Biochemical analysis of the human DMC1-I37N polymorphism Juri Hikiba 1 , Yoshimasa Takizawa 1 , Shukuko Ikawa 2 , Takehiko Shibata 2 and Hitoshi Kurumizaka 1 1 Laboratory of Structural Biology, Waseda University, Tokyo, Japan 2 Cellular & Molecular Biology Laboratory, RIKEN Advanced Science Institute, Saitama, Japan Two successive rounds of nuclear division, meiosis I and meiosis II, occur with a single round of DNA rep- lication during meiosis. These processes produce haploid gametes from diploid cells in eukaryotes [1]. Homologous recombination occurs between homolo- gous chromosomes during meiosis I. This process forms chiasma which ensure the correct segregation of homologous chromosomes into daughter cells at meiosis I [1–4]. The DMC1 protein was first discovered as a mei- osis-specific gene, whose mutants are defective in meiotic homologous recombination in the yeast Saccharomyces cerevisiae [5]. The DMC1 protein is a homolog of the bacterial RecA protein, and is highly conserved from yeast to human [6]. In eukaryotes, a second RecA homolog, the RAD51 protein, has also been found [7–12]. The RAD51 protein is produced in both meiotic and mitotic cells [9,10]. The knockout of the RAD51 gene results in early embryonic lethality in mice [13,14], and causes cell death, with the accumula- tion of spontaneous chromosome breaks, in chicken DT40 cells [15]. Therefore, the RAD51 protein is essential for the mitotic homologous recombinational repair of damaged DNA, as well as meiotic homolo- gous recombination. However, the DMC1 protein is produced only in meiotic cells [5,6], indicating its meio- sis-specific function. Consistent with this, DMC1 knockout mice are viable, but exhibit defects in meiotic recombination processes and sterility [16,17]. There- fore, the DMC1 protein is essential in meiotic homo- logous recombination. The DMC1 protein promotes homologous-pairing and strand-exchange reactions in the early stage of meiotic homologous recombination [18–21]. In bacteria, the RecA protein catalyzes the homologous- pairing and strand-exchange reactions [22–25]. These Keywords DMC1; DMC1-I37N; homologous recombination; meiosis; SNP Correspondence H. Kurumizaka, Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan Fax: +81 3 5367 2820 Tel: +81 3 5369 7315 E-mail: kurumizaka@waseda.jp (Received 5 September 2008, revised 6 November 2008, accepted 10 November 2008) doi:10.1111/j.1742-4658.2008.06786.x The DMC1 protein, a meiosis-specific DNA recombinase, promotes homol- ogous pairing and strand exchange. The I37N single nucleotide polymor- phism of the human DMC1 protein was reported as a result of human genome sequencing projects. In this study, we purified the human DMC1- I37N variant, as a recombinant protein. The DMC1 protein is known to require DNA for efficient ATP hydrolysis. By contrast, the DMC1-I37N variant efficiently hydrolyzed ATP in the absence of DNA. Like the con- ventional DMC1 protein, the DMC1-I37N variant promoted strand exchange, but it required a high Ca 2+ concentration (4–8 mm), a condition that inactivates the strand-exchange activity of the conventional DMC1 protein. These biochemical differences between the DMC1 and DMC1- I37N proteins suggest that the DMC1-I37N polymorphism may be a source of improper meiotic recombination, causing meiotic defects in humans. Abbreviations dsDNA, double-stranded DNA; ssDNA, single-stranded DNA. FEBS Journal 276 (2009) 457–465 ª 2008 The Authors Journal compilation ª 2008 FEBS 457 reactions occur between the 3¢ single-stranded DNA (ssDNA) tails generated at DSB sites and the double- stranded DNA (dsDNA) of the other, intact homo- logous chromosome. An autosomal-dominant dmc1 mutation, containing a single amino acid substitution, was isolated as a recombination-defective allele causing male-specific sterility in mice [26]. This indicates that a DMC1 polymorphism containing a single amino acid substitution may cause infertility by a meiotic defect in mammals. Supporting this hypothesis, the homozygous DMC1-M200V polymorphism has been identified by the sequencing of candidate genes from a set of infer- tile patients [27,28]. In addition, our previous study revealed that the human DMC1-M200V mutation or the corresponding Schizosaccharomyces pombe Dmc1 mutation partially impairs the recombination activities of the DMC1 protein in vitro and in vivo [29]. There- fore, a DMC1 single-nucleotide polymorphism has the potential to be a source of human infertility. In this study, we purified a human DMC1 polymor- phic variant, DMC1-I37N, in which the Ile37 residue is replaced by Asn. The DMC1-I37N variant hydro- lyzed ATP in the absence of DNA, and promoted strand exchange under high Ca 2+ concentrations, which were not suitable for the strand-exchange reac- tion by the conventional DMC1 protein. These find- ings provide important new insights into the biological consequences of the DMC1 polymorphic variant in human meiotic recombination. Results The human DMC1-I37N variant hydrolyzes ATP in the absence of DNA The DMC1-I37N polymorphism, in which the Ile37 residue is replaced by Asn, was found in human gen- ome-sequencing projects (NCBI rfSNP ID: rs1129426). To elucidate the biochemical consequences of this single amino acid substitution, we purified the human DMC1-I37N variant, as a bacterially expressed recom- binant protein, using a method including Ni-NTA agarose column chromatography, removal of the hexahistidine tag from the DMC1 portion with throm- bin protease and heparin Sepharose column chroma- tography (Fig. 1A). CD analysis revealed that the DMC1-I37N variant was thermally unstable, com- pared with the conventional DMC1 protein (Fig. 1B). We then tested the ATPase activity of the DMC1- I37N variant. The human DMC1 protein efficiently hydrolyzes ATP in the presence of ssDNA. In the 125 mm KCl concentration used in this assay, the ATPase activity of the DMC1 protein was not detected in the absence of ssDNA (Fig. 2A). By contrast, we found that the DMC1-I37N variant exhibited detect- able ATPase activity in the absence of ssDNA (Fig. 2B). To eliminate the possibility that the ATPase activity of the DMC1-I37N variant may be stimulated by contaminating DNA, we subjected the DMC1-I37N Markers I37N WT 132 A –12 –10 –8 –6 –4 –2 0 2 20 30 40 50 60 70 80 90 Temperature (ºC) CD (mdeg) I37N WT 66 30 17 116 42 200 B Fig. 1. Purification of the DMC1-I37N variant. (A) The human DMC1 protein and the DMC1-I37N variant were purified by a method including Ni-NTA agarose column chromatography, removal of the hexahistidine tag from the DMC1 portion with thrombin protease, and heparin Sepharose column chromatography. The purified DMC1 and DMC1-I37N proteins were analyzed by SDS ⁄ PAGE with Coomassie Brilliant Blue staining. Lane 1 indicates the molecular mass markers. Lanes 2 and 3 indicate the purified DMC1 and DMC1-I37N proteins, respec- tively. Asterisks indicate degradation products. (B) Temperature dependence of the CD effect at 222 nm, as a function of temperature, for the conventional DMC1 protein (closed circles with broken line) and the DMC1-I37N variant (open circles with solid line). Activity of the human DMC1-I37N variant J. Hikiba et al. 458 FEBS Journal 276 (2009) 457–465 ª 2008 The Authors Journal compilation ª 2008 FEBS variant sample used in the ATPase assay to agarose gel electrophoresis, and confirmed that detectable amounts of DNA were not present in the purified DMC1-I37N variant sample (data not shown). The A 280 ⁄ A 260 ratio (1.29) of the purified DMC1-I37N pro- tein was exactly the same as that of the conventional DMC1 protein, indicating that the DMC1-I37N prepa- ration did not contain contaminating nucleotides. In addition, DNaseI treatment did not affect the ATPase activity of the DMC1-I37N variant (Fig. 2C). There- fore, the DNA-independent ATPase activity of the DMC1-I37N variant may not be due to DNA contam- ination during protein purification. The ATPase activity of the DMC1 protein is also stimulated under high salt conditions without DNA. As shown in Fig. 2D, the DMC1 protein hydrolyzed ATP in the presence of salt concentrations > 0.5 m KCl, but did not under low salt conditions (0–0.3 m KCl). By contrast, the DMC1-I37N variant hydrolyzed ATP in the presence of low salt concentrations (< 0.5 m KCl), but did not under high salt conditions (> 1.0 m KCl) (Fig. 2D). These results indicated that the DMC1-I37N variant possesses ATPase activity, but it is very different from that of the conventional human DMC1 protein. Strand-exchange and homologous-pairing activities of the human DMC1-I37N variant We next tested the strand-exchange activity of the DMC1-I37N variant. In this assay, /X174 phage circular ssDNA (5386 bp) and linearized /X174 dsDNA (5386 bp) were used as DNA substrates. Both intermediate (joint molecule; JM) and complete strand- exchange products (nicked circular; NC) are detectable in this assay (Fig. 3A). We performed the strand-exchange reactions in the presence of 200 mm KCl, because the human DMC1 protein requires  200 mm KCl for the efficient pro- motion of strand exchange [20]. Under conditions with 200 mm KCl, the DMC1-I37N variant was sig- nificantly defective in the strand-exchange activity (Fig. 3B). The strand-exchange activity of the DMC1 protein is reportedly enhanced by Ca 2+ ions [21]. We found that the effects of the presence of Ca 2+ ions in the strand-exchange reactions were quite different between the DMC1 and DMC1-I37N proteins. As shown in Fig. 3C, the DMC1 protein efficiently pro- moted strand exchange under low Ca 2+ conditions (lanes 2, 4 and 6), but did not under high Ca 2+ con- ditions (lanes 8 and 10). By contrast, the DMC1- I37N variant promoted strand exchange only under high Ca 2+ conditions (Fig. 3C, lanes 9 and 11), but did not under low Ca 2+ conditions (Fig. 3C, lanes 3 and 5). Consistent with this, the DMC1-I37N variant promoted homologous pairing only under high Ca 2+ conditions, in contrast to the conventional DMC1 protein (Fig. 4B). In this assay, a single-stranded oli- gonucleotide 50-mer and supercoiled dsDNA were used as the substrates for homologous pairing (Fig. 4A). These results indicated that the optimal conditions for homologous pairing and strand 0 0.05 0.1 0.15 0.2 0.25 0.3 No DNA ssDNA Time (min) Phosphate (mM) Time (min) Phosphate (mM) 0 0.04 0.08 0.12 0.16 0.2 No DNA No DNA + DNaseI 0 0.05 0.1 0.15 0.2 WT I37N Phosphate (mM) KCl (M) 0 0.05 0.1 0.15 0.2 0.25 0.3 0 0.3 0.6 0.9 1.2 1.5 0 102030405060 0 102030405060 0 102030405060 Time (min) Phosphate (mM) No DNA ssDNA AB CD Fig. 2. The ATPase activity of the DMC1- I37N variant. (A) Time course experiments of ATP hydrolysis by the conventional DMC1 protein. Open and closed circles indi- cate the experiments in the presence and absence of ssDNA, respectively. (B) Time course experiments of ATP hydrolysis by the DMC1-I37N variant. Open and closed circles indicate the experiments in the pres- ence and absence of ssDNA, respectively. (C) DNaseI treatment. The ATPase assay with the DMC1-I37N variant was performed in the presence of DNaseI. Open and closed circles indicate the experiments in the pres- ence and absence of DNaseI, respectively. (D) KCl titration. The ATPase assays were performed with the indicated amounts of KCl. Open and closed circles indicate the experiments with the DMC1-I37N variant and the DMC1 protein, respectively. J. Hikiba et al. Activity of the human DMC1-I37N variant FEBS Journal 276 (2009) 457–465 ª 2008 The Authors Journal compilation ª 2008 FEBS 459 exchange are very different between the DMC1 and DMC1-I37N proteins. No synergistic or additive action between the DMC protein and the DMC1-I37N variant for strand exchange We then tested strand exchange under conditions where the conventional DMC1 and DMC1-I37N proteins co-existed in various stoichiometries. These conditions may mimic the heterozygous DMC1 ⁄ DMC1-I37N situation. As shown in Fig. 5, no syner- gistic or additive action in the strand-exchange reaction was observed when the conventional DMC1 and DMC1-I37N proteins co-existed. Under the conditions suitable for the conventional DMC1 pro- tein, the DMC1-I37N variant did not affect the strand-exchange reaction, because the reaction only depended on the DMC1 concentration (Fig. 5, lanes 1–6). Similarly, the conventional DMC1 protein did not affect the DMC1-I37N-mediated strand-exchange reaction under the conditions suitable for the DMC1-I37N variant (Fig. 5, lanes 7–12). These results suggested that the DMC1 and DMC1-I37N proteins may not functionally interact with each other, when they co-exist. DNA-binding activity of the human DMC1-I37N variant The Ile37 residue of the DMC1 protein is located in the N-terminal domain. The corresponding N-terminal domain of the RAD51 protein was suggested to inter- act directly with DNA [30,31]. Therefore, we tested the DNA-binding activity of the DMC1-I37N variant. The DMC1-I37N variant was completely proficient in the ssDNA binding (Fig. 6A). However, the dsDNA-bind- ing activity of the DMC1-I37N variant was clearly decreased, as compared to that of the conventional DMC1 protein (Fig. 6B). These results suggested that the N-terminal domain of the DMC1 protein is impor- tant in dsDNA binding. + ssDNA dsDNA JM (joint molecule) NC (nicked circular dsDNA) A + ssDNA No protein WT I37N dsDNA NC JM ssDNA B WT I37N 891011 1234567 1234567 CaCl 2 (mM) WT WT WT WT I37N I37N I37N I37N no protein 01248 C dsDNA NC JM ssDNA Fig. 3. The strand-exchange activity of the DMC1-I37N variant. (A) A schematic representation of the strand-exchange assay. (B) The DMC1 protein or the DMC1-I37N variant was incubated with /X174 circular ssDNA (20 l M) at 37 °C for 10 min. After the addition of RPA, /X174 linear dsDNA (20 l M) was added to initiate the reaction. The reactions were continued for the indicated times. The DNA products were then deproteinized, and were separated by 1% agarose gel electrophoresis in 1· TAE buffer at 3.3 VÆcm )1 for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecules and nicked circular DNA are indicated by JM and NC, respectively. Lane 1 indicates a negative control experiment without the DMC1 protein. Lanes 2–4 indicate experiments with the DMC1 protein, and lanes 5–7 indicate experiments with the DMC1-I37N variant. Protein concentrations were 3.75 l M (lanes 2 and 5), 7.5 lM (lanes 3 and 6) and 15 lM (lanes 4 and 7). (C) The strand-exchange assay with CaCl 2 . Reactions were performed in the presence of the DMC1 (6 lM) or DMC1-I37N (6 lM) protein in the same manner as described in (B), except for the presence of the indicated amounts of CaCl 2 . Activity of the human DMC1-I37N variant J. Hikiba et al. 460 FEBS Journal 276 (2009) 457–465 ª 2008 The Authors Journal compilation ª 2008 FEBS Discussion Human genome sequencing projects have identified many SNPs in the DMC1 gene locus. The DMC1- I37N polymorphism contains an amino acid substi- tution in the coding region of the DMC1 gene. The distribution of the DMC1-I37N polymorphism in the human population has not yet been elucidated. In this study, we purified the DMC1-I37N variant, and com- pared its biochemical properties with those of the con- ventional DMC1 protein. We have previously studied the human DMC1-M200V variant [29], which is suspected to be a source of infertility [27,28]. The DMC1-M200V variant was moderately defective in the recombinase activity in vitro and in vivo; however, its biochemical and structural characteristics are not sig- nificantly different from those of the DMC1 protein [29]. In contrast to the DMC1-M200V variant, in this study, we found that the DMC1-I37N variant pos- sesses quite different biochemical characteristics from those of the DMC1 protein. The DMC1 protein requires ssDNA or a high salt concentration for effi- cient ATP hydrolysis. We found that the DMC1-I37N variant hydrolyzed ATP in the absence of DNA or the presence of a low salt concentration. The DMC1-I37N variant promoted homologous pairing and strand exchange under high Ca 2+ conditions, but was com- pletely defective under the conditions suitable for the conventional DMC1 protein. Conversely, under such high Ca 2+ conditions, the DMC1 protein did not effi- ciently promote the strand-exchange reaction. There- fore, the DMC1-I37N variant has very different optimal conditions for its recombinase activity from those for the conventional DMC1 protein. Given that the conventional DMC1 protein has been evolutionally optimized for promoting proper meiotic recombination, the DMC1-I37N variant may cause seri- ous problems in meiosis. The I37N mutant, which requires a high Ca 2+ concentration for its recombinase activity, may be inactive in pachytene spermatocytes, because the Ca 2+ concentration of pachytene spermato- cytes is  30–40 nm in mammals [30]. Because the DMC1-I37N variant neither activated nor inhibited the B WT I37N CaCl 2 (mM) WT WT WT WT I37N I37N I37N I37N No protein 8 9 10 11 1234567 01248 ssDNA dsDNA D-loop A ssDNA D-loops Fig. 4. The homologous-pairing activity of the DMC1-I37N variant. (A) A schematic representation of the homologous-pairing assay. (B) The DMC1 (4 l M) or DMC1-I37N (4 lM) protein was incubated with the 32 P-labeled single-stranded oligonucleotide 50-mer in the presence of the indicated amounts of CaCl 2 (final concentration 1–8 m M), and the reaction was initiated by adding the supercoiled dsDNA. The reactions were conducted for 8 min, and the deprotei- nized products were resolved by 0.8% agarose gel electrophoresis in 1· TAE buffer at 3.3 VÆcm )1 for 2.5 h. Bands were visualized by an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). 89 10 11 12 WT I37N 1234567 0 0 6 06 3 3 4.5 1.5 0 4.5 1.5 0 0 6 06 3 3 4.5 1.5 0 4.5 1.5 200 m M KCl 50 m M KCl 8 m M CaCl 2 dsDNA NC JM ssDNA Fig. 5. The strand-exchange assay in the presence of various amounts of the DMC1 protein and the DMC1-I37N variant. The indicated amounts of the DMC1 and DMC1-I37N proteins (total 6 l M) were incubated with /X174 circular ssDNA at 37 °C for 10 min. After the addition of RPA, /X174 linear dsDNA was added to initiate the reaction. The reactions were continued for 60 min. J. Hikiba et al. Activity of the human DMC1-I37N variant FEBS Journal 276 (2009) 457–465 ª 2008 The Authors Journal compilation ª 2008 FEBS 461 strand-exchange reaction promoted by the DMC1 protein when it co-existed, the heterozygous DMC1 ⁄ DMC1-I37N situation may not cause serious meiotic defects, if a sufficient amount of the DMC1 pro- tein is produced. An infertile woman, who is homozy- gous for the human DMC1-M200V polymorphism, has been identified in a set of infertile patients [27,28], and a mouse DMC1 mutant containing a single amino acid substitution (Ala272 to pro, A272P) causes male-specific infertility [26]. These DMC1-M200V and DMC1-A272P proteins are moderately and significantly defective in the recombinase activity in vitro, respectively [26,29]. We report here that the DMC1-I37N protein is significantly defective in the recombinase activity under the condi- tions suitable for the DMC1 protein. Therefore, the DMC1-I37N polymorphism may have the potential to be a source of human infertility. Extensive SNP analyses of the DMC1-I37N polymorphism in the human popu- lation and in sets of infertile patients may be required to understand the relationship between the DMC1-I37N polymorphism and human infertility. The helical-filament structure is considered to be the active form for the recombinase activity in the DMC1 and RAD51 proteins and their orthologs. We and oth- ers [29,31–34] previously suggested that the N-terminal domain of the RAD51 and DMC1 proteins constitutes a DNA-binding path that runs between two consecu- tive N-terminal domains in the filament structure. This putative DNA-binding path may function to guide DNA into the DNA-binding loops, L1 and L2, which are located inside the filament [35–39]. We found that the DMC1-I37N variant is moderately defective in dsDNA binding. This result supports the idea that the N-terminal domain of the DMC1 protein functions as part of the DNA-binding path. Materials and methods Purification of the human DMC1 protein and the DMC1-I37N variant The human DMC1 protein and the DMC1-I37N variant were each overexpressed with the pET-15b plasmid system (Novagen, Darmstadt, Germany) in Escherichia coli strain BL21(DE3) Codon Plus (Stratagene, La Jolla, CA, USA), as hexahistidine-tagged proteins. The proteins were then puri- fied by the method described previously [40]. Briefly, cells producing the proteins were harvested and lyzed by sonica- tion in buffer A (50 mm Tris ⁄ HCl buffer pH 8.0, containing 0.5 m NaCl, 2 mm 2-mercaptoethanol, 10% glycerol and 5mm imidazole) on ice. The cell lysate was centrifuged at 27 700 g for 20 min, and the supernatant was gently mixed by the batch method with 4 mL of Ni-NTA agarose beads (QIAGEN, Hilden, Germany) at 4 °C for 1 h. The protein- bound beads were packed into an Econo-column (Bio-Rad Laboratories, Hercules, CA, USA) and washed with 30 col- umn volumes of buffer A. The human DMC1 protein was eluted in a 20 column volume linear gradient of 5–500 mm imidazole in buffer A. The peak fractions were collected, and thrombin protease (2 unitsÆmg )1 of the human DMC1 pro- tein; GE Healthcare Biosciences, Uppsala, Sweden) was added to remove the His-tag. The samples were then immedi- ately dialyzed overnight at 4 °C in buffer B (20 mm Tris ⁄ HCl buffer pH 8.0, containing 0.2 m KCl, 0.25 mm EDTA, 2 mm 2-mercaptoethanol and 10% glycerol). The human DMC1 protein, which now lacked the His-tag, was subjected to chromatography on a 4 mL Heparin-Sepharose (GE Health- care Biosciences) column. The column was washed with 20 column volumes of buffer B, and the protein was eluted with a 20 column volume linear gradient of 0.2–1.0 m KCl in buffer B. The purified proteins were concentrated with a centrifugal cartridge, and the buffer was exchanged with BA 1234567812345678 ssDNA dsDNA WT I37N No protein No protein WT I37N No protein No protein Fig. 6. The DNA-binding activity of the DMC1-I37N variant. (A) The /X174 circular ssDNA (20 lM) was mixed with the DMC1 protein (lanes 2–4) or the DMC1-I37N variant (lanes 6–8), and the reactions were conducted at 37 °C for 10 min. Samples were then analyzed by 0.8% agarose gel electrophoresis in 1· TAE buffer at 3.3 VÆcm )1 for 2.5 h. The bands were visualized by ethidium bromide staining. Protein con- centrations were 3.75 l M (lanes 2 and 6), 7.5 lM (lanes 3 and 7) and 15 lM (lanes 4 and 8). (B) The supercoiled /X174 dsDNA (10 lM) was used instead of the ssDNA. Activity of the human DMC1-I37N variant J. Hikiba et al. 462 FEBS Journal 276 (2009) 457–465 ª 2008 The Authors Journal compilation ª 2008 FEBS 20 mm Hepes–KOH buffer (pH 7.5), containing 500 mm KCl, 0.25 mm EDTA, 2 mm 2-mercaptoethanol and 10% glycerol. The protein concentration was determined using the Bradford method, with BSA as the standard. CD measurements CD spectra of the DMC1 proteins (4 lm) were recorded on a JASCO J-820 spectropolarimeter (JASCO, Tokyo, Japan). All CD experiments were performed in a buffer containing 20 mm potassium phosphate (pH 7.0) and 50 mm KCl. The strand-exchange assay The human DMC1 protein or the DMC1-I37N variant was incubated at 37 °C for 10 min with 20 lm /X174 circular ssDNA, in 10 lLof20mm Hepes–KOH buffer (pH 7.5), containing 1 mm ATP, 1 mm MgCl 2 , 0.1 mgÆmL )1 BSA, 20 mm creatine phosphate and 75 lgÆmL )1 creatine kinase. After this incubation, 2 lm RPA and the indicated amounts of KCl and CaCl 2 were added to the reaction mixture, which was incubated at 37 °C for 10 min. The reactions were then initiated by the addition of 20 lm /X174 linear dsDNA, and were continued for 1 h. The reactions were stopped by the addition of 0.1% SDS and 1.7 mgÆmL )1 proteinase K (Roche Applied Science, Basel, Switzerland), and the samples were further incubated at 37 °C for 20 min. The deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1· TAE buffer (ice- cold, 40 mm Tris-acetate and 1 mm EDTA) at 3.0 VÆcm )1 for 4 h. The products were visualized by SYBR Gold (Invi- trogen, Carlsbad, CA, USA) staining. The D-loop assay The reactions were conducted in 10 l Lof20mm Tris ⁄ HCl buffer (pH 8.0), containing 1 mm MgCl 2 ,1mm ATP, 2 mm creatine phosphate, 75 lgÆmL )1 creatine kinase, 0.1 mgÆmL )1 BSA and the indicated amounts of CaCl 2 (final concentration 1–8 mm), and were started by incubating either the DMC1 protein (4 lm) or the DMC1-I37N variant (4 lm) with 1 lm of 32 P-labeled ssDNA 50-mer at 37 °C for 5 min. For the ssDNA substrate, the following HPLC-purified oligonucleo- tide was purchased from Roche Applied Science: 50-mer, 5¢-ATTTCATGCTAGACAGAAGAATTCTCAGTAACTT CTTTGTGCTGTGTGTA-3¢. The 5¢-ends of the oligo- nucleotides were labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) in the presence of [ 32 P]ATP[cP] at 37 °C for 30 min. Afterwards, the super- coiled dsDNA (pGsat4; final concentration of 30 lm) was added, and the reaction mixtures were further incubated for 8 min. The reactions were terminated by the addition of 1% SDS and 1.7 mgÆmL )1 proteinase K (Roche Applied Science), and the samples were further incubated at 37 °C for 15 min. The products were resolved by 0.8% agarose gel elec- trophoresis in 1· TAE buffer at 3.3 VÆcm )1 for 2.5 h, and were visualized and quantitated by an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). Assays for DNA binding The /X174 circular ssDNA (20 lm) or the supercoiled /X174 dsDNA (10 lm) was mixed with the human DMC1 protein or the DMC1-I37N variant in 10 lL of a standard reaction solution, containing 20 mm Hepes–KOH (pH 7.5), 1mm dithiothreitol, 0.1 mgÆmL )1 BSA, 1 mm MgCl 2 , 250 mm KCl, 5% glycerol and 1 mm ATP. The reaction mixtures were incubated at 37 °C for 10 min, and then analyzed by 0.8% agarose gel electrophoresis in 1· TAE buffer at 3.3 VÆcm )1 for 2.5 h. The bands were visualized by ethidium bromide staining. ATPase activity The human DMC1 protein or the DMC1-I37N variant was incubated in 20 mm Hepes–KOH (pH 7.5), 125 mm KCl, 1mm MgCl 2 ,1mm dithiothreitol and 0.1 mgÆmL )1 BSA in the presence or absence of ssDNA. For the experiments with DNaseI treatment, 1 unit of DNaseI (Roche Applied Science) was added to the reaction mixture, which was fur- ther incubated at 37 °C for 30 min. The reaction was per- formed at 37 °C. 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The corresponding N-terminal domain of the RAD51