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Originally published In Press as doi:10.1074/jbc.M410101200 on September 30, 2004

J. Biol. Chem., Vol. 279, Issue 50, 51973-51980, December 10, 2004
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Multiple Interactions with the Rad51 Recombinase Govern the Homologous Recombination Function of Rad54*

Markus Raschle{ddagger}§, Stephen Van Komen§, Peter Chi¶, Tom Ellenberger{ddagger}||, and Patrick Sung¶**

From the {ddagger}Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115-5730 and the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520

Received for publication, September 2, 2004 , and in revised form, September 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {Delta}129 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Homologous recombination (HR)1 is the major pathway for the error-free repair of DNA double strand breaks (1, 2). Impaired HR leads to genetic instability and the cancer phenotype in humans (3), as aptly illustrated in certain familial breast cancer patients who harbor mutations in the tumor suppressors BRCA1 and BRCA2 (4). Defective HR is also associated with a marked cellular sensitivity toward ionizing radiation, DNA cross-linking agents, and other DNA-damaging agents. More recently, a role for HR in the restart of collapsed replication forks has been suggested. In mitotic cells, the homologous DNA from the sister chromatid is most often used to direct the repair of damaged DNA by HR. During meiosis, recombination between homologous chromosomes from the maternal and paternal genomes is the basis for genetic diversity. Moreover, meiotic recombination leads to the physical linkage of homologous chromosomes that is indispensable for their proper segregation (5).

The genetic requirement for double strand break repair by HR is best understood in the budding yeast Saccharomyces cerevisiae (reviewed in Refs. 1, 2, 6, and 7). Mutant analyses in this organism have led to the identification of the RAD52 epistasis group of genes. Current models suggest that DNA double strand breaks are processed by a 5' -> 3' exonuclease and that the resulting single-stranded tails are initially occupied by the single-stranded DNA-binding protein RPA, which is subsequently replaced by a helical protein filament of the Rad51 recombinase. The assembly of the Rad51 single-stranded DNA nucleoprotein filament, also known as the presynaptic filament, is facilitated by several HR factors, namely Rad52, the Rad55-Rad57 complex, and possibly Rad54. Once assembled, the presynaptic filament conducts a search for a homologous double-stranded donor and invades the donor to yield a displacement loop (D-loop) (7), the length of which is extended by branch migration. The D-loop is finally resolved by one of several pathways (reviewed in Ref. 6).

Rad54 has been implicated in several distinct steps of the recombination reaction. Chromatin immunoprecipitation analyses suggest that Rad54 is recruited early to the site of a double strand break before synapsis of the recombining DNAs occurs (8). Rad54 promotes the assembly and enhances the stability of the presynaptic filament in vitro (8–10), but it is debatable whether these activities of Rad54 are germane for the targeting of Rad51 to recombination sites in cells (8, 11). Rad54 physically associates with Rad51 in vitro and in vivo (12–15). The amino acid sequence of Rad54 places it in the Swi2/Snf2 family of proteins (16). Rad54 hydrolyzes ATP in the presence of DNA, and the free energy derived from ATP hydrolysis fuels its translocation on double-stranded DNA, which induces dynamic topological changes in the DNA. Biochemical and scanning force microscopic analyses have revealed that both positive and negative supercoils are introduced by the translocating Rad54 (17–20). Moreover, negative supercoiling by Rad54 leads to transient DNA strand separation, as evidenced by an increased sensitivity of double-stranded DNA toward nicking by the single strand-specific nuclease P1 (19). It therefore appears that Rad54 is capable of separating the strands of the donor duplex molecule, rendering it accessible for strand invasion by the Rad51 presynaptic filament. Alternatively, or in addition, Rad54 may facilitate the search of DNA homology by pumping DNA along the presynaptic filament (9, 19). The intimate collaboration between Rad54 and Rad51 in DNA joint formation is readily demonstrated in the D-loop reaction (12). The specificity of this interaction is indicated by the fact that only proteins from the same species cooperate in D-loop formation (9, 21). Rad54 has recently been shown to remodel chromatin and to promote D-loop formation with chromatinized DNA templates (22–24). The biochemical and genetic experiments described above provide ample evidence for functional and physical interactions between Rad54 and the Rad51 recombinase. To begin dissecting the interactions between the two HR factors, we have carried out a deletion analysis of the S. cerevisiae Rad54 protein. Our results show that truncated forms of Rad54 that are compromised for physical interaction with Rad51 retain normal levels of ATPase and DNA supercoiling activities but are unresponsive to Rad51. The attenuated Rad51 binding activity coincides with an impairment of the ability of Rad54 to promote the D-loop reaction. Furthermore, our results provide evidence that Rad54 contacts Rad51 via separable epitopes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Partial Proteolysis and Amino-terminal Sequencing—Rad54 (15 µg) purified from a yeast strain tailored to overexpress this protein (12) was incubated with the indicated amount of chymotrypsin or elastase in 30 µl of buffer (20 mM Hepes, pH 7.5, 200 mM NaCl, 2 mM 2-mercaptoethanol, 10% glycerol) at 25 °C. Aliquots (5 µl) were taken at the indicated times, and the digestion was stopped by adding 5 µl of phenylmethylsulfonyl fluoride (50 µM) and 10 µl of 2x SDS loading buffer (30 mM Tris-HCl, pH 6.8, 5% glycerol, 1% SDS, 360 mM 2-mercaptoethanol). Samples were heated for 1 min at 70 °C, separated on 10% SDS-polyacrylamide gels, and stained with Coomassie Blue. For amino-terminal sequencing, proteins were blotted onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were briefly stained with Coomassie Blue (30 s) and destained with 10% acetic acid and 45% methanol, and the protein bands of interest were excised and analyzed by amino-terminal sequencing in the Molecular Biology Core Facility of the Dana Farber Cancer Institute (Boston, MA).

Purification of Full-length and Truncated Forms of Rad54 from Escherichia coli—DNA fragments encoding full-length and amino-terminally truncated variants of S. cerevisiae Rad54 protein were amplified by PCR and cloned into the BglII and XhoI restriction sites of pET32a (Novagen). Upstream primers include the consensus sequence for the Rhinovirus PreScission protease (Amersham Biosciences) and were designed to allow expression of in-frame fusion proteins that harbor a cleavable thioredoxin-His6 affinity tag at their amino terminus. BL21 Rosetta cells (Novagen) were transformed with the recombinant plasmids and grown at 30 °C to an A600 of 0.6. The cultures were shifted to 16 °C and induced with 0.1 mM isopropyl-1-thio-{beta}-D-galactopyranoside for 15 h. Cells were harvested by centrifugation and washed with phosphate-buffered saline containing 2 mM EDTA and protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml, 1 µg/ml aprotinin, 1 µg/ml E64, 5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 156 µg/ml benzamidine, and 1 µg/ml chymostatin) before being frozen in liquid nitrogen and stored at –80 °C. The thawed cells (100 g) were resuspended in 300 ml of buffer A (20 mM KH2PO4, pH 7.6, 10% glycerol, 10 mM 2-mercaptoethanol, 0.5 mM EDTA, and protease inhibitors as above) containing 150 mM KCl and lysed by passing twice through a cell disruptor (Avestin). Lysates were clarified for 30 min at 40,000 x g, and the supernatant was loaded onto a P11 phosphocellulose column (120 ml; Whatman). The P11 column was developed with a gradient of 100–1000 mM KCl in buffer A, with Rad54 eluting at ~400 mM KCl. Rad54 and other proteins were precipitated with 0.4 g/ml ammonium sulfate, redissolved in 40 ml of buffer B (20 mM KH2PO4, pH 8.0, 600 mM KCl, 10 mM imidazole, 10% glycerol, 5 mM 2-mercaptoethanol, 0.01% Nonidet P-40, and protease inhibitors as above), and loaded onto a 10-ml Ni2+-NTA-agarose (Qiagen) column. The Ni2+-NTA matrix was washed with 200 ml of buffer B containing 300 mM KCl, and bound proteins were eluted by a gradient of 10–250 mM imidazole in buffer B. After adding dithiothreitol (1 mM final) and EDTA (2 mM final) to the imidazole eluate that contained Rad54, the thioredoxin-His6 affinity tag was cleaved off by an overnight incubation at 4 °C with recombinant Rhinovirus PreScission protease (1:200 (w/w) of protease to Rad54). The released protein was diluted with buffer A to a conductivity reading corresponding to 100 mM KCl and loaded onto a Mono S column (8 ml), which was developed with a KCl gradient of 100–1000 mM KCl in buffer A. Peak fractions were pooled and applied onto a Superdex S-200 column (330 ml), which was developed with buffer C (20 mM Hepes, pH 7.5, 150 ml NaCl, 1 mM EDTA, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride). We also purified Rad54 and its truncated variants with the thioredoxin His6 affinity tag intact for the pull-down experiment presented in Fig. 4B.



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  		FIG. 4.
Physical interaction of full-length and truncated Rad54 proteins with Rad51. A, purified Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 were mixed with Affi-Gel 15 beads containing either Rad51 (Affi-Rad51) or bovine serum albumin (Affi-BSA) in buffer containing 150 mM KCl. The beads were collected by centrifugation, washed with buffer, and then eluted with SDS. The supernatant (S), wash (W), and SDS-eluate (E) were run in a 7.5% denaturing polyacrylamide gel followed by staining with Coomassie Blue. B, an ammonium sulfate precipitate of E. coli extract containing Rad51 was dissolved in buffer with either 25 or 200 mM KCl and mixed with glutathione-Sepharose beads containing GST-Rad54-(1–107) or GST. Beads were washed three times and then treated with SDS to elute bound Rad51. The supernatant (S) and SDS eluate (E) were analyzed by 12.5% SDS-PAGE and staining with Coomassie Blue. The arrow marks either GST or the GST-Rad54-(1–107) protein. Rad51 is also marked. C, purified Rad51 was incubated in buffer containing 15 mM KCl with Rad54, Rad54 {Delta}113, Rad54 {Delta}129, or alone and then mixed with Ni2+-NTA-agarose beads. The beads were collected by centrifugation, washed with buffer, and then eluted with imidazole. The supernatant (S), wash (W), and imidazole-eluate (E) were run in a 12.5% denaturing polyacrylamide gel and immunoblotted with anti-Rad51 antibodies.

 
DNA Substrates—{phi}X174 replicative form I DNA and viral (+)-strand DNA were from Invitrogen and New England Biolabs, respectively. The {phi}X replicative form I DNA was relaxed by treatment with calf thymus topoisomerase I (Invitrogen), as described previously (21). pBluescript form I double-stranded DNA was prepared using standard methods. For the DNA mobility shift assays, the 83-mer oligonucleotide 3 (21) was 5'-end-labeled with T4 polynucleotide kinase (Promega) and [{gamma}-32P]ATP (Amersham Biosciences). After removing the unincorporated nucleotide in a Spin 30 column (Bio-Rad), the radiolabeled oligonucleotide 3 was annealed to its complement by heating the mixture of the two oligonucleotides at 75 °C for 10 min and slow cooling to 23 °C. The resulting duplex was purified from a 10% polyacrylamide gel by overnight diffusion at 4 °C into TAE buffer (40 mM Tris acetate, pH 7.5, 0.5 mM EDTA), as described (21). The 90-mer oligonucleotide D1 used in the D-loop reaction is complementary to pBluescript SK DNA from positions 1932–2022 (25). The oligonucleotide was 5'-end-labeled with T4 polynucleotide kinase (Promega) and [{gamma}-32P] ATP and then purified using the MERmaid Spin Kit (Bio101). This substrate was stored in TE (10 mM Tris-HCl, pH 7.0, 0.5 mM EDTA).

ATPase Assay—Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 proteins (75 nM each) were incubated with {phi}X174 replicative form I DNA (30 µM base pairs) in 10 µl of buffer D (30 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM dithiothreitol, and 100 µg/ml bovine serum albumin (BSA)) with 1.5 mM [{gamma}-32P]ATP and the indicated amounts of KCl at 23 °C. To examine the effect of Rad51 on ATP hydrolysis by the full-length and truncated Rad54 proteins, Rad51 (300 nM) was mixed with Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 (75 nM each) at 0 °C for 20 min prior to the addition of {phi}X174 replicative form I DNA and incubation at 23 °C. The reaction was terminated by the addition of EDTA to 250 mM and then subject to thin layer chromatography in polyethyleneimine plates (J. T. Baker), as described (21). The amount of labeled phosphate released as a result of ATP hydrolysis was quantified by phosphorimaging analysis in a Personal FX phosphor imager with Quantity One software (Bio-Rad).

DNA Mobility Shift—Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 (50–200 nM each) and the 32P-labeled 83-mer duplex substrate (1.25 µM nucleotides) were incubated for 10 min at 23 °C in 10 µl of buffer D containing 60 mM KCl with or without 2.5 mM ATP and an ATP-regenerating system consisting of 10 mM creatine phosphate and 30 µg/ml creatine kinase. Where indicated, the reactions were deproteinated with 0.5% SDS and 0.5 mg/ml proteinase K at 37 °C for 5 min. After adding 2 µl of loading buffer (30 mM Tris-HCl, pH 7.4, 2 mM EDTA, 0.1% orange G, 50% glycerol), the reaction mixtures were analyzed in 10% native polyacrylamide gels run in TAE buffer at 4 °C. The gels were dried and analyzed in the phosphor imager.

Rad51 and Rad54 Complex Formation—Rad51 and BSA were coupled to Affi-Gel 15 beads (Bio-Rad) according to the manufacturer's instructions to create matrices containing 3 mg/ml Rad51 and 12 mg/ml BSA. To examine binding of Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 to the affinity beads, 5 µg of these proteins were mixed with 6 µl of Affi-Rad51 or Affi-BSA beads in 30 µl of binding buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.01% Nonidet P-40, 0.5 mM dithiothreitol, and 150 mM KCl). The reactions were incubated for 45 min on ice with gentle mixing every 2 min. The beads were collected by centrifugation, and the supernatant was removed. After being washed twice with 100 µl of binding buffer, the Affi-Rad51 and Affi-BSA beads were treated with 30 µl of 3% SDS at 37 °C for 10 min to elute bound Rad54. The supernatant containing unbound Rad54 (10 µl), the second wash (15 µl), and the SDS eluate (10 µl) were subject to SDS-PAGE in a 7.5% denaturing polyacrylamide gel to determine their content of Rad54. For affinity pull-down through the His6 tag on Rad54, Rad51 (3 µg) was incubated with His6-Rad54, His6-Rad54 {Delta}113, or His6-Rad54 {Delta}129 (1.25 µg) in 30 µl of buffer E (20 mM KH2PO4, pH 7.4, 0.01% Nonidet P-40, 0.5 mM EDTA, and 1 mM 2-mercaptoethanol) containing 15 mM KCl and 10 mM imidazole. After 30 min of incubation at 0 °C, 10 µl of nickel-NTA-agarose beads (Qiagen) were added, and the reaction mixtures were left at 0 °C for another 60 min, with gentle mixing every 2 min. The beads were washed twice with 30 µl of buffer D containing 15 mM imidazole before eluting the bound proteins from the nickel matrix with 30 µl of 200 mM imidazole in buffer D. The supernatant containing unbound Rad51 (10 µl), the second wash (15 µl), and the 200 mM imidazole eluate (10 µl) were subjected to SDS-PAGE in a 12.5% gel and immunoblot analysis with anti-Rad51 antibodies.

Interaction of GST-Rad54-(1–107) with Rad51—A DNA fragment encoding residues 1–107 of Rad54 was amplified by PCR and introduced into a modified version of pCDFDuet-1 (Novagen) that expresses the Rad54 fragment as a GST fusion in the E. coli Rosetta strain (Novagen). When the cell density reached an A600 of 0.6, 0.4 mM isopropyl-1-thio-{beta}-D-galactopyranoside was added, and cells were harvested after 3 h of incubation at 37°C. The cell pellet from 50 ml of culture was resuspended in 2 ml of buffer A containing 200 mM KCl, 1 mg/ml lysozyme (Sigma), and protease inhibitors, as above. Cells were lysed by sonication, and extracts were clarified by centrifugation (14,000 x g, 30 min), and 400 µl of the extract was mixed with 100 µl of glutathione-Sepharose beads (Amersham Biosciences) for 12 h at 4 °C. E. coli extract containing GST was similarly treated. The beads containing bound GST-Rad54-(1–107) or GST at 0.1 µg of protein/µl of beads were washed four times with 300 µl of buffer A containing 0.1% Nonidet P-40 (Sigma) and 25 or 200 mM KCl and stored in 100 µl of the buffer at 4 °C. To express Rad51 in E. coli, the RAD51 gene was amplified by PCR and introduced into pLant2B (RIL). This plasmid was transformed into BL21 cells. When the cell density reached A600 of 0.6, cells were shifted to 16 °C and induced with 0.1 mM isopropyl-1-thio-{beta}-D-galactopyranoside for 15 h. Cells from 6-liter cultures were harvested by centrifugation and resuspended in 150 ml of buffer A containing 200 mM KCl, 1 mg/ml lysozyme (Sigma), and protease inhibitors, as above. Cells were lysed by sonication, and extracts were clarified by centrifugation (40,000 x g, 30 min). The extract was treated with ammonium sulfate (0.22 g/ml), and the protein pellet was stored at 4 °C. For GST pull-down experiments, a small fraction of this ammonium sulfate pellet was dissolved in buffer A containing 0.1% Nonidet P-40 and protease inhibitors and then adjusted to either 25 mM KCl or 200 mM KCl. The protein solutions (30 µl) were added to 20 µl of glutathione-Sepharose beads containing either GST or GST-Rad54-(1–107) followed by a 1-h incubation at 4 °C with gentle mixing. After washing the beads three times with 50 µl of buffer containing either 25 or 200 mM KCl, bound proteins were eluted with 15 µl of 2x SDS-PAGE loading buffer. The supernatants (15 µl) and SDS eluates (15 µl) were resolved by 15% SDS-PAGE, and proteins were stained with Coomassie Blue.

Topoisomerase I-linked DNA Supercoiling Assay—The indicated amounts of Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 were incubated with topologically relaxed {phi}X174 DNA (18.5 µM base pairs) for 3 min at 23 °C in 9.5 µl of buffer D with the indicated concentrations of KCl, followed by the addition of 150 ng of E. coli topoisomerase I in 0.5 µl of buffer D. Reactions were incubated at 23 °C for 10 min and processed for agarose gel electrophoresis, as described (21). To examine the effect of Rad51 on Rad54-catalyzed supercoiling of DNA, Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 (40 nM each) were incubated with Rad51 (300 nM) in buffer D containing 20, 60, or 100 mM KCl at 4 °C for 20 min prior to the addition of the DNA substrate and incubation with topoisomerase at 23 °C.

D-loop Assay—D-loop reactions were performed by incubating radio-labeled 90-mer oligonucleotide substrate (3.6 µM nucleotides) with Rad51 (1.5 µM) in 10.5 µl of buffer D containing an ATP-regenerating system (20 mM creatine phosphate and 30 µg/ml creatine kinase) and the indicated concentrations of KCl for 5 min at 37 °C. Rad54, Rad54 {Delta}113, or Rad54 {Delta}129 (260 nM) was then added in 1 µl, and the mixture was incubated for 2 min at 23 °C. The D-loop reaction was initiated by adding pBluescript replicative form I DNA (35 µM base pairs) in 1 µl. The reaction mixtures were incubated at 23 °C for 4 min and processed for electrophoresis in 0.9% agarose gels in TAE buffer at 23 °C, as previously described (25). The gels were dried, and the radiolableled DNA species were visualized and quantified in the phosphor imager. The percentage of D-loop refers to the fraction of the oligonucleotide substrate being incorporated into the replicative form I substrate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Architecture of the Rad54 Protein—Several motifs (Fig. 1) are highly conserved among Rad54 homologues and other Swi2/Snf2-related enzymes (26). These motifs harbor consensus sequences that are related to the superfamily II helicases (SFII). Based on the crystal structures of the SFII helicases eIF4a, UvrB, and RecG, it can be predicted that the central region encompassing the seven helicase-like motifs constitutes the catalytic core of the enzyme that is likely to fold into a tandem repeat of two RecA-type subdomains (27). The regions being appended to the catalytic core are conserved only among the Rad54 homologues. These flanking regions probably mediate interactions with other recombination proteins. In fact, the amino terminus of Rad54 is involved in an interaction with the Rad51 protein (12–15) (this work).



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  		FIG. 1.
Architecture of Rad54 orthologues. Upper panel, schematic overview depicting the conserved regions of Rad54 (see "Results"). The helicase-like motifs that define the Swi2/Snf2 family of proteins are shown in gray (26), whereas sequences that are conserved only in Rad54 homologues are shown in black. The sites of proteolytic cleavage are indicated by the arrows (A and B), and the amino-terminal truncations of Rad54 are indicated below. Lower panel, sequence alignment showing the starting amino acid of the yeast Rad54 truncations {Delta}113 and {Delta}129 (hs, Homo sapiens; gg, Gallus gallus; dm, D. melanogaster; ce, Caenorhabditis elegans; sp, Schizosaccharomyces pombe; sc, S. cerevisiae).

 
The Amino Terminus of S. cerevisiae Rad54 Is Prone to Proteolytic Attack—Our results with full-length S. cerevisiae Rad54 overexpressed in yeast cells or bacteria indicated that the amino terminus of Rad54 is prone to proteolytic degradation. We used controlled proteolysis to more specifically examine the domain structure of Rad54. Chymotrypsin cleaves Rad54 at two distinct sites to yield proteolytic fragments of ~89 and 75 kDa, respectively (A and B in Fig. 1 and Fig. 2). Bands of very similar sizes were generated in digests with various other proteases (e.g. elastase (Fig. 2A) and proteinase K) (data not shown), indicating that an unstructured region near the amino terminus of Rad54 was being targeted for cleavage. Peptide sequencing unambiguously identified the amino termini of the chymotryptic fragments A and B (Fig. 2A) as amino acid residues 126 and 228 of Rad54, in agreement with the apparent molecular masses of these proteolytic fragments in SDS-PAGE analysis (Fig. 2A).



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  		FIG. 2.
Partial proteolysis of purified ScRad54. A, full-length Rad54 (15 µg) was incubated in a volume of 30 µl with chymotrypsin (Chym) or elastase (Elast) at 25 °C. Samples (5 µl each) were taken at the indicated time points, resolved by SDS-PAGE, and stained with Coomassie Blue. B, expression of a series of amino-terminally truncated Rad54 starting around the proteolytically sensitive sites (Figs. 1, A and B, and 2A) as thioredoxin-His6 fusions in E. coli. Cell lysates were analyzed by SDS-PAGE with Coomassie Blue staining. The gel contained the soluble (S) and insoluble (P) fraction for each construct.

 
Expression and Purification of Amino-terminally Truncated Variants of Rad54 —Based on the results of the partial proteolysis experiments described above and on sequence alignments, a number of amino-terminally truncated Rad54 proteins were expressed as thioredoxin-His6 fusion proteins in E. coli cells (Fig. 2B). In these constructs, a site that can be cleaved by the PreScission protease is present between the thioredoxin His6 tag and the Rad54 protein. Overexpression of all of the Rad54 proteins was contingent upon supplementation with rare tRNAs encoded by the Rosetta plasmid (data not shown). With the exception of Rad54 {Delta}321, which was entirely insoluble, varying amounts of all of the truncated Rad54 species were found in the soluble fraction (Fig. 2B). Rapid protein degradation has thus far prevented us from purifying a significant amount of Rad54 {Delta}227. Although full-length Rad54 and Rad54 {Delta}113 were prone to proteolysis during purification, it was possible to completely separate the degradation products away. Using a procedure that we have devised (Fig. 3A), 1-mg quantities of highly purified Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 (Fig. 3B) could be obtained from 24 liters of E. coli culture. During purification, we cleaved the thioredoxin His6 tag from Rad54 using the PreScission protease (Fig. 3A).



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  		FIG. 3.
Purification of full-length and truncated variants of Rad54. A, scheme for Rad54 purification. B, purified full-length Rad54 (lane 1), Rad54 {Delta}113 (lane 2), and Rad54 {Delta}129 (lane 3)(1 µg each) were resolved by SDS-PAGE and stained with Coomassie Blue.

 
Salt-sensitive Physical Interaction of Rad54 {Delta}113 and Rad54 {Delta}129 with Rad51—Rad54 was previously shown to interact with Rad51 in vivo and in vitro (12–15). In one study (15), a fragment of Rad54 encompassing the amino-terminal 113 residues was shown to interact with Rad51 in a Far Western blot analysis. We wanted to know whether the Rad54 {Delta}113 and Rad54 {Delta}129 proteins retain the ability to interact with Rad51. To determine this, purified Rad54 and the two truncated proteins were incubated with Affi-gel 15 beads containing covalently conjugated Rad51 or BSA. After washing with buffer containing 150 mM KCl, bound proteins were eluted with SDS and then analyzed by SDS-PAGE. As expected, full-length Rad54 was efficiently pulled-down by the Affi-Rad51 beads (Fig. 4A) but not in the control reaction by the Affi-BSA beads (Fig. 4A). Under these conditions, reproducibly, a significant amount of Rad54 {Delta}113 was retained on the Affi-Rad51 beads, whereas little or no Rad54 {Delta}129 was pulled down by the affinity beads (Fig. 4A). As expected, neither of the truncated Rad54 proteins bound to the control Affi-BSA beads. Taken together, the results reveal that with a relatively high level of KCl (150 mM), the deletion of the first 113 amino acid residues of Rad54 (Rad54 {Delta}113) attenuates its interaction with Rad51, whereas an additional deletion of 16 amino acid residues, as in Rad54 {Delta}129, abolishes complex formation with Rad51. That the N-terminal region of Rad54 harbors a free-standing Rad51 binding domain is indicated from the observations that (i) a soluble GST fusion protein containing the N-terminal 107 residues of Rad54, but not GST alone, bound Rad51 in a pull-down assay (Fig. 4B) and (ii) an insoluble GST fusion protein that harbors the N-terminal 113 residues of Rad54 was shown to interact with Rad51 in a far Western assay after in situ renaturation on a nitrocellulose membrane (15).

Since the Rad54 {Delta}129 protein is active in functional assays with Rad51 when the ionic strength is low (see below), it was of interest to see whether it would form a complex with Rad51 at a reduced KCl concentration. Because the Affi-Gel 15 beads bind Rad54 nonspecifically at low KCl concentrations, we used nickel-NTA-agarose beads to capture complexes of Rad51 and the full-length and truncated variants of Rad54 through the His6 affinity tag on the latter. For this purpose, we purified full-length Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 with the thioredoxin-His6 tag intact. Rad51 alone and Rad51 preincubated with full-length His6-Rad54, His6-Rad54 {Delta}113, or His6-Rad54 {Delta}129 were mixed with Ni2+-NTA-agarose beads in buffer containing 15 mM KCl. After washing with buffer, the immobilized Rad51-Rad54 complexes were eluted with SDS and analyzed by SDS-PAGE. As expected, Rad51 was found associated with full-length His6-Rad54 and His6-Rad54 {Delta}113 (Fig. 4C). Interestingly, at the low salt concentration used, a significant fraction of Rad51 bound to His6-Rad54 {Delta}129 as well (Fig. 4C). The control experiments confirmed that retention of Rad51 on the Ni2+-NTA-agarose beads required the presence of the Rad54 proteins. The results thus reveal the presence of a Rad51 binding epitope in Rad54 {Delta}129.

The Amino Terminus of Rad54 Is Dispensable for DNA Binding and DNA-dependent ATPase Activity—Based on the sequence conservation among Rad54, other Swi2/Snf2-like proteins, and a subset of DNA helicases, the region of Rad54 comprising amino acid residues 230–810 of Rad54 probably constitutes the catalytic core domain (Fig. 1). Because this region is left intact in the Rad54 {Delta}113 and Rad54 {Delta}129 proteins, we expected these proteins to be proficient in the DNA binding and enzymatic activities of Rad54. We conducted a series of biochemical experiments to validate this expectation. In DNA mobility shift assays, Rad54 {Delta}113 and Rad54 {Delta}129 were just as proficient as full-length Rad54 in binding radiolabeled double-stranded DNA, both at 60 and 100 mM KCl (Fig. 5A and data not shown). Consistent with this result, the two truncated Rad54 variants bind {phi}X174 (+) strand DNA as well as the full-length protein at various KCl concentrations (data not shown). Likewise, Rad54 {Delta}113 and Rad54 {Delta}129 showed a level of DNA-dependent ATPase activity indistinguishable from that of the full-length protein at several KCl concentrations (Fig. 6). These results argue that the amino-terminal deletions, {Delta}113 and {Delta}129, do not compromise the overall fold of Rad54, consistent with a modular architecture of Rad54 in which the catalytic function and the ability to interact with other recombination factors reside within distinct structural domains.



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  		FIG. 5.
DNA binding by Rad54, Rad54 {Delta}113, and Rad54 {Delta}129. A, the 32P-labeled 83-mer duplex (1.5 µM nucleotides) was incubated at 23 °C for 10 min with Rad54 (I), Rad54 {Delta}113 ({Delta}113; II), and Rad54 {Delta}129 ({Delta}129; III) at 50, 100, 150, and 200 nM (lanes 2–5) in the presence of ATP and 60 mM KCl. In lane 6, 200 nM full-length or truncated Rad54 was incubated with the DNA substrate in the absence of ATP. In lane 7, 200 nM full-length or truncated Rad54 was incubated with the DNA substrate in the presence of ATP, but the reaction was treated with SDS and proteinase K prior to gel analysis. No protein was added in lane 1. The reaction mixtures were resolved in 10% nondenaturing polyacrylamide gels, which were dried and subjected to phosphorimaging analysis. dsDNA, double-stranded DNA. B, graphical representation of the results from lanes 2–5 of A.

 



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  		FIG. 6.
ATP hydrolysis by Rad54, Rad54 {Delta}113, and Rad54 {Delta}129. To examine the effect of Rad51 on ATPase hydrolysis by full-length and truncated Rad54 proteins, 75 nM each of these proteins were incubated with radiolabeled ATP and {phi}X replicative form I DNA and with or without 300 nM Rad51 in reaction buffer containing either 20 mM (I), 60 mM (II), or 100 mM (III) KCl for the indicated times. Rad51 (300 nM) alone was also incubated with ATP and DNA. The data represent the average of at least three independent experiments.

 
Functional Interactions of Rad54{Delta}113 and Rad54{Delta}129 with Rad51 in ATP Hydrolysis and DNA Supercoiling—It was previously shown that Rad51 stimulates the DNA-dependent ATPase activity of Rad54 (9, 19, 22). We therefore asked whether Rad51 could stimulate the ATPase activity of the amino-terminally truncated Rad54 proteins. Since our pull-down experiments (Fig. 4) suggested that the residual Rad51 binding affinity of the truncated Rad54 mutants is sensitive to the ionic strength, ATPase activity was measured at 20, 60, and 100 mM KCl. In agreement with our other results, the Rad54 {Delta}113 and Rad54 {Delta}129 proteins showed essentially the same level of ATPase activity as the full-length protein at the three salt concentrations. As expected, Rad51 enhanced the ATPase activity of full-length Rad54 regardless of the ionic strength (Fig. 6). At the lowest KCl level, Rad51 also stimulated the ATPase activity of both Rad54 {Delta}113 and Rad54 {Delta}129 (Fig. 6). However, the stimulation of these Rad54 mutants by Rad51 was diminished with increasing salt concentrations (Fig. 6). Repeated experiments showed that the Rad51-dependent stimulation of the ATPase activity of Rad54 {Delta}129 is significantly more salt-sensitive than Rad54 {Delta}113 (Fig. 6).

Biochemical and scanning force microscopy studies have shown that Rad54 utilizes the free energy derived from ATP hydrolysis to translocate along double-stranded DNA, with positive supercoils being generated in front of the translocating Rad54 and negative supercoils in its wake (9, 18, 19). In order to analyze the degree of DNA supercoiling catalyzed by the truncated Rad54 proteins, topoisomerase I-linked supercoiling assays were carried out (Fig. 7) (19). Briefly, the negative supercoils generated by Rad54 are removed by E. coli topoisomerase I, resulting in the accumulation of a positively supercoiled product termed Form OW (overwound) that can be separated from the relaxed DNA substrate by agarose gel electrophoresis (Fig. 7A). As shown in Fig. 7B, both Rad54 {Delta}113 and Rad54 {Delta}129 showed the same level of DNA supercoiling activity as full-length Rad54 (Fig. 7B). The results thus confirm that both Rad54 {Delta}113 and Rad54 {Delta}129 are as capable as full-length Rad54 in translocating on and supercoiling DNA, and they agree with other results showing a wild type level of DNA binding and ATPase activities in these truncated proteins.



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  		FIG. 7.
Functional interaction of Rad54 {Delta}113 and Rad54 {Delta}129 proteins with Rad51 as revealed by a topoisomerase-linked DNA supercoiling assay. A, schematic of the topoisomerase I-linked DNA supercoiling assay (18, 19). Briefly, translocation of oligomeric Rad54 generates positive supercoils and negative supercoils in the DNA template, as depicted. In the assay, the negative supercoils are removed by E. coli topoisomerase I, resulting in the formation of a positively supercoiled species called Form OW. B, Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 (150 nM in lanes 2, 5, and 8; 300 nM in lanes 3, 6, and 9; 450 nM in lanes 4, 7, and 10) were incubated with topologically relaxed DNA (9 µM base pairs) and E. coli topoisomerase I in the presence of 60 mM KCl at 23 °C for 10 min. C, stimulation of DNA supercoiling by Rad51. Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 (40 nM each) were incubated with or without Rad51 (300 nM) in the presence of 20 mM KCl (lanes 2, 3, 6, 7, 10, and 11), 60 mM KCl (lanes 4, 8, and 12), or 110 mM KCl (lanes 5, 9, and 13) with topologically relaxed DNA (9 µM base pairs) and E. coli topoisomerase I at 23 °C for 10 min. In lane 1, topologically relaxed DNA was incubated in buffer containing 20 mM KCl with E. coli topoisomerase I but without any recombination protein. In lane 14, topologically relaxed DNA (9 µM base pairs) was incubated in buffer containing 20 mM KCl with Rad51 (300 nM) and E. coli topoisomerase I. Following deproteination, the reaction mixtures were analyzed in a 0.9% agarose gel, followed by staining with ethidium bromide.

 
The DNA supercoiling activity of Rad54 is also stimulated markedly by Rad51 (9, 19, 22). In order to analyze the stimulatory effect of Rad51, topoisomerase I-linked assays were carried with an amount (40 nM) of Rad54, Rad54 {Delta}113, and Rad54 {Delta}129 that by themselves produced only a small amount of Form OW product (Fig. 7C). In agreement with published results (9, 19, 22), the addition of Rad51 to full-length Rad54 enhanced the supercoiling activity severalfold, and a similar degree of stimulation was observed over a range of salt concentrations (Fig. 7C). By contrast, the Rad51-dependent stimulation of the two amino-terminally truncated Rad54 proteins was notably sensitive to the salt concentration. Specifically, although stimulation of both truncated proteins to the level seen with full-length Rad54 was observed at 20 mM KCl (Fig. 7C, lanes 7 and 11), the stimulatory effect gradually diminished with increasing salt concentrations, with Rad54 {Delta}129 being more salt-sensitive than Rad54 {Delta}113 in this regard. The results from the DNA supercoiling experiments, together with those from the affinity pull-down and ATPase assays (Figs. 4 and 6), reveal that the amino-terminal truncations in Rad54 compromise but do not eliminate the interaction with Rad51.

The Ionic Strength Modulates the Ability of Rad54 {Delta}113 and Rad54 {Delta}129 to Promote the Rad51-mediated D-loop Reaction— Whereas Rad51 alone has only a modest ability to form the D-loop, a specific interaction with Rad54 leads to efficient D-loop formation (9, 12, 19, 25). We examined the ability of Rad54 {Delta}113 and Rad54 {Delta}129 to promote the Rad51-mediated D-loop reaction by monitoring the incorporation of a radiolabeled oligonucleotide into a homologous duplex molecule (Fig. 8A). With full-length Rad54, the amount of D-loop increased slightly as the KCl concentration was raised from 20 to 50 or 80 mM KCl and remained nearly as high at 110 mM KCl (Fig. 8, B and C). Although both Rad54 {Delta}113 and Rad54 {Delta}129 are capable of promoting the Rad51-mediated D-loop reaction at 20 mM KCl, this ability is attenuated by raising the KCl concentration, with Rad54 {Delta}129 being significantly more sensitive to the salt (Fig. 8B). For instance, when the KCl concentration was elevated from 20 to 110 mM, the level of D-loop decreased by slightly more than 2-fold in the case of Rad54 {Delta}113, whereas little D-loop was seen with Rad54 {Delta}129 at this KCl concentration. Thus, just as we have observed in other assays (Figs. 6 and 7), the ability of the N-terminally truncated Rad54 proteins to function in the Rad51-mediated D-loop reaction is salt-sensitive, with Rad54 {Delta}129 being more so than Rad54 {Delta}113.



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  		FIG. 8.
Behavior of the Rad54 {Delta}113 and Rad54 {Delta}129 proteins in the Rad51-dependent D-loop reaction. A, schematic of the D-loop assay. Homologous pairing between a radiolabeled 90-mer oligonucleotide and supercoiled pBluescript DNA yields a D-loop. ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. B, D-loop reactions containing 1.5 µM Rad51 and 260 nM Rad54, Rad54 {Delta}113 ({Delta}113), or Rad54 {Delta}129 ({Delta}129) in buffer containing 20 mM KCl (lanes 2, 6, and 10), 50 mM KCl (lanes 3, 7, and 11), 80 mM KCl (lanes 4, 8, and 12), or 110 mM KCl (lanes 5, 9, and 13). Lane 14 contains a reaction with Rad51 (1.5 µM) in reaction buffer containing 20 mM KCl. In lane 1, the DNA substrates were incubated in buffer containing 20 mM KCl without any recombinant protein. All of the reaction mixtures were incubated at 23 °C for 4 min. The reaction mixtures were deproteinated and resolved in a 0.9% agarose gel, which was dried and subject to phosphorimaging analysis. C, graphical representation of the results from the experiment in B.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Whereas bacterial RecA forms D-loops efficiently without the aid of another protein factor, ScRad51 protein does not make an appreciable amount of D-loop unless ScRad54 is also present (12). Rad54 appears to be unique to eukaryotes, and several published studies employing two-hybrid, immunoprecipitation, in vitro pull-down with purified proteins, and far Western analysis have shown its physical interaction with Rad51 (12–15). Importantly, as a result of complex formation with Rad51, the ATPase and DNA supercoiling activities of Rad54 are greatly enhanced (9, 19, 22). Consistent with its homology to other Swi2/Snf2-like protein factors, Rad54 has a chromatin remodeling activity; this Rad54 function is also enhanced by Rad51 (22–24). In the in vitro setting and in chromatin immunoprecipitation experiments, a positive influence of Rad54 on the assembly of the Rad51 presynaptic filament has been noted (8–10). Furthermore, Rad54 enhances the DNA strand exchange rate by severalfold and can remove Rad51 from duplex DNA (28); the latter activity is thought to promote the recycling of Rad51 and facilitate the DNA synthesis reaction primed from the end of the invading single strand in the D-loop (28). From these published data, it seems clear that Rad51 and Rad54 functionally cooperate in multiple steps of HR and that the physical interaction between these two factors is important for their HR role.

In this study, we have used biochemical approaches to begin examining in greater detail how Rad54 contacts Rad51 and also addressing the functional significance of the Rad51-Rad54 complex. By controlled proteolysis, we have presented evidence that the N terminus of Rad54 is particularly sensitive to proteolytic attack, consistent with this region forming a domain separate from the core catalytic domain that imparts a DNA translocase activity to Rad54. Previously, Jiang et al. (15) demonstrated that an insoluble fragment of Rad54 encompassing the amino-terminal 113 residues, after its in situ renaturation on a nitrocellulose membrane following SDS-PAGE, is capable of interacting with Rad51 in a far Western analysis. In agreement with this finding, we have shown that Rad54 {Delta}113 is attenuated for Rad51 interaction and that a soluble GST fusion protein harboring the N-terminal 107 residues of Rad54 binds Rad51. Importantly, we have demonstrated that an additional deletion of the sequence spanning residues 114 and 129, as in Rad54 {Delta}129, further compromises the ability to bind Rad51. Accordingly, whereas Rad54 {Delta}113 functionally interacts with Rad51 in different assays at physiological ionic strength, Rad54 {Delta}129 is defective in this respect. Unexpectedly, when the ionic strength was reduced, the Rad54 {Delta}129 protein exhibits an easily detectable ability to physically and functionally interact with Rad51. Thus, our results confirm the presence of a Rad51 binding motif in the N-terminal portion in Rad54 from residue 1 to 107 and reveal the presence of other epitopes that contribute to Rad51 interaction.

Our results demonstrating that Rad54 {Delta}113 and Rad54 {Delta}129 proteins retain the ability to bind and synergize with Rad51 in functional assays (albeit being dependent on the ionic strength) appear to differ significantly from those recently reported for the Drosophila melanogaster Rad54 protein (DmRad54) (29). In the latter study, a deletion of as few as nine of the most N-terminal residues greatly impaired the ability of DmRad54 to functionally interact with DmRad51 in several assays, although the DmRad51 binding activities of the truncated Dm-Rad54 proteins were not examined. Additional experiments will be required to determine whether differences between our results with yeast Rad54 and those reported for DmRad54 (29) reflect real differences in how functional complexes of Rad51 and Rad54 are assembled in these organisms or if they are instead caused by different reaction conditions employed in vitro.


   FOOTNOTES
 
* This work was supported in part by Department of Energy Grant DE-FG02-01E263071 and National Institutes of Health (NIH) Grant GM52504 (to T. E.) and NIH Grants ES07061 and GM57814 (to P. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

|| The Hsien Wu and Daisy Yen Wu Professor at Harvard Medical School. To whom correspondence may be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115-5730. Tel.: 617-432-0458; Fax: 617-432-3880; E-mail: tome{at}hms.harvard.edu. ** To whom correspondence may be addressed: Dept. of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar St., C130 Sterling Hall of Medicine, New Haven, CT 06520. Tel.: 203-785-4552; Fax: 203-785-6404; E-mail: Patrick.Sung{at}yale.edu.

1 The abbreviations used are: HR, homologous recombination; NTA, nitrilotriacetic acid; BSA, bovine serum albumin; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Mike O'Donnell for the pLant2B vector.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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