Holliday Junction Binding Activity of the Human Rad51B Protein*

Hiroshi YokoyamaDagger §, Hitoshi KurumizakaDagger ||, Shukuko Ikawa**, Shigeyuki YokoyamaDagger §DaggerDagger, and Takehiko Shibata||**§§

From the Dagger  RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan, the  Cellular Signaling Laboratory, RIKEN Harima Institute at SPring-8, 1-1-1 Kohto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, the § Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the ** Cellular & Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, and the || Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 1637 Yana, Kisarazu, Chiba 292-0812, Japan

Received for publication, October 24, 2002, and in revised form, November 15, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human Rad51B protein is involved in the recombinational repair of damaged DNA. Chromosomal rearrangements of the Rad51B gene have been found in uterine leiomyoma patients, suggesting that the Rad51B gene suppresses tumorigenesis. In the present study, we found that the purified Rad51B protein bound to single-stranded DNA and double-stranded DNA in the presence of ATP and either Mg2+ or Mn2+ and hydrolyzed ATP in a DNA-dependent manner. When the synthetic Holliday junction was present along with the half-cruciform and double-stranded oligonucleotides, the Rad51B protein only bound to the synthetic Holliday junction, which mimics a key intermediate in homologous recombination. In contrast, the human Rad51 protein bound to all three DNA substrates with no obvious preference. Therefore, the Rad51B protein may have a specific function in Holliday junction processing in the homologous recombinational repair pathway in humans.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chromosomes are continuously subjected to attacks by exogenous and endogenous mutagens, which damage the genomic DNA. Chromosomal double strand breaks, which are potential inducers of chromosome aberrations and tumorigenesis, are caused by ionizing radiation, oxygen free-radicals, DNA cross-linking reagents, and DNA replication failure (1, 2). Homologous recombinational repair (HRR)1 is an accurate pathway for repair without base substitutions, deletions, and insertions, and therefore, it is important to maintain chromosomal integrity (3-5). In the HRR pathway, a single-stranded DNA (ssDNA) tail, which is produced at the site, invades a homologous region of the intact sister chromatid. Through this "homologous-pairing" step, an intermediate structure, the Holliday junction, in which two double-stranded DNA (dsDNA) molecules form a four-way junction (6), is generated between the damaged and intact chromatids (7). The Holliday junction can move along the DNA and has the ability to expand a heteroduplex region. This step, termed "branch migration," is essential for the HRR pathway.

In humans, genes involved in the HRR pathway, including Rad51, Rad52, Rad54, Rad54B, Brca1, Brca2, Xrcc2, Xrcc3, Rad51B/Rad51L1/hRec2 (Rad51B), Rad51C/Rad51L2 (Rad51C), and Rad51D/Rad51L3 (Rad51D), have been identified (8, 9). Mutations in several of these genes have been found in cancer patients but not in healthy individuals (2). Notably, gross chromosomal rearrangements of the Rad51B gene have been reported in uterine leiomyomas, involving translocations with the HMG1C gene (10, 11), and in a pulmonary chondroid hamartoma (12). In mice, disruption of the Rad51B gene results in early embryonic lethality (13), suggesting that the Rad51B gene product is essential for development. The Rad51B knockout in chicken DT40 cells impaired the HRR pathway and caused sensitivity to DNA-damaging agents such as cisplatin, mitomycin C, and gamma -rays (14). Therefore, the Rad51B gene product plays an essential role in the HRR pathway in vertebrates. The human Rad51B protein is composed of 350 amino acid residues (15, 16) and has about 20% amino acid identity with the human Rad51 protein, like other human proteins such as the Xrcc2, Xrcc3, Rad51C, and Rad51D proteins (17). Therefore, these five proteins, Xrcc2, Xrcc3, Rad51B, Rad51C, and Rad51D, which have similarity with Rad51, have been classified as Rad51 paralogs.

In the present study, we purified the human Rad51B protein as a recombinant protein and biochemically characterized it. The purified Rad51B protein bound to ssDNA and dsDNA in the presence of ATP and Mg2+ or Mn2+ and hydrolyzed ATP in a DNA-dependent manner. When the Holliday structure coexisted with the replication-fork-like structure and B-form DNA, Rad51B only bound to the synthetic Holliday junction, suggesting that Rad51B may play a role in Holliday junction processing in the HRR pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Overexpression and Purification of Rad51B-- The human RAD51B gene was isolated from a brain cDNA library (purchased from Clontech) by the polymerase chain reaction and was cloned in the NdeI site of the pET15b vector (Novagen). In this construct, the His6 tag sequence was fused at the N terminus of the gene. The Rad51B protein was expressed in the Escherichia coli JM109(DE3) strain, which also carried an expression vector for the minor tRNAs (Codon(+)RIL, Novagen). The cells producing the Rad51B protein were resuspended in a buffer containing 50 mM sodium phosphate (pH 6.7), 0.3 M NaCl, 2 mM 2-mercaptoethanol, 5 mM imidazole, and 10% glycerol and were disrupted by sonication. The cell debris was removed by centrifugation for 20 min at 30,000 × g, and the lysate was mixed gently by the batch method with ProBond beads (Invitrogen) at 4 °C for one hour. The Rad51B-bound ProBond beads were then packed into an Econo-column (Bio-Rad) and were washed with 30-column volumes of a buffer containing 50 mM sodium phosphate (pH 6.7), 0.3 M NaCl, 2 mM 2-mercaptoethanol, 60 mM imidazole, and 10% glycerol at a flow rate of about 0.3 ml/min. The His6-tagged Rad51B was eluted in a 30-column volume linear gradient from 60 to 400 mM imidazole in 50 mM sodium phosphate (pH 6.7), 0.3 M NaCl, 2 mM 2-mercaptoethanol, and 10% glycerol. The His6 tag was uncoupled from Rad51B by a digestion with 10 units of thrombin protease (Amersham Biosciences) per mg of Rad51B, and the protein was immediately dialyzed against a buffer containing 20 mM sodium phosphate (pH 7.2), 100 mM NaCl, 5 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol at 4 °C. Then, the fractions containing Rad51B were mixed with 7 ml of Heparin-Sepharose (Amersham Biosciences) at 4 °C for one hour and were packed into an Econo-column (Bio-Rad). The Heparin-Sepharose beads with Rad51B were washed with 10-column volumes of a buffer containing 20 mM sodium phosphate (pH 7.2), 100 mM NaCl, 5 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol, and the Rad51B was eluted in a 10-column volume linear gradient from 0.1 to 1.5 M NaCl in this buffer. The Rad51B was dialyzed against a buffer containing 20 mM sodium phosphate (pH 7.2), 100 mM NaCl, 5 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol and was subjected to Mono-Q (Amersham Biosciences) column chromatography. The Rad51B was eluted in a 4-column volume linear gradient from 0.1 to 0.3 M NaCl in buffer containing 20 mM sodium phosphate (pH 7.2), 5 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol. The concentration of the purified Rad51B protein was determined with a Bio-Rad protein assay kit, using bovine serum albumin (BSA) as the standard.

Purification of the Human Rad51 Protein-- The Rad51 expression vector was constructed as described (18). His6-tagged Rad51 was overexpressed in E. coli strain JM109(DE3), as described above. Harvested cells were disrupted by sonication in a buffer containing 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM 2-mercaptoethanol, 10 mM imidazole, 10% glycerol, and protease inhibitors (Complete EDTA-free, Roche Molecular Biochemicals). Lysates were mixed gently by the batch method with nickel-nitrilotriacetic acid agarose beads (Qiagen) at 4 °C for one hour. The Rad51-coupled nickel-nitrilotriacetic acid agarose beads were then packed into an Econo-column (Bio-Rad) and were washed with 30-column volumes of a buffer containing 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM 2-mercaptoethanol, 60 mM imidazole, and 10% glycerol at a flow rate of about 0.3 ml/min. The His6-tagged Rad51 was eluted in a 30-column volume linear gradient from 60 to 400 mM imidazole in the aforementioned buffer. The Rad51, which eluted in a broad peak, was collected and treated with 1 unit of thrombin protease (Amersham Biosciences) per mg of Rad51. The Rad51 protein was immediately dialyzed against a buffer containing 50 mM Tris-HCl (pH 8.0), 0.2 M KCl, 0.5 mM EDTA, 2 mM 2-mercaptoethanol, and 10% glycerol at 4 °C. The Rad51 protein precipitated overnight but re-dissolved after changing the dialysis buffer the following day. After more than 24 h of dialysis, the Rad51 protein was collected and filtered to remove the residual precipitates. About 20 mg of HsRad51, which was more than 99% pure as judged by SDS-PAGE and Coomassie Brilliant Blue staining, were then applied to a 1-ml Mono Q column (Amersham Biosciences) for concentration. The Mono Q column was washed with buffer (50 mM Tris-HCl (pH 8.0), 0.2 M KCl, 0.5 mM EDTA, 2 mM 2-mercaptoethanol, and 10% glycerol), and the HsRad51 protein was eluted with a 20-ml linear gradient from 0.2 to 0.6 M KCl in this buffer. The Rad51 protein eluted sharply between 0.3 and 0.4 M KCl, and the peak fractions had concentrations of about 2 mg/ml. These fractions were dialyzed against a buffer containing 20 mM Tris-HCl (pH 8.0), 0.1 M KCl, 0.5 mM EDTA, 2 mM 2-mercaptoethanol, and 10% glycerol and were used for subsequent studies. Protein concentrations were determined using the Bio-Rad protein assay kit with BSA as the standard.

Oligonucleotides-- An HPLC-purified 50-mer oligonucleotide (5'-ATTTC ATGCT AGACA GAAGA ATTCT CAGTA ACTTC TTTGT GCTGT GTGTA-3') was used for the D-loop formation assay. This sequence is derived from a clone of human alpha -satellite DNA. The DNA substrates used in the DNA-binding assays were identical to the synthetic Holliday junction (I) designed by Iwasaki et al. (19). The synthetic Holliday junction was composed of four 49-mer single-stranded oligonucleotides, 1, 2, 3, and 4, with the sequences 5'-ATCGA TGTCT CTAGA CAGCT GCTCA GGATT GATCT GTAAT GGCCT GGGA-3', 5'-GTCCC AGGCC ATTAC AGATC AATCC TGAGC ATGTT TACCA AGCGC ATTG-3', 5'-TGATC ACTTG CTAGC GTCGC AATCC TGAGC AGCTG TCTAG AGACA TCGA-3', and 5'-CCAAT GCGCT TGGTA AACAT GCTCA GGATT GCGAC GCTAG CAAGT GATC-3', respectively. The resulting junction contained a 12-bp homologous region at the center, and the four 5' ends had one base overhangs. The half-cruciform, which was produced by oligonucleotides 1 and 2, was composed of a 30-bp duplex region, and two 18-bp and 19-bp heterologous single strands. The dsDNA was produced by annealing oligonucleotide 2 with its complementary oligonucleotide. All of the oligonucleotides were purified by HPLC, and the DNA concentrations are expressed in moles of nucleotides.

Preparation of Closed Circular Double-stranded DNA and Circular Single-stranded DNA-- To avoid irreversible denaturation of the dsDNA, we prepared plasmid DNA without any treatment that would potentially cause denaturation, such as alkaline treatment, as described previously (20). The plasmid DNA (pGsat4; 3216 base pairs) containing the human alpha -satellite sequence was introduced into the E. coli DH5alpha strain, and the cells were cultured for 12-16 h at 37 °C. The cells were harvested, mildly disrupted with 0.5 mg/ml lysozyme and 0.1% sarkosyl, and centrifuged at 28,000 rpm for one hour. The supernatant containing the pGsat4 plasmid DNA was extracted with phenol/chloroform three times, and the pGsat4 DNA in the aqueous phase was precipitated by ethanol. The pellet was dissolved in 1 ml of TE buffer and was treated with 0.15 mg/ml RNaseA at 37 °C for 30 min. The pGsat4 DNA was purified by 5-20% sucrose gradient centrifugation at 24,000 rpm for 18 h. Phage circular ssDNAs (phi X174 and M13mp19) were prepared as described previously (20). DNA concentrations are expressed in moles of nucleotides.

Single-stranded DNA-binding Assay-- phi X174 ssDNA (40 µM) was mixed with the indicated amounts of Rad51B or Rad51 in 10 µl of standard reaction buffer containing 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM ATP, 1 mM dithiothreitol, 100 µg/ml BSA, 1 mM MgCl2, and 6% glycerol. The reaction mixtures were incubated at 37 °C for 10 min and were analyzed by 0.8% agarose gel electrophoresis in TAE buffer (40 mM Tris acetate, 1 mM EDTA). The bands were visualized by ethidium bromide staining.

Double-stranded DNA-binding Assay-- phi X174 superhelical dsDNA (10 µM) was mixed with the indicated amounts of Rad51B or Rad51 in 10 µl of standard reaction buffer containing 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM ATP (or dATP), 1 mM dithiothreitol, 100 µg/ml BSA, 1 mM MgCl2, and 6% glycerol. MgCl2 and ATP were omitted from the DNA-binding experiments testing for the requirements of MgCl2 and ATP. In the experiments testing for the requirements of divalent cations in the DNA binding, MgCl2 was substituted for the indicated amounts of MnCl2 or ZnCl2 in the standard reaction buffer. The reaction mixtures were incubated at 37 °C for 10 min, and the samples were electrophoresed on a 0.8% agarose gel for 4 h at 3 V/cm in 0.5 × TBE buffer. The bands were visualized by ethidium bromide staining.

Synthetic Holliday Junction, Half-cruciform, Single-stranded Oligonucleotide, and Double-stranded Oligonucleotide-binding Assays-- The indicated amounts of Rad51B were incubated with 2 µM single-stranded 49-mer oligonucleotide, 1 µM double-stranded 49-mer oligonucleotide, 1 µM half-cruciform, or 1 µM synthetic Holliday junction in 10 µl of standard reaction buffer containing 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM ATP, 1 mM dithiothreitol, 100 µg/ml BSA, 1 mM MgCl2, and 6% glycerol at 37 °C for 15 min. The samples were analyzed by electrophoresis on a 2.75% Nusieve GTG agarose gel in TBE buffer (90 mM Tris, 90 mM boric acid, and 2 mM EDTA (pH 8.3)). In the competitive DNA-binding assay, the indicated amounts of Rad51B were incubated at 37 °C for 15 min in the presence of 33 nM of each DNA substrate, the synthetic Holliday junction, the half-cruciform, and the dsDNA. The products were analyzed by 10% polyacrylamide gel electrophoresis in TBE buffer.

Assay for Homologous Pairing of ssDNA and dsDNA-- A 32P-labeled single-stranded 50-mer oligonucleotide (1 µM) was mixed with the indicated amounts of Rad51B or RecA in 10 µl of standard reaction buffer containing 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM ATP, 1 mM dithiothreitol, 100 µg/ml BSA, 6% glycerol, and 1 mM MgCl2. The mixtures were incubated at 37 °C for 5 min, and the reaction was started by the addition of pGsat4 form I DNA (20 µM). After an incubation at 37 °C for 15 min, the reaction was terminated by the addition of 0.5% SDS, and the proteins were removed from the DNA by treatment with proteinase K (0.7 mg/ml) followed by an incubation at 37 °C for 15 min. The products of homologous pairing (D-loops) were separated from the unreacted DNA substrates by 0.8% agarose gel electrophoresis in 0.5 × TBE buffer.

ATPase Assay-- The ATPase activity of Rad51B was analyzed by the release of 32Pi from [gamma -32P]ATP. The reaction mixtures contained 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM MgCl2, 10 µM ATP, 50 nCi [gamma -32P]ATP, 60 mM NaCl, 5 mM dithiothreitol, 6% glycerol, 100 µg/ml BSA, and 3 µM Rad51B. After an incubation at 37 °C for 1 h, the reactions were stopped by adding one-half volume of 0.5 M EDTA. The samples were separated by thin layer chromatography on polyethyleneimine-cellulose (Sigma) in a 0.5 M LiCl and 1.0 M formic acid solution and were quantified by a Fuji BAS2500 image analyzer.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of the Human Rad51B Protein-- To study the function of Rad51B, we overexpressed Rad51B in the E. coli recA- JM109(DE3) strain as a fusion protein with an N-terminal hexahistidine tag (His6 tag) containing a cleavage site for thrombin protease. The His6-tagged Rad51B protein was expressed by induction with isopropyl-1-thio-beta -D-galactopyranoside (Fig. 1, lane 3) and was purified by chromatography on a Ni2+-chelating column (ProBond, Invitrogen) (Fig. 1, lane 4). The His6 tag was uncoupled with thrombin protease (Amersham Biosciences) from the Rad51B portion, which migrated slightly faster than the His6-tagged Rad51B protein upon SDS-PAGE (15-25%) (Fig. 1, lane 5). Then, Rad51B was further purified by Heparin-Sepharose column chromatography (Amersham Biosciences) (Fig. 1, lane 6) followed by Mono Q column chromatography (Amersham Biosciences) (Fig. 1, lane 7).


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Fig. 1.   Purification of the human Rad51B protein. Proteins (2 µg) from each purification step were analyzed by 15-25% SDS-PAGE with Coomassie Brilliant Blue staining. Lane 1 indicates the molecular mass markers. Lanes 2 and 3 indicate the whole cell lysates of the E. coli JM109(DE3) cultures before and after induction with isopropyl-1-thio-beta -D-galactopyranoside, respectively. Lanes 4-7 indicate the samples from the peak ProBond fraction, the fraction after the removal of the hexahistidine tag, the peak Heparin-Sepharose fraction, and the peak MonoQ fraction, respectively.

The ATPase Activity of Rad51B-- The Rad51B protein contains the Walker ATPase motifs (17). As shown in Fig. 2, the purified Rad51B protein hydrolyzed ATP. The ATPase activity of Rad51B is enhanced in the presence of ssDNA, whereas superhelical dsDNA slightly stimulated it (Fig. 2). Therefore, Rad51B is a DNA-dependent ATPase like the bacterial RecA and eukaryotic Rad51 proteins. It has been reported that Rad51B directly interacts with Rad51C (21-28), and the Rad51B·Rad51C complex hydrolyzes ATP. The ATPase activity of the Rad51B·Rad51C complex is also significantly enhanced in the presence of ssDNA and is moderately stimulated in the presence of dsDNA (24). These characteristics of the Rad51B·Rad51C complex in the ATP hydrolysis are similar to those of Rad51B alone.


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Fig. 2.   The ATPase activity of Rad51B. A, Rad51B (3 µM) was incubated for 1 h with or without 75 µM of the M13mp19 circular ssDNA (lane 3) or 50 µM of the phi X174 dsDNA (lane 4) in the presence of [gamma -32P]ATP. The samples were separated by thin layer chromatography and were visualized with the BAS2500 image analyzer. Lanes 1 and 2 indicate the control experiments without protein and DNA, respectively. B, graphic representation of the time course experiments for the ATPase activities of Rad51B. The amounts of hydrolyzed Pi at the indicated time points are presented. Open circles and open triangles indicate the experiments with ssDNA and dsDNA, respectively, and closed circles and closed squares indicate the control experiments without protein and DNA, respectively.

The Homologous-pairing Activity of Rad51B-- We previously reported that the other Rad51 paralogs, the Xrcc2·Rad51D and Xrcc3·Rad51C complexes, catalyze homologous pairing, by which homologous joint molecules between ssDNA and dsDNA are formed during the HRR pathway (29, 30). A phylogenetic analysis has shown that Rad51B is evolutionally close to the other Rad51 paralogs (17). Therefore, we next examined whether Rad51B catalyzes the homologous pairing. To examine the homologous pairing activity of Rad51B, we employed the D-loop formation assay (Fig. 3A), which is a standard assay to evaluate the homologous-pairing activity of the bacterial RecA, RecT, and RecO proteins (31-33) and their eukaryotic homologs (34-39). In contrast to the previous observations with Xrcc2-Rad51D and Xrcc3-Rad51C, Rad51B did not catalyze homologous pairing by itself (Fig. 3B). This result suggests that Rad51B may have a different role from the other Rad51 paralogs in the HRR pathway.


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Fig. 3.   The homologous-pairing activity of Rad51B. A, schematic representation of the D-loop formation assay. In the reaction, an oligonucleotide 50-mer, labeled by [gamma -32P]ATP and T4 polynucleotide kinase at the 5' end, invades the homologous region of the superhelical dsDNA and forms joint molecules (D-loops). B, Rad51B and RecA were preincubated with the 32P-labeled oligonucleotide 50-mer (1 µM) at 37 °C for 5 min, and the reactions were started by the addition of the superhelical dsDNA (20 µM). The reactions were incubated at 37 °C for 15 min and were terminated by the addition of 0.5% SDS. Then, the proteins were removed by a treatment with proteinase K (0.7 mg/ml) at 37 °C for 15 min. The samples were separated by 0.8% agarose gel electrophoresis in 0.5 × TBE buffer and were analyzed with the BAS2500 image analyzer. Lane 1 indicates the negative control experiment without protein. Lanes 2 and 3 indicate the experiments with Rad51B, and lanes 4 and 5 indicate the experiments with RecA. The concentrations of Rad51B and RecA were 5.2 µM (lanes 2 and 4) and 7.8 µM (lanes 3 and 5).

The DNA-binding Activity of Rad51B-- We next examined the DNA-binding ability of Rad51B. phi X174 phage circular ssDNA (5386 bases) and superhelical dsDNA, containing 30% nicked circular dsDNA, were used for the ssDNA-binding and dsDNA-binding assays, respectively. As shown in Fig. 4A, Rad51B bound to the phage ssDNA (lanes 2-5), but its binding ability was extremely low as compared with that of Rad51 (lanes 7-9). In contrast, Rad51B clearly bound to both superhelical and nicked circular dsDNAs (Fig. 4B, lanes 2-6). The dsDNA binding by Rad51B was strongly dependent on the presence of both ATP and Mg2+, in contrast to the Rad51-dsDNA binding, which required neither ATP nor Mg2+ (Fig. 4C). Rad51B bound to dsDNA in the presence of Mn2+ instead of Mg2+; however, Zn2+ did not support its dsDNA binding (Fig. 4D). Therefore, these results indicate that DNA binding by Rad51B requires ATP together with either Mg2+ or Mn2+.


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Fig. 4.   The DNA-binding activities of Rad51B. A, circular ssDNA binding. phi X174 ssDNA (40 µM) was incubated with Rad51B or Rad51 at 37 °C for 15 min. The concentrations of Rad51B and Rad51 used in the ssDNA-binding experiments were 0.5, 1, 2, 4, and 6 µM (lanes 2-6), and 0.5, 1, and 2 µM (lanes 7-9), respectively. The samples were separated by 0.8% agarose gel electrophoresis in TAE buffer and were visualized by ethidium bromide staining. Lane 1 indicates the negative control experiments without protein. B, superhelical and nicked circular dsDNA binding. phi X174 superhelical dsDNA containing nicked circular dsDNA (10 µM) was incubated with Rad51B or Rad51 at 37 °C for 15 min. The concentrations of Rad51B and Rad51 used in the dsDNA-binding experiments were 0.5, 1, 2, 4, and 6 µM (lanes 2-6), and 0.5, 1, and 2 µM (lanes 7-9), respectively. The samples were separated by 0.8% agarose gel electrophoresis in 0.5 × TBE buffer and were visualized by ethidium bromide staining (B-D). NC and SC indicate nicked circular dsDNA and superhelical dsDNA, respectively. Lane 1 indicates the negative control experiment without protein. C, ATP and Mg2+ requirements for dsDNA binding. phi X174 dsDNA (10 µM) was incubated with Rad51B (4.4 µM) or Rad51 (4.4 µM) at 37 °C for 15 min in the presence of 1 mM MgCl2 and 1 mM ATP (lanes 4 and 7, respectively). Lane 1 indicates the negative control experiment without protein. Lanes 2-4 indicate the experiments with Rad51B, and lanes 5-7 indicate those with Rad51. Lanes 2 and 5 indicate the experiments without Mg2+ and ATP, and lanes 3 and 6 indicate those without ATP. D, Mg2+, Mn2+, and Zn2+ requirements for dsDNA binding. phi X174 dsDNA (10 µM) was incubated with Rad51B (3.3 µM) at 37 °C for 15 min in the presence of 1 mM MgCl2 (lane 3), 10 mM MgCl2 (lane 4), 1 mM MnCl2 (lane 5), 10 mM MnCl2 (lane 6), 1 mM ZnCl2 (lane 7), or 10 mM ZnCl2 (lane 8). Lane 1 indicates the negative control experiment without protein. Lane 2 indicates a negative control experiment without divalent cation.

Rad51B Specifically Binds to the Synthetic Holliday Junction-- Next, we tested the DNA binding of Rad51B to various DNA structures, including a single-stranded oligonucleotide 49-mer (ssDNA 49-mer), a double-stranded oligonucleotide 49-mer (dsDNA 49-mer), a replicational fork-like structure (half-cruciform), and a synthetic Holliday junction. The dsDNA, half-cruciform, and synthetic Holliday-junction structures were made with 49-mer oligonucleotides. The Holliday junction, a four-way junction of dsDNA molecules, is a key intermediate in the HRR pathway (6). As shown in Fig. 5, Rad51B efficiently bound to the synthetic Holliday junction (panel A), half-cruciform (panel B), and dsDNA (panel C) structures. Rad51B also bound to the ssDNA 49-mer (Fig. 5D), but its ability was weak, consistent with the experiment with the phage ssDNA (Fig. 4A).


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Fig. 5.   Structure-specific DNA binding of Rad51B. The indicated amounts of Rad51B were incubated with 1 µM of the 32P-labeled synthetic Holliday junction (A), half-cruciform (B), dsDNA 49-mer (C), or 2 µM of ssDNA 49-mer (D). Lane 1 indicates a negative control experiment without protein. The samples were separated by 2.75% agarose gel electrophoresis in TBE buffer and were analyzed by the BAS2500 image analyzer.

Then, we next examined the DNA-structure specificity of Rad51B in DNA binding. When the same amounts of the synthetic Holliday junction, half-cruciform, and dsDNA structures were incubated together, Rad51B only bound to the synthetic Holliday junction in the presence of these three substrates (Fig. 6A). In contrast, Rad51 bound to all three substrates without an obvious preference (Fig. 6B). Therefore, we conclude that Rad51B has specificity to the Holliday junction, and this specificity is not conserved in Rad51.


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Fig. 6.   Rad51B specifically binds to the synthetic Holliday junction. Increasing amounts of Rad51B (A) or Rad51 (B) were incubated with the DNA mixture containing the three 32P-labeled DNA substrates, the synthetic Holliday junction (33 nM), the half-cruciform (33 nM), and dsDNA (33 nM). The samples were separated by electrophoresis on a 10% non-denaturing polyacrylamide gel in TBE buffer and were analyzed by the BAS2500 image analyzer. The concentrations of Rad51B and Rad51 used in the DNA-binding experiments were 0.44, 0.88, 1.3, 1.8, 2.2, 2.6, and 3.1 µM (panel A, lanes 2-8), and 0.025, 0.05, 0.1, 0.25, 0.5, 1, and 2 µM (panel B, lanes 2-8), respectively. Lane 1 indicates the negative control experiment without protein.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As bacterial Holliday junction-binding proteins, the RuvA·RuvB complex (RuvAB), which promotes branch migration of the Holliday junction, has been found (19, 40-42). Crystallographic and electron micrographic analyses have shown that RuvA forms a tetramer, whereas RuvB forms hexameric and heptameric ring structures (43-53). Both RuvA and RuvB are ATP-binding proteins, and the branch migration promoted by the RuvAB complex requires ATP (19, 40, 41, 54). In addition, the E. coli RecG protein is a factor that creates a four-way junction by reversing the stalled replication fork (55, 56). In the present study we found that Rad51B specifically binds to the synthetic Holliday junction, suggesting that Rad51B may have a role in processing the Holliday-junction intermediate in HRR. However, we could not detect branch migration activity with Rad51B (data not shown). Helicase activity, which is required to promote efficient branch migration by the RuvAB complex or RecG, also was not detected with Rad51B (data not shown). Rad51B may require a subunit(s), like the bacterial RuvAB complex, if it is involved in the branch migration process in the HRR pathway.

Thus far, a eukaryotic nuclease, Mus81, which resolves the Holliday junction into two duplex DNAs, has been suggested to be a functional homolog of the bacterial Holliday-junction resolvase, the RuvC protein (57, 58). On the other hand, an ATP-dependent branch migration activity, which co-fractionates with the Holliday junction resolving activity, has been detected in mammalian cell-free extracts (59, 60), suggesting that the Holliday junction migrase and resolvase may form a complex in mammals, like the bacterial RuvA, RuvB, and RuvC proteins (61). Rad51B, which can target the Holliday junction structure, may be a subunit of the human Holliday junction-processing complex. In humans, the TIP60 complex, which is composed of fourteen subunits, was found to bind the Holliday junction structure and exhibited helicase activity, but not branch-migration activity (62). The TIP60 complex contains TIP49a (also known as RUVBL1, NMP238, or Pontin52) and TIP49b, which have similarity to RuvB (63-67). Rad51B may function in the Holliday junction processing with the TIP60 complex. Further studies are required to identify the factors that process the Holliday junction during HRR in mammals.

    ACKNOWLEDGEMENTS

We thank Dr. Hiroshi Iwasaki (Yokohama City University) for discussions. We also thank Takashi Kinebuchi, Wataru Kagawa, and Rima Enomoto (RIKEN Genomic Sciences Center) for technical assistance.

    FOOTNOTES

* This work was supported in part by the Bioarchitect Research Program (RIKEN), Core Research for Evolutional Science and Technology of Japan Science and Technology, and also by a grant-in-aid from the Ministry of Education, Sports, Culture, Science, and Technology, Japan.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 Dagger To whom correspondence may be addressed. E-mail: yokoyama@biochem.s.u-tokyo.ac.jp.

§§ To whom correspondence may be addressed. E-mail: tshibata@postman.riken.go.jp.

Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M210899200

    ABBREVIATIONS

The abbreviations used are: HRR, homologous recombinational repair; ss, single-stranded; ds, double-stranded; BSA, bovine serum albumin; HPLC, high pressure liquid chromatography; TAE, Tris-acetate/EDTA; TBE, Tris-borate/EDTA; SDS, sodium lauryl sulfate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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