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Genes to Cells (2004) 9, 781-790. doi:10.1111/j.1365-2443.2004.00772.x
© 2004 Blackwell Publishing or its licensors
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Mutational analyses of the human Rad51-Tyr315 residue, a site for phosphorylation in leukaemia cells

Yoshimasa Takizawa1,2,3, Takashi Kinebuchi3, Wataru Kagawa3, Shigeyuki Yokoyama3,4,5, Takehiko Shibata1,6,7 and Hitoshi Kurumizaka1,2,3,7,*

1 Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
2 Waseda University School of Science and Engineering, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan
3 RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
4 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
5 RIKEN Harima Institute at SPring-8, 1-1-1 Kohto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
6 Cellular & Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
7 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 1637 Yana, Kisarazu, Chiba 292-0812, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The human Rad51 protein, which plays a central role in homologous recombination, catalyses homologous pairing. The Rad51-Tyr315 residue is known to be constitutively phosphorylated in leukaemia cells and is thought to reside within the subunit-subunit interface of the Rad51 filament. To study the function of the Tyr315 residue, we purified five Rad51 mutants, Y315D, Y315E, Y315R, Y315A and Y315F, in which the Tyr315 residue was replaced by Asp, Glu, Arg, Ala and Phe, respectively. Biochemical studies of these Rad51 mutants revealed that the Y315D and Y315E mutants are defective in homologous pairing due to their impaired ssDNA binding, but their dsDNA binding remained unaffected. The Y315D, Y315E and Y315R mutants are defective in dsDNA unwinding, which depends on Rad51-filament formation, suggesting that these mutants are defective in filament formation on dsDNA. Therefore, the Rad51-Tyr315 residue plays important roles in ssDNA binding and filament formation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Homologous recombinational repair (HRR) is an accurate pathway for correcting chromosomal double-strand breaks (DSBs) that arise from exposure to DNA damaging agents (Pierce et al. 2001; Thompson & Schild 2001; Ferguson & Alt 2001; van Gent et al. 2001). Homologous recombination is also required in the replication process, to re-establish stalled or broken replication forks (Cox et al. 2000; Cox 2001; Aguilera 2001; West 2003). When the HRR pathway is defective, spontaneous chromosomal DSBs accumulate in cells (Sonoda et al. 2001), indicating that the HRR pathway is essential for chromosome maintenance.

A key step in the HRR pathway is homologous pairing, in which a 3' single-stranded DNA (ssDNA) tail derived from a DSB site invades the homologous double-stranded DNA (dsDNA) to form a heteroduplex (West 2003). In Escherichia coli, the RecA protein catalyses homologous pairing (McEntee et al. 1979; Shibata et al. 1979) and the Rad51 protein has been identified as a eukaryotic homologue of RecA (Shinohara et al. 1992, 1993). Like RecA, the Rad51 protein has been shown to promote homologous pairing in vitro (Sung 1994; Bauman et al. 1996; Gupta et al. 1997). The Rad51-gene knockout results in early embryonic lethality in mice (Lim & Hasty 1996; Tsuzuki et al. 1996) and causes cell death with the accumulation of spontaneous chromosome breaks in chicken DT40 cells (Sonoda et al. 1998). Mutations in the Rad51 gene have been identified in several tumours (Kato et al. 2000; Levy-Lahad et al. 2001; Wang et al. 2001; Blasiak et al. 2003; Jakubowska et al. 2003), suggesting the involvement of Rad51 in tumour suppression mechanisms.

In homologous pairing, RecA forms a helical filament on DNA. The RecA crystal structure revealed that a helical filament with six monomers per turn is organized by extensive interactions between the neighbouring RecA monomers (Story et al. 1992). Electron microscopic analyses showed that Rad51 forms a similar helical-filament (Ogawa et al. 1993; Benson et al. 1994), suggesting that this may be the functional form of Rad51 for homologous pairing, like RecA. Fluorescence spectroscopic analyses revealed that the Tyr191 and Tyr315 residues are probably located in the monomer-monomer interface of the Rad51 filament (Selmane et al. 2004; Conilleau et al. 2004). Interestingly, the Tyr315 residue of the human Rad51 protein is constitutively phosphorylated by the BCR/ABL fusion protein, which is derived from the translocation of the c-ABL gene from chromosome 9 to the BCR gene locus on chromosome 22 (Philadelphia chromosome) in leukaemia patients (Slupianek et al. 2001). The Tyr315 residue is also phosphorylated by c-Abl in the ATM-dependent manner under ionizing radiation conditions (Chen et al. 1999).

In order to study the function of the Tyr315 residue, we constructed five human Rad51 mutants, in which the Tyr315 residue was replaced by acidic amino acid residues, Asp and Glu, a basic amino acid residue, Arg, a small amino acid residue, Ala, or an aromatic amino acid residue, Phe. These Rad51 mutants were purified nearly to homogeneity and were tested for their biochemical activities in vitro.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Purification and ATPase activities of the Rad51 mutants

In the present study, we designed five Rad51-Tyr315 mutants, Y315D, Y315E, Y315R, Y315A and Y315F, in which the Tyr315 residue was replaced by Asp, Glu, Arg, Ala and Phe, respectively. Asp, Glu and Arg, which are negatively (Asp and Glu) and positively (Arg) charged hydrophilic residues, may change the hydrophobic environment between the Rad51 monomers. Ala, which contains the smallest side chain, may reduce the hydrophobic surface area around the 315th residue without adding the negative and positive charges. As a control, the Y315F mutant was constructed, because Phe is an aromatic amino acid residue that does not significantly change the hydrophobic environment created by Tyr315.

All five Rad51 mutants designed in this study were over-expressed in the E. coli recA- JM109 (DE3) strain, as fusion proteins with an N-terminal hexahistidine tag (His6 tag) and were purified by chromatography on nickel-nitrilotriacetic acid (Ni-NTA) agarose (Invitrogen). The His6 tag was then cleaved with thrombin protease (Amersham Biosciences) from the Rad51 portion (Fig. 1A) and the Rad51 mutants were further purified by MonoQ column chromatography (Amersham Biosciences) (Fig. 1B).



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  		Figure 1  Purification and ATPase activities of the Rad51-Tyr315 mutants. (A) The Y315D mutant (2 µg) was analysed by 15-25% SDS-PAGE with Coomassie Brilliant Blue staining. Lane 1 shows the molecular mass markers. Lanes 2 and 3 indicate the Y315D mutant after hexahistidine tag removal and the His6-tagged Y315D mutant purified by Ni-column chromatography, respectively. (B) The purified wild-type Rad51 protein (2 µg) and the Rad51-Tyr315 mutants (2 µg) were fractionated by 15–25% gradient SDS-PAGE. Lanes 1 and 2 indicate the molecular mass markers and the wild-type Rad51 protein, respectively. Lanes 3–7 indicate the Y315D, Y315E, Y315R, Y315A and Y315F mutants, respectively. (C, D) Graphic representations of the ATPase activities of the Rad51-Tyr315 mutants. The averages of three independent experiments with the wild-type Rad51 protein and the Rad51 mutants are shown with the SD values. Protein titration (C) and time course (D) experiments are shown. Closed and open symbols indicate experiments with the wild-type Rad51 protein and the Rad51-Tyr315 mutants, respectively. Circles indicate experiments in the presence of ssDNA; squares indicate control experiments in the absence of ssDNA. The corresponding mutants, Y315D, Y315E, Y315R, Y315A and Y315F, are indicated in the panels.

 
Rad51 contains Walker-type ATPase motifs (Thacker 1999) and hydrolyses ATP in the presence of ssDNA (Sung 1994; Benson et al. 1994). We tested the ssDNA-dependent ATP hydrolysis by the Rad51-Tyr315 mutants. Protein titration and time course experiments were performed. As shown in Fig. 1C,D, all five mutants purified in this study hydrolysed ATP as efficiently as the wild-type Rad51 protein. The efficiency of ATP hydrolysis may be reduced, if the mutations cause large structural changes in the Rad51 monomer. Therefore, these results suggested that the mutations at the Tyr315 residue do not significantly affect the Rad51 monomeric structure.

The homologous-pairing activity of the human Rad51 protein

To test the homologous-pairing activities of the Rad51-Tyr315 mutants, we employed the assay described in Fig. 2A (Lio et al. 2003). In this assay, a 63-mer ssDNA and a homologous 32-mer dsDNA were used as substrates. The strand containing a sequence identical to that of the 63-mer ssDNA in the dsDNA was labelled by 32P. In the reaction, the 32P-labelled 32-mer strand is displaced from the dsDNA, as a consequence of the homologous-pairing and subsequent strand-exchange reaction by Rad51. The displaced 32P-labelled 32-mer strand can be separately detected by 15% polyacrylamide gel electrophoresis. As shown in Fig. 2B, Rad51 catalysed homologous pairing between the 63-mer ssDNA and the homologous 32-mer dsDNA (lane 2). The displaced 32P-labelled 32-mer product was not detected, when either Rad51 or the 63-mer ssDNA was omitted from the reaction mixture (Fig. 2B, lanes 1 and 3, respectively), or when a heterologous ssDNA was used instead of the homologous 63-mer ssDNA (Fig. 2B, lane 4). Rad51 required ATP and a high Mg2+ ion concentration (more than 5 mM) for efficient homologous pairing and strand exchange (Fig. 2C). These results confirmed that Rad51 promotes homologous pairing and strand exchange in an ATP and Mg2+ dependent manner.



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  		Figure 2  The homologous-pairing assay. (A) Schematic representation of the homologous-pairing assay. The 32P-labelled strand is marked by an asterisk. (B) The wild-type Rad51 protein (10 µM) was incubated with a 63-mer ssDNA (15 µM) in 10 µL of standard reaction buffer, containing 20 mM Tris-HCl (pH 8.0), 1 mM ATP, 1 mM DTT, 100 µg/mL BSA, 20 mM MgCl2, 2 mM creatine phosphate, 75 µg/mL creatine kinase and 2% glycerol, at 37 °C for 10 min. The homologous-pairing reaction was initiated by the addition of the 32-mer dsDNA (1.5 µM), which contained a sequence homologous to that of the 63-mer ssDNA. After a 30 min incubation at 37 °C, the reaction was terminated and the samples were deproteinized. The DNA products were analysed by 15% polyacrylamide gel electrophoresis and were visualized by autoradiography of the dried gel. Lane 1 shows the control experiment without Rad51; lane 2 shows the experiment with homologous ssDNA and dsDNA. Lanes 3 and 4 indicate controls without ssDNA and with heterologous ssDNA, respectively. (C) Mg2+ and ATP are required for the Rad51-dependent homologous pairing. Lane 1 shows the control experiment without Rad51. Lanes 2–6 indicate the experiments with 1 mM, 2 mM, 5 mM, 10 mM and 20 mM Mg2+ concentrations, respectively. Lane 7 shows the experiment without ATP.

 
The homologous-pairing activities of the Rad51-Tyr315 mutants

We next performed a protein titration experiment for the homologous pairing of the Rad51-Tyr315 mutants. As shown in Fig. 3A, the homologous-pairing activity of the Y315D mutant was only slightly detected (lanes 5–7), but those of the Y315E, Y315R, Y315A and Y315F mutants were clearly detected in this assay (lanes 8–10 for Y315E, lanes 11–13 for Y315R, lanes 14–16 for Y315A and lanes 17–19 for Y315F). Mutant proteins often have different optimal pH values, because the pI of the protein may be changed by the amino acid replacement, especially with acidic and basic amino acid residues. However, none of the homologous-pairing abilities of the Rad51-Tyr315 mutants tested here were significantly changed in the four pH values, 6.0, 7.0, 8.0 and 9.0, tested in this assay (data not shown). Time course experiments revealed that the Y315D and Y315E mutants exhibited about 20% and 47% activities relative to the wild-type Rad51 protein in 30 minute reactions (Fig. 3B,C). In 10 minute reactions, the Y315D and Y315E mutants exhibited about 10% and 58% activities relative to the wild-type Rad51 protein. These results indicate that the Y315D mutant was significantly defective in homologous pairing and the Y315E mutant was moderately defective in it.



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  		Figure 3  The homologous-pairing activities of the Rad51-Tyr315 mutants. (A) Protein titration experiments. Reactions were performed as indicated in Figure 2. Lane 1 shows a control experiment without Rad51. The protein concentrations used in the homologous-pairing experiments were 1 µM (lanes 2, 5, 8, 11, 14 and 17), 5 µM (lanes 3, 6, 9, 12, 15 and 18) and 10 µM (lanes 4, 7, 10, 13, 16 and 19). (B–F) Time course experiments. The protein concentration was 5 µM in the time course experiments. Graphic representations of the homologous-pairing activities of the Rad51-Tyr315 mutants are shown. Closed circles indicate the experiments with the wild-type Rad51 protein; open circles indicate the experiments with the Rad51-Tyr315 mutants. Panels (B–F) show experiments with Y315D, Y315E, Y315R, Y315A and Y315F, respectively. The averages of three independent experiments with the wild-type Rad51 protein and the Rad51 mutants are shown with the SD values.

 
On the other hand, the Y315R, Y315A and Y315F mutants exhibited 64%, 64% and 71% homologous-pairing activities relative to the wild-type Rad51 protein, respectively, in 30 minute reactions (Fig. 3D–F). In 10 minute reactions, the Y315R, Y315A and Y315F mutants exhibited 73%, 72% and 77% homologous-pairing activities relative to the wild-type Rad51 protein, respectively. Therefore, the Y315A, Y315R and Y315F mutants promote homologous pairing with slightly reduced rates.

The ssDNA-binding abilities of the Rad51-Tyr315 mutants

We next tested the ssDNA-binding ability of the Rad51-Tyr315 mutants by a gel mobility shift assay with the M13mp18 ssDNA. As shown in Fig. 1(C,D), all of the mutants were proficient in the ssDNA-dependent ATPase activity, indicating that these Rad51-Tyr315 mutants bind to ssDNA. However, in the ssDNA-binding assay, we found that the Y315D and Y315E mutants were clearly defective in ssDNA binding (Fig. 4A, lanes 5–8 for Y315D and lanes 9–12 for Y315E). Therefore, the Y315D and Y315E mutants can bind to ssDNA, but their affinities for ssDNA are significantly reduced. In contrast, the positive control mutant, Y315F, was proficient in ssDNA binding (Fig. 4A, lanes 21–24). The reduced affinities of the Y315D and Y315E mutants for ssDNA are consistent with their defective homologous-pairing abilities.



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  		Figure 4  The DNA-binding activities of the Rad51-Tyr315 mutants. (A) The ssDNA-binding experiments. The M13mp18 ssDNA (20 µM) was incubated with the wild-type Rad51 protein or the Rad51-Tyr315 mutants at 37 °C for 10 min. (B) The dsDNA-binding experiments. The pGsat4 dsDNA (20 µM) was incubated with the wild-type Rad51 protein or the Rad51-Tyr315 mutants at 37 °C for 10 min. The samples were analysed by 0.8% agarose gel electrophoresis in 1 x  TAE buffer. Lanes 1, 5, 9, 13, 17 and 21 indicate control experiments without Rad51. The bands were visualized by ethidium bromide staining. The protein concentrations used in the ssDNA-binding experiments were 1 µM (lanes 2, 6, 10, 14, 18 and 22), 2 µM (lanes 3, 7, 11, 15, 19 and 23) and 4 µM (lanes 4, 8, 12, 16, 20 and 24).

 
Interestingly, the ssDNA binding of Rad51 was not affected when the Tyr315 residue was replaced by a basic amino acid residue, Arg, or a small amino acid residue, Ala (Fig. 4A, lanes 13–16 for Arg and lanes 17–20 for Ala), in contrast to the mutants in which the Tyr315 residue was replaced by an acidic amino acid residue, Asp or Glu. These results indicate that a negative charge, but not a positive charge (Y315R) or a methyl group (Y315A), interferes with the Rad51-ssDNA binding. The negatively charged phosphate backbone of ssDNA may bind near the 315th position of Rad51 and may be repulsed by the acidic amino acid residue introduced in this position of the Y315D and Y315E mutants.

The dsDNA-binding abilities of the Rad51-Tyr315 mutants

Next, we tested the dsDNA-binding ability of the Rad51-Tyr315 residue. In the dsDNA-binding assay, we used superhelical dsDNA (form I) containing nicked circular DNA (form II). In contrast to the ssDNA binding, all five of the mutants testedhere efficiently bound to dsDNA (Fig. 4B). These results suggest that dsDNA binds different sites from ssDNA on Rad51. It should be noted that, the wild-type Rad51 protein and the positive control mutant, Y315F, preferentially bound to the form I DNA rather than the form II DNA (Fig. 4B, lanes 1–4 for Rad51 and lanes 21–24 for Y315F). However, the Y315D, Y315E, Y315R and Y315A mutants did not show preferential binding to the form I DNA (Fig. 4B, lanes 5–8 for Y315D, lanes 9–12 for Y315E, lanes 13–16 for Y315R and lanes 17–20 for Y315A). The aromatic ring of the Tyr315 residue may function in specific binding to superhelical dsDNA, through an unknown mechanism.

The dsDNA-unwinding abilities of the Rad51-Tyr315 mutants

It has been reported that Rad51 extensively unwinds dsDNA in the Rad51-dsDNA filament, in an ATP-dependent manner (Benson et al. 1994). When a covalently closed dsDNA was used as the substrate for Rad51 binding, the ATP-dependent dsDNA unwinding by Rad51 resulted in highly negatively supercoiled DNA (form X), which can be detected by a topological assay after treatment with topoisomerase I and subsequent deproteination (Fig. 5A) (Benson et al. 1994; Solinger et al. 2002). The formation of form X depends on Rad51-filament formation, in which the dsDNA is unwound by the cooperative binding of Rad51. Therefore, using this assay, it is possible to detect defective filament formation on dsDNA by the Rad51 mutants.



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  		Figure 5  The dsDNA-unwinding activity of the Rad51-Tyr315 mutants. (A) Schematic representation of the topological assay for dsDNA unwinding. Supercoiled dsDNA is relaxed by eukaryotic topoisomerase I. The dsDNA is unwound by Rad51 binding and compensatory positive supercoils (+) are introduced. Highly negative supercoiled DNA (form X) is formed by relaxation of the dsDNA bound to Rad51 with topoisomerase I, followed by deproteination. (B) The dsDNA-unwinding activity of the Rad51-Tyr315 mutants. The {phi}X174 dsDNA (form I, 20 µM) was incubated with Rad51 (4 µM) at 23 °C for 30 min. Then, topoisomerase I (10 units) was added and the reaction mixture was incubated at 23 °C for 60 min. The reaction was stopped by adding 1 µL of 5% SDS followed by the immediate addition of 1 µL of 8.2 mg/mL proteinase K. The reaction products were separated on a 0.8% agarose gel in 1 x  TAE buffer with 5 µg/mL chloroquine and the bands were visualized by ethidium bromide staining. Lane 1 shows the supercoiled DNA (form I DNA); lane 2 shows the control experiment without Rad51. The experiments with Rad51 (lanes 3 and 4), Y315D (lanes 5 and 6), Y315E (lanes 7 and 8), Y315R (lanes 9 and 10), Y315A (lanes 11 and 12) and Y315F (lanes 13 and 14) are presented. Lanes 3, 5, 7, 9, 11 and 13 indicate the experiments in the absence of ATP and lanes 2, 4, 6, 8, 10, 12 and 14 indicate the experiments in the presence of ATP.

 
To distinguish the form X DNA product from the form I DNA, we analysed the reaction products with an agarose gel containing chloroquine, which changes the superhelical density of the plasmid DNA. As shown in Fig. 5B, the wild-type Rad51 protein formed a significant amount of form X DNA (lane 4), indicating that the dsDNA was extensively unwound through the Rad51-filament formation. When ATP was omitted from the reaction mixture, no form X DNA was detected (Fig. 5B, lane 3). These results confirmed that the dsDNA unwinding by Rad51 is an ATP-dependent reaction. In contrast, the Y315D mutant was significantly defective in the form X DNA formation (Fig. 5B, lane 6); the Y315E and Y315R mutants were moderately defective in it (lanes 8 and 10). While the Y315D, Y315E and Y315R mutants still retained the dsDNA-binding abilities and no free dsDNAs were observed in the reaction conditions used for the topological assay (Fig. 4B). Therefore, the Y315D, Y315E and Y315R mutants were defective in the dsDNA-unwinding ability, probably due to the defective filament formation.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The Rad51-Tyr315 residue is phosphorylated by the BCR/ABL tyrosine kinase in leukaemia cells (Slupianek et al. 2001). Constitutive Tyr315 phosphorylation is thought to enhance the recombination frequency in the cells. In the present study, we revealed that Tyr315 plays an essential role in ssDNA binding and in the filament formation on dsDNA, both being critical activities of Rad51. The role of Tyr315 in ssDNA binding has been demonstrated by the lack of ssDNA binding activity of the two point mutants (Y315D and Y315E). Whether Tyr315 directly interacts with ssDNA is unknown. The Tyr residue is capable of engaging in hydrophobic stacking interactions with the bases of ssDNA. If Tyr315 is located on the ssDNA-binding path on the Rad51 filament, then by mutating Tyr to acidic amino acid residues (Asp or Glu) would repulse with the negatively charged phosphate backbone of ssDNA.

In contrast, we found that the Y315D and Y315E mutants are proficient in dsDNA binding, suggesting that the ssDNA and dsDNA binding sites on the Rad51 filament is different. Interestingly, the structure-based mutational analysis of the human meiotic homologue of Rad51, Dmc1, has shown two distinct DNA binding sites of which one is specific for ssDNA binding (Kinebuchi et al. 2004). Furthermore, Shin et al. (2003) has proposed two DNA binding sites: One along the axis of the helical filament that binds ssDNA and the other along the groove of the filament that binds dsDNA. These observations suggest that the separation of ssDNA and dsDNA binding sites is one of the key features of Rad51 and its homologues.

The other finding was that Tyr315 is likely to play an essential role in Rad51 filament formation. Point mutants (Y315D, Y315E and Y315R) proficient in dsDNA binding but defective in unwinding supercoiled DNA were identified, suggesting that these mutations inhibit the formation of functional Rad51 nucleoprotein filaments. Consistent with our findings, previous fluorescence spectroscopic analysis of the Y315W mutant has suggested that the Tyr315 residue is directly involved in the Rad51 filament formation (Conilleau et al. 2004).

Based on the present observations, the Tyr315 residue appears to play a pivotal role on how Rad51 function. Hence, the modulation of the Rad51 function by the phosphorylation of the Tyr315 residue can be easily imagined. Our results suggest that the phosphorylation may affect ssDNA binding and filament formation by Rad51. How these activities are related to the enhanced recombination frequencies in leukaemia cells must be resolved by further analyses using the phosphorylated Rad51 protein.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Over-expression and purification of the Rad51 protein and the Rad51-Tyr315 mutants

The human Rad51 protein was prepared as previously described (Kurumizaka et al. 1999; Kagawa et al. 2001). The pET15b expression vectors, containing the Rad51-Tyr315 mutant genes inserted at the NdeI site, were constructed using the Quik-Change kit (Stratagene). The hexahistidine-tagged Rad51 mutants were over-expressed in the E. coli strain JM109 (DE3), which also carried an expression vector for the minor tRNAs (Codon(+)RIL, Novagen). The Rad51-Tyr315 mutants expressed in the E. coli strain were purified by the same method as that used for the wild-type Rad51 protein. Protein concentrations were determined using the Bio-Rad protein assay kit, with bovine serum albumin (Pierce) as the standard protein.

DNAs

The M13mp18 phage ssDNA used in the ssDNA-binding and ATPase assays was purchased (New England Biolabs). High-pressure liquid chromatography-purified oligonucleotides for strand exchange were purchased (Nihon Gene Research Laboratory). The DNA sequences used in this assay are as follows (Lio et al. 2003):

63-mer 5'-TCC TTT TGA TAA GAG GTC ATT TTT GCG GAT GGC TTA GAG CTT AAT TGC TGA ATC TGG TGC TGT-3'

32-mer top strand 5'-CCA TCC GCA AAA ATG ACC TCT TAT CAA AAG GA-3'

32-mer bottom strand 5'-TCC TTT TGA TAA GAG GTC ATT TTT GCG GAT GG-3'.

The 5' end of the oligonucleotide was labelled by T4 polynucleotide kinase (New England Biolabs) in the presence of [{gamma}-32P]ATP. The pGsat4 dsDNA used in the dsDNA-binding assay was prepared by the method previously described (Kagawa et al. 2001). All DNA concentrations are expressed in moles of nucleotides.

ATP hydrolysis assay

The ATPase activities of the Rad51 protein and the Rad51-Tyr315 mutants were analysed by the release of 32Pi from [{gamma}-32P]ATP. The 10 µL reaction mixtures contained 20 mM Tris-HCl (pH 8.0), 100 µM ATP, 50 nCi [{gamma}-32P]ATP, 1 mM DTT, 100 µg/mL BSA, 1 mM MgCl2, 2% glycerol, 30 µM M13mp18 ssDNA (7249 bases; New England Biolabs) and the indicated amounts of the Rad51 protein or the Rad51-Tyr315 mutants. The reactions proceeded for the indicated times and were stopped by adding 5 µL of 0.5 M EDTA. The 32Pi released from [{gamma}-32P]ATP was separated by thin layer chromatography on polyethyleneimine-cellulose (Sigma) in a 0.5 M LiCl and 1.0 M formic acid solution and was quantified by a Fuji BAS2500 image analyser.

Assays for ssDNA and dsDNA binding

The M13mp18 ssDNA (20 µM) was mixed with the Rad51 protein or the Rad51-Tyr315 mutants in 10 µM of standard reaction buffer, containing 20 mM Tris-HCl (pH 8.0), 100 µM ATP, 1 mM DTT, 100 µg/mL BSA, 1 mM MgCl2 and 2% glycerol. The reaction mixtures were incubated at 37 °C for 10 min and were then analysed by 0.8% agarose gel electrophoresis in 1 x  TAE buffer (40 mM Tris-acetate and 1 mM EDTA) at 3.3 V/cm for 1 h. The bands were visualized by ethidium bromide staining. For the dsDNA binding, Rad51 was incubated with the pGsat4 dsDNA (20 µM). The reaction conditions were the same as those for ssDNA binding.

Assay for homologous pairing

The indicated amounts of the Rad51 protein or the Rad51-Tyr315 mutants were incubated with a 63-mer ssDNA (15 µM) in 10 µL of standard reaction buffer, containing 20 mM Tris-HCl (pH 8.0), 1 mM ATP, 1 mM DTT, 100 µg/mL BSA, 20 mM MgCl2, 2 mM creatine phosphate, 75 µg/mL creatine kinase and 2% glycerol, at 37 °C for 10 min. The homologous-pairing reaction was initiated by the addition of the 32-mer dsDNA (1.5 µM), which shared sequence homology with the 63-mer ssDNA. After incubations for the indicated times at 37 °C, the reactions were stopped by adding 1 µL of 5% SDS, followed by the immediate addition of 1 µL of 6.7 mg/mL proteinase K (Roche Molecular Biochemicals). The reaction mixtures were further incubated for 10 min at 37 °C. After sixfold loading dye was added, the reaction mixtures were subjected to 15% polyacrylamide gel electrophoresis in 1 x TBE buffer (90 mM Tris, 90 mM boric acid and 2 mM EDTA) at 10 V/cm for 5 h. The products were visualized by autoradiography of the dried gel.

dsDNA unwinding assay

The Rad51 protein (4 µM) or a Rad51-Tyr315 mutant was incubated with the supercoiled dsDNA ({phi}X174; 20 µM) in 10 µL of standard reaction buffer, containing 20 mM Tris-HCl (pH 8.0), 100 µM ATP, 100 µg/mL BSA, 1 mM MgCl2, 2 mM creatine phosphate (Roche Molecular Biochemicals), 75 µg/mL creatine kinase (Roche Molecular Biochemicals) and 2% glycerol at 23 °C for 30 min. After the incubation, 10 units of wheat germ topoisomerase I (Promega) were added and the dsDNA was allowed to relax for 1 h at 23 °C. The reaction was stopped by adding 1 µL of 5% SDS, followed by the immediate addition of 1 µL of 8.2 mg/mL proteinase K (Roche Molecular Biochemicals). The reaction mixtures were further incubated at 23 °C for 20 min. Then, the reaction mixtures were subjected to 0.8% agarose gel electrophoresis, with or without 5 µg/mL chloroquine, in 1 x TAE buffer (40 mM Tris-acetate and 1 mM EDTA) at 3.3 V/cm for 2 h. The bands were visualized by ethidium bromide staining.


   Acknowledgements
 
We thank Dr M. Takahashi (Nantes University) for useful discussions. This work was supported by the Bioarchitect Research Program (RIKEN), CREST of JST (Japan Science and Technology), the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses and also by Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Shu Narumiya

* Correspondence: E-mail: kurumizaka{at}waseda.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 23 April 2004
Accepted: 28 June 2004




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