients, and linearized with BsaI. The oligonucleotides (83-mer) used in strand exchange were: oligo 1, 5'-AAATGAACATAAGATAAATAAGTATAAGGATAATACAAAATAAGTAAATGAATAAACATAGAAAATAAAGTAAAGGATATAAA; oligo 2, the exact complement of oligo 1. Oligo 2 was labeled at the 5' end with [gamma -32P]ATP and T4 polynucleotide kinase and then annealed to oligo 1. The labeled duplex was purified from 10% polyacrylamide gels by overnight diffusion at 4 °C into TAE buffer (40 mM Tris acetate, pH 7.4, 0.5 mM EDTA). DNA substrates were stored in TE (10 mM Tris-HCl, pH 7.5, with 0.5 mM EDTA).

Plasmids-- Plasmid pRh51.1 consists of human RAD51 K313 cDNA under the control of the T7 promoter in vector pET11 (Novagen). Plasmid pRh51.1 was then subject to in vitro mutagenesis using the QuikChange kit (Stratagene), to change Lys313 (AAA codon) to a glutamine residue (CAA codon). The resulting plasmid, pRh51.2, was sequenced to ensure that no other undesired change has occurred in the RAD51 sequence. Plasmid p11d-tRPA (13), which coexpresses all three subunits of hRPA, was used for purification of this factor.

Cell Growth-- Plasmids pRh51.1 and pRh51.2 were introduced into the RecA-deficient E. coli strain BLR (DE3) with pLysS. Following transformation, single clones were picked and grown for 15 h in 30 ml of Luria broth. The starter culture was diluted 200 times with fresh Luria broth and incubated at 37 °C. When the A600 of the cultures reached 0.6 to 1, isopropyl-1-thio-beta -D-galactopyranoside was added to 0.4 mM and the induction of hRad51 continued at 37 °C for 4 h. Cells were harvested by centrifugation and stored frozen at -70 °C. Plasmid p11d-tRPA was introduced into E. coli strain BL21 (DE3) and the induction of hRPA was carried out as described previously (13).

Protein Purification-- All the following steps were carried out at 4 °C. For the purification of hRad51 Lys313 and hRad51 Gln313 proteins, E. coli cell paste, 30 g from 20 liters of culture, was suspended in 150 ml cell breakage buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 200 mM KCl, 2 mM dithiothreitol, 10% sucrose, and the following protease inhibitors: aprotinin, chymostatin, leupeptin, and pepstation A at 3 µg/ml each, and 1 mM phenylmethylsulfonyl fluoride) and then passed through a French press once at 20,000 p.s.i. The crude lysate was clarified by centrifugation (100,000 × g, 120 min), and the supernatant (Fraction I) was treated with ammonium sulfate at 0.23 g/ml to precipitate hRad51 and about 20% of the total extract protein. The ammonium sulfate pellet was dissolved in 300 ml of T buffer (30 mM Tris-HCl, pH 7.4, 10% glycerol, 0.5 mM EDTA, 0.5 mM dithiothreitol) with the set of protease inhibitors used in extract preparation, and then clarified by centrifugation (10,000 × g for 30 min). The cleared protein solution (Fraction II) was then applied onto a column of Q-Sepharose (2.6 × 6 cm; total 30-ml matrix) equilibrated in T buffer with 100 mM KCl and eluted with a 400 ml of gradient of 100 to 600 mM KCl in T buffer. The peak of hRad51 (Fraction III), eluting at about 330 mM KCl (60 ml), was dialyzed against T buffer with 100 mM KCl and then fractionated in a column of Affi-Gel Blue (Bio-Rad; 1.6 × 5 cm; total 10-ml matrix) with a 100-ml gradient of 100 to 2000 mM KCl in T buffer. The hRad51 protein eluted from Affi-Gel Blue at 800 to 1200 mM KCl, and the pool of which (Fraction IV; 20 ml) was dialyzed against T buffer with 100 mM KCl and fractionated in a column of Macro hydroxyapatite (Bio-Rad; 1 × 7.5 cm; total 6-ml matrix) with a 100-ml 30 to 320 mM KH2PO4 gradient in buffer T. hRad51 eluted from 150 to 220 mM KH2PO4, and the peak fractions were pooled (Fraction V; 15 ml containing 7.5 mg of hRad51), dialyzed against T buffer with 50 mM KCl, and applied onto a Mono S column (HR5/5), which was developed with a 30-ml 100 to 400 mM KCl gradient in buffer T. The Mono S fractions containing the peak of hRad51, eluting at about 250 mM KCl, were pooled (Fraction VI; 4 ml containing 6 mg of hRad51), diluted with an equal volume of 10% glycerol and then fractionated in Mono Q (HR 5/5) with a 30-ml 100 to 600 mM KCl gradient. The final pool of hRad51 (Fraction VII; 3 ml containing 5 mg of hRad51 in ~350 mM KCl) was concentrated in Centricon-30 microconcentrators and stored at -70 °C. The hRad51 concentration was determined using the calculated molar extinction coefficient of 12,800 M-1 cm-1 at 280 nM (10).

For the purification of hRPA, extract was made from E. coli BL21 (DE3) harboring the plasmid p11d-tRPA (13) and subjected to the purification procedure we have used for yRPA (14). The concentration of hRPA was determined by comparison of multiple loadings of hRPA against known amounts of bovine serum albumin and ovalbumin in a Coomassie Blue R-stained polyacrylamide gel.

DNA Strand Exchange System That Uses phi X174 DNA-- All the reaction steps were carried out at 37 °C. In Fig. 2, the reaction (50 µl final volume) was assembled by mixing hRad51 (7.5 µM) added in 2 µl of storage buffer and phi X174 viral (+)-strand (30 µM nucleotides) added in 2 µl in 40 µl of buffer R (40 mM Tris-HCl, pH 7.8, 2 mM ATP, 1 mM MgCl2, 1 mM dithiothreitol, and an ATP regenerating system consisting of 8 mM creatine phosphate and 28 µg/ml creatine kinase). After a 5-min incubation, hRPA (2 µM) in 2 µl of storage buffer was added, followed by a 5-min incubation, and then 5 µl of ammonium sulfate (1 M stock, final concentration of 100 mM), followed by another 1-min incubation. To complete the reaction, linear phi X174 replicative form I DNA (30 µM nucleotides) in 3 µl of TE and 4 µl of 50 mM spermidine (4 mM) were incorporated. At the indicated times, 4.5-µl portions were withdrawn, mixed with 7 µl of 0.8% SDS and 800 µg/ml proteinase K, incubated for 15 min before electrophoresis in 0.9% agarose gels in TAE buffer. The gels were stained in ethidium bromide (2 µg/ml in H2O) for 1 h, destained for 12 to 18 h in a large volume of water, and then subjected to image analysis in a NucleoTech gel documentation station equipped with a CCD camera using Gel Expert for quantification of the data. Unless stated otherwise, the reaction mixtures in other experiments were assembled in the same manner with the indicated amounts and order of addition of reaction components, except that they were scaled down two and one-half times.

DNA Strand Exchange System That Employs Oligonucleotides-- The reaction mixture had a final volume of 12.5 µl and the steps were carried out at 37 °C. hRad51 (7.5 µM) was incubated with oligonucleotide 2 (30 µM nucleotides) in 10 µl of buffer R. The reaction mixture was completed by adding ammonium sulfate in 1 µl, 1 µl of 50 mM spermidine, and the radiolabeled duplex (30 µM nucleotides) in 0.5 µl. At all the times indicated, a 3-µl portion of the reaction mixture was deproteinized as described above and then subjected to electrophoresis in 10% polyacrylamide gels run in TAE buffer. The level of DNA strand exchange was determined by PhosphorImager analysis of the dried gels.

Examination of Interaction between hRad51-ssDNA Filament and Duplex DNA-- Oligonucleotides F1 and F1b (Midland) both have the sequence 5'-TGGCTTGAACGCGTCATGGAAGCGATAAAACTCTGCAGGTTGGATACGCCAATCATTTTTATCGAAGCGCGCCGCCC-3', except that the latter also contains a biotin molecule positioned at the 5' terminus. In these oligonucleotides, nucleotide residues 11 to 72 are complementary to positions 5348 to 23 of the phi X (+)-strand DNA. These oligonucleotides were hybridized to phi X (+)-strand by incubating a 3 M excess of the oligonucleotide with the latter in buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, and 1 mM dithiothreitol. The F1-phi X (+)-strand and F1b-phi X (+)-strand hybrids (30 µM nucleotides) were mixed with 10 µl of magnetic beads containing streptavidin (Roche Molecular Biochemicals) in binding buffer containing 10 mM Tris-HCl, pH 7.5, 100 mM KCl, and 1 mM EDTA for 10 min at 37 °C. About 70% of the F1b-(+)-strand hybrid was immobilized on the beads, whereas, as expected, less than 5% of the F1-(+)-strand hybrid was retained. To assemble hRad51 filament on the immobilized phi X (+)-strand, magnetic beads preloaded with the F1b-phi X (+)-strand hybrid were incubated with 4 µM hRad51 in 20 µl of buffer R. Reproducibly, ~85% of the hRad51 was bound to the magnetic beads under the stated conditions, as determined by eluting the bound hRad51 with 2% SDS followed by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue; this procedure gave an immobilized hRad51-ssDNA nucleoprotein complex of ~3 nucleotides/hRad51 monomer. The magnetic beads containing hRad51-ssDNA complex was then washed once each with 20 µl of buffer R with 0.01% Nonidet P-40 and 20 µl buffer R, before being incubated with linear phi X duplex (8 µM nucleotides) for 3 min at 37 °C in 20 µl of buffer R containing 4 mM spermidine and the indicated concentrations of ammonium sulfate. The beads were treated with 20 µl of 2% SDS at 37 °C for 5 min to elute bound duplex and hRad51. The supernatants and SDS eluates were analyzed in agarose gels followed by staining with ethidium bromide to quantify DNA and in polyacrylamide gels with Coomassie Blue staining to determine the amount of hRad51. As controls, magnetic beads alone, magnetic beads preincubated with the F1-phi X (+)-strand hybrid, and magnetic beads preincubated with F1b but without phi X (+)-strand were similarly incubated with the linear phi X duplex and then processed for analyses.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recombination Factors-- The cDNA for hRad51 was amplified from a human B-cell cDNA library. Sequencing of the hRAD51 cDNA insert revealed that it was identical to one of the published hRAD51 sequences (15) but differed from the other sequence (16) at amino acid residue 313; the former has a lysine (an AAA codon) while the latter has a glutamine (a CAA codon) at this position. The cloned cDNA was subjected to targeted mutagenesis to change lysine 313 to glutamine. Both hRad51 Lys313 and hRad51 Gln313 variants were expressed in E. coli and purified to near homogeneity (Fig. 1A). The two hRad51 isoforms behaved identically during purification and gave indistinguishable results in all the enzymatic assays described here. Only the results with hRad51 Lys313 are shown. We presume that the two hRad51 isoforms correspond to naturally occurring polymorphic variants. For DNA strand exchange experiments, the human ssDNA binding factor replication protein A (hRPA), a heterotrimer of 70-, 32-, and 14-kDa subunits, was also purified to near homogeneity (Fig. 1B) from E. coli cells harboring a plasmid which coexpresses all three subunits of this factor (13).


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Fig. 1.   Recombination factors. A, purified hRad51 Lys313 (lane 2) and hRad51 Gln313 (lane 3), 3 µg each, were analyzed in an 11% SDS-polyacrylamide gel and stained with Coomassie Blue. B, purified hRPA, 3 µg in lane 2, was analyzed in a 12.5% SDS-polyacrylamide gel and stained with Coomassie Blue. The three subunits of hRPA are denoted by the arrows.

System for ATP-dependent Homologous DNA Pairing and Strand Exchange-- For characterizing the homologous DNA pairing and strand exchange activity of hRad51, we used as substrates phi X 174 viral (+)-strand and linear duplex that are 5.4 kilobase pairs in length (Ref. 6; schematic shown in Fig. 2A). In this system, hRad51 is preincubated with the (+)-strand, followed by the addition of hRPA, and the linear duplex is incorporated last. Pairing between the DNA substrates yields a joint molecule, and branch migration, if successful, over 5.4 kilobase pairs produces nicked circular duplex as product (Fig. 2A). We have examined a variety of reaction conditions including the levels of magnesium, pH, and various types of salt on the hRad51 recombinase activity. As documented below, the most dramatic effects were seen with the addition of salts.


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Fig. 2.   Homologous DNA pairing and strand exchange by hRad51 and hRPA. A, schematic of the reaction. The phi X174 viral (+)-strand (ss) is paired with the linear homologous duplex (ds) to form joint molecules (jm), and strand exchange over the length of the DNA (5.4 kilobase pairs) yields nicked circular duplex (nc) and linear ssDNA as products. B, hRad51-mediated homologous DNA pairing and strand exchange. Panel I shows reactions carried out with ammonium sulfate as salt and with ATP (lanes 1-9) or without ATP (lanes 10-12). The reactant concentrations were: 7.5 µM hRad51, 2 µM hRPA, 30 µM (in nucleotides) each of ssDNA and dsDNA. Panel II shows reactions carried out as described by Baumann and West (9, 10), with ATP (lanes 1-10) or without it (lanes 11-13). The reactant concentrations were: 5 µM hRad51, 1 µM hRPA, and 30 µM (in nucleotides) each of ssDNA and dsDNA. C, in panel I, the time courses of conversion of the input linear duplex into nicked circular duplex in the experiments in panels I () and II (open circle ) of B are graphed. In panel II, the time courses of conversion of the input linear duplex into total products (joint molecules and nicked circular duplex) in the experiments in panels I () and II (open circle ) of B are graphed.

We tested the effects of increasing concentrations of potassium acetate, potassium chloride, potassium phosphate, potassium sulfate, ammonium chloride, and ammonium sulfate, and found that while all of these salts were stimulatory, ammonium sulfate produced the most stimulation, followed by potassium sulfate. Panel I in Fig. 2B shows a time course experiment in which 7.5 µM hRad51 was used with 2 µM hRPA, phi X (+)-strand (30 µM nucleotides), and phi X linear duplex (30 µM nucleotides) in pH 7.8 buffer and 100 mM ammonium sulfate. Following the published conditions of Baumann and West (9, 10), another reaction was also carried out in which hRad51, at 5 µM, was used with 1 µM hRPA and the same concentrations of DNA substrates and 80 mM potassium acetate at pH 7.5 (Fig. 2B, panel II). The results showed a much higher level of DNA strand exchange under the new conditions. Specifically, whereas no full strand exchange product (nicked circular duplex) was detected under the published conditions (Fig. 2, B, panel II, and C, panel I) (9, 10), the inclusion of ammonium sulfate resulted in conversion of ~30 and ~60% of the linear duplex to nicked circular duplex after 30 and 60 min, respectively (Fig. 2, B, panel I, and C, panel I). Overall, there was a 3-4-fold increase in total products (joint molecules plus nicked circular duplex) in the reaction that employed ammonium sulfate (Fig. 2C, panel II). Even though 100 mM ammonium sulfate was found to be optimal, highly significant levels of homologous DNA pairing and complete DNA strand exchange were seen at reduced concentrations (50 and 75 mM) of the salt (data not shown).

In published studies (9, 10), 80 mM potassium acetate was employed. In agreement with the published work (9, 10), neither higher (up to 200 mM in 20 mM increments) nor lower concentrations of potassium acetate would improve homologous DNA pairing and strand exchange efficiency beyond the level seen in panel II of Fig. 2B. As expected from published work (8), omission of ATP from the reaction abolished strand exchange, either under our reaction conditions (Fig. 2B, panel I, lanes 10-12) or the published conditions (Fig. 2B, panel II, lanes 11-13) (9, 10).

Thus, the inclusion of ammonium sulfate renders hRad51-mediated ATP-dependent homologous DNA pairing and strand exchange highly efficient. Under the new reaction conditions, the optimal levels of hRad51 for pairing and strand exchange were found to be between 2 and 4 nucleotides/protein monomer. Increasing hRad51 above 2 nucleotides/protein monomer resulted in gradual inhibition (data not shown), which was likely due to binding of hRad51 to the duplex and its sequestration from pairing with the hRad51-ssDNA complex (7, 9).

Dependence on hRPA-- In the yRad51-mediated DNA strand exchange reaction that uses plasmid length DNA substrates, a strong dependence of the reaction efficiency on yRPA has been observed (2, 4). However, in the published work, when hRad51 was used at the optimal ratio of 3 nucleotides of ssDNA/hRad51 monomer, hRPA has no stimulatory effect on the reaction efficiency, and relatively high levels of hRPA were in fact strongly inhibitory (9, 10). We have examined whether under the newly devised reaction conditions, hRPA is required for strand exchange efficiency. Fig. 3 summarizes the results obtained with 7.5 µM hRad51, 30 µM nucleotides of ssDNA, 100 mM ammonium sulfate, and increasing concentrations of hRPA, from 0.4 to 4.0 µM. Whereas only negligible pairing and strand exchange was seen in the absence of hRPA, increasing concentrations of hRPA gave progressively higher levels of products (Fig. 3, panels I and II). The optimal level of hRPA was ~2 µM, although addition of as little as 0.4 µM hRPA gave highly notable stimulation. Importantly, increasing the hRPA concentration to 4 µM did not lower the level of products, which is very different from published studies (10) in which concentrations of hRPA >= 0.8 µM were found to be strongly inhibitory.


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Fig. 3.   Dependence of homologous DNA pairing and strand exchange on hRPA. hRad51 at 7.5 µM was incubated with 30 µM phi X ssDNA with or without increasing concentrations of hRPA, and the resulting hRad51-ssDNA nucleoprotein filaments were reacted with linear phi X dsDNA for 20 (), 40 (open circle ), and 60 (triangle ) min. In panel I, the level of full DNA strand exchange, as measured by the percent conversion of the input linear duplex to nicked circular duplex, was graphed. In panel II, the level of total products, joint molecules plus nicked circular duplex, was graphed.

Additional experiments revealed that at levels of hRad51 higher (2 nucleotides/hRad51 monomer) or lower (6 nucleotides/hRad51 monomer) than that (4 nucleotides/hRad51 monomer) used in Fig. 3, there is also a similar dependence of homologous pairing and strand exchange on hRPA. Likewise, at ammonium sulfate levels higher and lower than that used in prior experiments, we have also observed a similar dependence of the strand exchange reaction on hRPA. Control experiments confirmed that hRPA by itself does not have homologous pairing and strand exchange activity under the new conditions (data not shown). In summary, under our reaction conditions, there is a uniform dependence of DNA strand exchange on hRPA, regardless of the amount of hRad51 used.

Effect of Order of Addition of Salt and Heterologous DNA-- In the experiments described thus far, ammonium sulfate was added to the reaction mixture after hRad51 had already nucleated onto the ssDNA but before the incorporation of hRPA. We have also examined whether the addition of ammonium sulfate at other stages would affect the reaction efficiency, as such an endeavor could yield important clues as to the basis of stimulation. As shown in Fig. 4, similar levels of homologous DNA pairing and strand exchange were observed when ammonium sulfate was added at the beginning with hRad51, after hRad51 but before the incorporation of hRPA (as in the standard reaction), and after hRPA but before the incorporation of the duplex. Interestingly, when ammonium sulfate was incorporated a few minutes after the duplex, there was little product formed even at the reaction end point of 60 min (Fig. 4A, lanes 11-13). Since dsDNA coated with hRad51 or yRad51 has been found to be inactive in the DNA strand exchange reaction (7, 9), we considered the possibility that perhaps the suppression of DNA strand exchange seen with ammonium sulfate being added after the duplex might have stemmed from free hRad51 binding to the duplex (7). However, two lines of evidence strongly suggest that this was not the main reason for the lack of strand exchange stimulation. First, even at levels of hRad51 (6 nucleotides and 9 nucleotides of ssDNA/hRad51 monomer) lower than that (4 nucleotides ssDNA/hRad51 monomer) used in Fig. 4 and with much longer preincubation of hRad51 with ssDNA to minimize the level of free hRad51, addition of ammonium sulfate before the duplex molecule is still necessary to see significant strand exchange (data not shown). Second, an excess of a heterologous duplex (pBluescript) added together with the homologous duplex or before the homologous duplex, in an attempt to titrate out any free hRad51, also did not compensate for the stimulatory effect of adding ammonium sulfate before the homologous duplex (see below). Taken together, the results strongly suggested that the lack of DNA strand exchange when ammonium sulfate was incorporated after the homologous duplex was due to a reason other than free hRad51 coating the duplex molecule.


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Fig. 4.   Order of addition of salt is critical for reaction efficiency. A, ammonium sulfate (100 mM) was added at the same time as hRad51 to phi X ssDNA (1o), after phi X ssDNA had been preincubated with hRad51 (2o), after phi X ssDNA had been preincubated first with hRad51 and then with hRPA (3o), or added 3 min after the duplex had already been incorporated into the reaction (4o). The complete reaction mixtures were incubated for 20, 40, and 60 min and processed for gel analysis. In lane 1, DNA substrates were incubated in the absence of recombination proteins. The reactant concentrations were: hRad51 at 7.5 µM, phi X ssDNA at 30 µM nucleotides, hRPA at 2 µM, and phi X dsDNA at 30 µM nucleotides. B, the levels of total products, joint molecules plus nicked circular duplex, were graphed.

Interestingly, whereas the incorporation of increasing amounts of the heterologous pBluescript duplex in the presence of ammonium sulfate lowered the reaction efficiency only slightly (Fig. 5, A, panel I, and B, panel I), the addition of pBluescript duplex before ammonium sulfate resulted in much more pronounced inhibition (Fig. 5, A, panel II, and B, panel II). These results, coupled with those presented above, indicated that binding of duplex to the hRad51-ssDNA nucleoprotein filament, regardless of whether the duplex is homologous to the ssDNA situated in the hRad51 filament, has a strong suppressive effect on pairing and strand exchange, unless salt is already present.


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Fig. 5.   Effect of heterologous duplex. A, increasing concentrations of pBluescript (1 to 3 times the concentration of phi X dsDNA) was added in the presence of ammonium sulfate (panel I) or 3 min before the incorporation of ammonium sulfate (panel II) to presynaptic complex assembled with hRad51 (7.5 µM), hRPA (2 µM), and phi X ssDNA (30 µM nucleotides). Following the incorporation of phi X duplex (30 µM nucleotides), the reaction mixtures were incubated for the indicated times. The concentration of ammonium sulfate was 100 mM. In lane 1 of both panels, DNA substrates were incubated in the absence of recombination proteins. B, the levels of total products, joint molecules plus nicked circular duplex, in A were graphed. Panel I shows the levels of products (joint molecules and nicked circular duplex) when pBluescript was added after ammonium sulfate and panel II shows the levels of products when pBluescript was added before ammonium sulfate.

In summary, the results have revealed a strict dependence of homologous DNA pairing and strand exchange efficiency on ammonium sulfate being incorporated into the reaction prior to the duplex, and they suggest that ammonium sulfate exerts its stimulatory effect via modulation of the interactions between the hRad51-ssDNA nucleoprotein complex and the incoming duplex molecule. This premise is further tested and verified in the experiments below.

Dependence of Strand Exchange Efficiency on Interactions between Duplex and hRad51-ssDNA Complex-- Extensive biochemical studies conducted with RecA have revealed that the incoming duplex molecule is bound only transiently within the RecA-ssDNA nucleoprotein filament (4, 5, see "Discussion"). We reasoned that if the hRad51-ssDNA filament has a relatively high affinity for the duplex, then the DNA homology search process might occur efficiently only when the association of the duplex molecule and the hRad51-ssDNA nucleoprotein filament is rendered transient, which could conceivably be realized by salt inclusion.

Intrinsic to this hypothesis are two predictions. First, it might be expected that the salt dependence of the homologous DNA pairing and strand exchange process would be lessened with reduction in the length of the DNA substrates, such as when oligonucleotides are used (11) (see Fig. 6A for schematic). This is because the extent of interactions of a short duplex with the limited length of hRad51-ssDNA nucleoprotein filament assembled on a short single-strand would not be extensive. Furthermore, the search for DNA homology with short DNA substrates would not be as rate-limiting as when phi X DNA substrates are used, because the probability of productive collisions between two short substrates leading to their homologous registry should be considerably higher. As predicted, when the DNA substrates used were based on 83-mer oligonucleotides, the rate of homologous pairing between the substrates was the same in the absence of salt as when ammonium sulfate was present at 25 mM, and higher levels of ammonium sulfate were in fact inhibitory (Fig. 6, B and C).


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Fig. 6.   Homologous pairing with oligonucleotide-based substrates is independent of salt. A, pairing and strand exchange between the unlabeled single-stranded oligonucleotide with the 32P-labeled duplex results in displacement of the radiolabeled single strand. B, hRad51 was incubated with the 83-mer oligonucleotide and the resulting nucleoprotein filament was reacted with the homologous duplex in the absence of salt or with increasing concentrations of ammonium sulfate, as indicated. In lane 1, DNA substrates were incubated in the absence of hRad51. C, the results from PhosphorImager analysis of the gel in B were plotted.

Second, if salt indeed acted to weaken the interaction between the duplex and the hRad51-ssDNA nucleoprotein complex, then we would expect to see lessened binding of the duplex molecule to the hRad51-ssDNA complex when ammonium sulfate was present. To test this premise experimentally, we examined the interaction of phi X linear dsDNA with hRad51-phi X ssDNA complex immobilized on streptavidin magnetic beads via a short biotinylated oligonucleotide, called F1b, which is complementary to a portion of the phi X (+)-strand (see schematic in panel I of Fig. 7A and "Experimental Procedures"). Analysis of the SDS eluate of the magnetic beads allowed us to determine the amount of duplex DNA that had bound to the immobilized hRad51-ssDNA complex (Fig. 7A, panel I). As shown in Fig. 7A, lanes 1 and 2 in panel II, incubation of the duplex with bead-immobilized hRad51-ssDNA complex in the absence of ammonium sulfate resulted in >90% retention of the duplex on the beads. Binding of the duplex to the magnetic beads was due to its interaction with the immobilized hRad51-ssDNA complex, because little retention of the duplex DNA occurred with magnetic beads pretreated with hRad51 and phi X (+)-strand hybridized to an oligonucleotide, F1, that had identical sequence to F1b but lacked the biotin tag of the latter (Fig. 7A, lanes 3 and 4 in panel II). As expected, only the background level of duplex retention was seen with beads containing the F1b-phi X (+)-strand hybrid but without hRad51, with DNA-free beads preincubated with hRad51, and with beads that contained only the F1b oligonucleotide and preincubated with hRad51 (Fig. 7A, lanes 5-10 in panel II).


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Fig. 7.   Salt lessens interactions between duplex and hRad51-ssDNA nucleoprotein filament. A, panel I, the assay scheme is summarized. Briefly, phi X (+)-strand hybridized to the biotinylated oligonucleotide F1b is immobilized on streptavidin magnetic beads via the biotin tag. hRad51 filament is assembled on the (+)-strand and then mixed with phi X duplex DNA. The bound duplex and hRad51 are eluted by SDS and analyzed. To verify the utility of the assay (panel II), beads containing phi X (+)-strand-F1b hybrid (phi X-F1b; lanes 1 and 2), beads preincubated with phi X (+)-strand hybridized to the nonbiotinylated oligonucleotide F1 (phi X-F1; lanes 3 and 4), beads with F1b but no phi X (+)-strand (F1b only; lanes 5 and 6), and beads that contained neither phi X (+)-strand nor F1b (beads only; lanes 7 and 8) were incubated with hRad51 and then mixed with phi X duplex. As an additional control, phi X duplex DNA was incubated with beads containing the phi X (+)-strand-F1b hybrid without hRad51 (phi X/F1b, No hRad51; lanes 9 and 10). The supernatants (S) and the SDS eluates (E) from the reactions were analyzed in an agarose gel for their content of duplex DNA. B, salt weakens interaction of duplex with the hRad51-ssDNA filament. Duplex phi X DNA was incubated with the hRad51 filament assembled on the immobilized phi X (+)-strand with increasing concentrations of ammonium sulfate. The supernatants (S) and SDS eluates (E) from the binding reactions were analyzed for their contents of DNA duplex (upper panel) and hRad51 (lower panel). C, the results in B are graphed (), as are results from binding reactions in which potassium acetate was used (open circle ). D, effect of order of addition of ammonium sulfate. Duplex DNA was added to binding reactions 3 min before or immediately after the incorporation of increasing levels of ammonium sulfate (panel I). The results with adding ammonium sulfate before (shaded bar) and after (dark bar) the incorporation of duplex are presented in the histogram in panel II.

Once the utility of the assay system was verified, we proceeded to test the effect of ammonium sulfate on the interactions of duplex DNA with the immobilized hRad51-ssDNA complex. The results revealed gradual weakening of the duplex/hRad51-ssDNA complex interactions by increasing levels of ammonium sulfate. Specifically, whereas greater than 90% retention of the duplex occurred in the absence of ammonium sulfate, less than 10% of the duplex was bound at 100 mM of the salt (Fig. 7, B, upper panel, and C). Analysis of the amount of hRad51 in the various SDS eluates showed that even the highest concentration of ammonium sulfate did not cause significant turnover of hRad51 from the bound ssDNA (Fig. 7B, lower panel). Taken together, we concluded that ammonium sulfate indeed weakens the binding of duplex DNA to the hRad51-ssDNA complex. Other experiments revealed that pBluescript duplex also binds to the immobilized hRad51-phi X ssDNA complex in a manner that is reduced by ammonium sulfate (data not shown), indicating that the hRad51-ssDNA complex can interact with both homologous and heterologous duplex molecules.

Interestingly, potassium acetate lessened the interaction between the duplex and the immobilized hRad51-ssDNA complex only slightly (Fig. 7C), and as expected, the hRad51-ssDNA complex was stable to potassium acetate (data not shown). Since potassium acetate is much less effective in the homologous pairing and strand exchange reaction (Fig. 2) (9, 10), the observation in Fig. 7C is again consistent with the suggestion that ammonium sulfate stimulates homologous pairing and strand exchange by attenuating the affinity of the hRad51-ssDNA nucleoprotein filament for the incoming duplex.

In the DNA strand exchange experiments, we found that the order of addition of ammonium sulfate relative to duplex DNA was important for ensuring strand exchange efficiency, such that if duplex DNA was added before ammonium sulfate, only negligible pairing and strand exchange was observed (see Fig. 4). Given this observation, we wanted to test whether the level of duplex retention by bead-immobilized hRad51-ssDNA complex would change with the order of addition of ammonium sulfate. To examine this, we used three different concentrations of ammonium sulfate (25, 50, and 100 mM) and added the salt either before the incorporation of duplex or after the duplex had already been preincubated with the bead-immobilized hRad51-ssDNA complex. The results from this experiment revealed that more of the duplex becomes associated with the hRad51-ssDNA complex with preincubation of duplex and Rad51-ssDNA complex prior to salt addition (Fig. 7D).

The experiments in Fig. 7 were conducted with hRad51-ssDNA nucleoprotein complex assembled in the absence of hRPA. We have obtained similar results when hRPA was included in the binding reaction (data not shown).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Homologous DNA Pairing and Strand Exchange by hRad51 and hRPA-- Both hRad51 and yRad51 are related in amino acid sequence and biological function to E. coli RecA. Like RecA, yRad51 forms nucleoprotein filaments on ssDNA and dsDNA in an ATP-dependent manner. Biochemical studies have indicated that the search for DNA homology in the incoming duplex DNA molecule and formation of heteroduplex joints with the duplex occur within the confines of the RecA-ssDNA and yRad51-ssDNA nucleoprotein filaments, which are also referred to presynaptic filaments. The assembly of the recombinase-ssDNA nucleoprotein filaments and the efficiency of subsequent homologous pairing and strand exchange are stimulated by the single-strand binding factor, SSB for RecA and yRPA for yRad51 (2, 4, 5). In the presence of ATP, hRad51 also forms a filament on ssDNA similar in structure to the equivalent nucleoprotein filaments assembled with RecA and yRad51 (3-5). However, published studies have suggested that hRad51 has only a modest ability to make DNA joints and an even lower capacity to promote DNA strand exchange. These published observations have suggested that either hRad51 participates in making DNA joints without catalyzing much DNA strand exchange, as discussed before (3), or that other reaction conditions are in fact required to reveal the strand exchange activity in hRad51. To entertain the latter possibility, we have explored a variety of reaction parameters for their effect on the hRad51 recombinase activity, and have shown that when ammonium sulfate is included, an avid capability of hRad51 to catalyze DNA joint formation and strand exchange is revealed. Moreover, formation of nicked circular duplex, the product of full DNA strand exchange, becomes completely dependent on hRPA. The dependence of homologous DNA pairing and strand exchange on hRPA is seen over a wide range of hRad51 concentrations, from below and above the optimal level. The requirement for hRPA in homologous DNA pairing and strand exchange is very likely due to its ability to minimize secondary structure in DNA, thus facilitating the assembly of a contiguous hRad51-ssDNA filament, as suggested in previous studies (2-5).

In summary, the results presented here demonstrate an intrinsic ability of hRad51 to form DNA joints efficiently and to catalyze a substantial amount of DNA strand exchange. These findings also reveal the functional dependence of hRad51 recombinase on the ssDNA binding factor hRPA. The ability of hRad51 and hRPA to promote DNA joint formation and extension of nascent heteroduplex joints by strand exchange is likely to be indispensable for various recombination reactions in vivo.

Possible Basis for Salt Stimulation of Homologous DNA Pairing and Strand Exchange-- Extensive biochemical studies have revealed the presence of two distinct DNA-binding sites in the RecA protein filament, with the initiating ssDNA substrate viewed as being situated within the "primary" site, while the incoming duplex molecule is bound within the "secondary" site of the presynaptic filament (4, 5). The search for DNA homology in the duplex DNA occurs by way of reiterative binding and release of the duplex until homology is located. For this random collision mode of DNA homology search to work efficiently, the incoming duplex molecule must be retained only transiently within the secondary site of the recombinase-ssDNA filament. Consistent with this deduction, evidence has been presented to suggest that the RecA-ssDNA filament has modest affinity for duplex DNA (4).

Given the structural and functional similarities between hRad51 and RecA, it seems reasonable to suggest that DNA homology conducted by the hRad51-ssDNA presynaptic filament also occurs by means of random association/dissociation of the duplex molecule with the former, with efficient homology search to be dictated by transient, rather than stable, association of the incoming duplex with the presynaptic filament. We have presented two lines of evidence to support the notion that salt exerts its remarkable stimulatory effect by weakening the interactions of the duplex molecule with the hRad51-ssDNA filament, thereby enhancing the rate of turnover and the efficiency at which the duplex can be sampled for homology. First, we have shown that the dependence of the homologous DNA pairing and strand exchange process on salt is alleviated if the length of the DNA substrates is reduced, as when oligonucleotides are used. This is likely due to the increased probability for productive collisions between the two oligonucleotide substrates that lead to homologous registry and also because the interactions between the duplex and the short presynaptic filament of hRad51 assembled on the ss oligonucleotide are similarly minimized. Furthermore, we have provided direct evidence that binding of a duplex molecule to hRad51 presynaptic filament is weakened by the inclusion of ammonium sulfate.

In summary, we surmise from the biochemical results that the hRad51-ssDNA filament binds duplex DNA in the available secondary site with high affinity, thus limiting efficient sampling of the duplex molecule for DNA homology. Accordingly, stimulation of homologous DNA pairing is realized by salt addition, with the degree of stimulation being dependent on the effectiveness of a particular salt to weaken the affinity of the hRad51-ssDNA nucleoprotein filament for the duplex molecule. We further suggest that the stimulation of strand exchange efficiency by ammonium sulfate is also due to increased turnover of the duplex DNA from the secondary DNA-binding site, as the ease with which the initial DNA joint can be extended by branch migration may be expected to be critically dependent on the hRad51-ssDNA nucleoprotein filament being free of stably bound duplex DNA molecules as well. These suggestions are summarized in our working model in Fig. 8.


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Fig. 8.   Model for hRad51-mediated DNA strand exchange. The results suggest that duplex DNA molecules stably bound to the hRad51-ssDNA nucleoprotein filament present a strong impediment to the different reaction steps, including DNA homology search, DNA pairing, and branch migration of the nascent DNA joint, that lead to successful recombination between the DNA substrates. Efficient DNA pairing and strand exchange is realized by lessening the duplex/hRad51-ssDNA interactions, achieved by the inclusion of salt.

It is reasonable to ask whether the requirement for a high level of salt for revealing the catalytic potential of the hRad51 recombinase is physiological. Concerning this point, it is important to note that the in vivo salt concentration is between 0.17 and 0.24 M (17). The specific stimulation of hRad51 strand exchange activity by salts could also be reflecting the requirement for a small molecule (a polyanion, for instance), a certain post-translational modification of hRad51 (phosphorylation, for instance), or the involvement of other recombination factors (see discussion below) in modulating the affinity of the hRad51-ssDNA filament for the incoming duplex DNA molecule.

Significance of the in Vitro DNA Strand Exchange System-- Studies on yRad51 using plasmid length DNA molecules as substrates have been instrumental for formulating biochemical models for understanding the functions of various RAD52 group proteins. For instance, in addition to the well documented stimulatory role of yRPA in yRad51-mediated DNA strand exchange, experiments which varied the order of addition of reaction components have revealed that yRPA, if added with or before yRad51 to the ssDNA substrate, can also compete with yRad51 for binding sites on the ssDNA and consequently suppress the assembly of yRad51-ssDNA nucleoprotein filament (2). The yeast RAD52 encoded product and the heterodimeric molecule of yRad55 and yRad57 proteins, referred to as recombination mediators, promote the assembly of the yRad51-ssDNA filament and help overcome the suppression of DNA strand exchange caused by coaddition of yRPA with yRad51 to the ssDNA substrate or by preincubation of ssDNA with yRPA (2, 18).

We have verified that preincubation of ssDNA with hRad51 before the incorporation of hRPA is in fact critical for homologous pairing and strand exchange efficiency,2 providing evidence that hRPA competes with hRad51 for binding sites on ssDNA. This observation suggests the existence of specific mediators in the human recombination machinery for promoting hRad51-ssDNA filament assembly when there is the need for hRad51 to compete with other single-strand binding factors for sites on the initiating ssDNA substrate. A possible mediator function may exist in various human recombination factors including hRad52 and a number of Rad55/Rad57-like proteins, namely XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D, which are all known to be involved in recombination and either directly, or through another recombination factor, physically interact with hRad51 (3, 19). Furthermore, it remains a distinct possibility that some of these other recombination factors are in fact integral components of the presynaptic filament, and as such, may modulate the dynamics of the presynaptic filament to facilitate sampling of duplex DNA for homology and to promote the formation of DNA joints once homology is located. The in vitro DNA strand exchange system with the defined biochemical parameters described herein should be well suited for examining the function of various recombination factors and the role of post-translational modifications of these recombination factors in the DNA strand exchange reaction.

Recently, hRad51 was shown to interact with the breast tumor suppressor BRCA2 (12). In the Capan-1 cell line defective in BRCA2 function, the DNA damage-induced formation of hRad51 nuclear foci is defective, suggesting the possibility that BRCA2 helps deliver hRad51 to the DNA substrate (12). Whether or not BRCA2 functions as a mediator to promote hRad51 nucleoprotein filament assembly can be tested with the in vitro DNA strand exchange system described herein.

    ACKNOWLEDGEMENTS

We are very grateful to Wen-Hwa Lee and Phang-Lang Chen for kindly providing the hRAD51 K313 cDNA and Marc Wold for the gift of plasmid p11d-tRPA.

    FOOTNOTES

* This work was supported by United States Public Health Service Grants RO1 ES07061, RO1GM57814, and PO1 CA81020, Army Research Grant DAMD 17-98-1-8247, and Army Training Grant DAMD17-99-1-9402.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 210-567-7216; Fax: 210-567-7277; E-mail: sung@uthscsa.edu.

Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M010011200

2 S. Sigurdsson and P. Sung, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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