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DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange.

Ishida T, Takizawa Y, Kainuma T, Inoue J, Mikawa T, Shibata T, Suzuki H, Tashiro S, Kurumizaka H - Nucleic Acids Res. (2009)

Bottom Line: We found that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) efficiently inhibited the RAD51-mediated strand exchange.A gel mobility shift assay showed that DIDS significantly inhibited the DNA-binding activity of RAD51.Therefore, DIDS may bind near the DNA binding site(s) of RAD51 and compete with DNA for RAD51 binding.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.

ABSTRACT
RAD51, an essential eukaryotic DNA recombinase, promotes homologous pairing and strand exchange during homologous recombination and the recombinational repair of double strand breaks. Mutations that up- or down-regulate RAD51 gene expression have been identified in several tumors, suggesting that inappropriate expression of the RAD51 activity may cause tumorigenesis. To identify chemical compounds that affect the RAD51 activity, in the present study, we performed the RAD51-mediated strand exchange assay in the presence of 185 chemical compounds. We found that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) efficiently inhibited the RAD51-mediated strand exchange. DIDS also inhibited the RAD51-mediated homologous pairing in the absence of RPA. A surface plasmon resonance analysis revealed that DIDS directly binds to RAD51. A gel mobility shift assay showed that DIDS significantly inhibited the DNA-binding activity of RAD51. Therefore, DIDS may bind near the DNA binding site(s) of RAD51 and compete with DNA for RAD51 binding.

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DIDS efficiently inhibits RAD51-mediated strand exchange. (A) DIDS titration experiments in the absence of 0.2 M KCl. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) in the presence of DIDS at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecule is indicated by jm. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 and 5% methanol in the absence of DIDS. DIDS concentrations were 0.01 μM (lane 3), 0.1 μM (lane 4), 1 μM (lane 5) and 10 μM (lane 6). Lane 7 indicates an experiment with 10 μM DIDS in the absence of RAD51. (B) Graphic representation of the experiments shown in (A). The band intensities of the jm product were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (A, lane 2) were plotted against the DIDS concentration. (C) The strand-exchange assay in the presence of 0.2 M KCl. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate control experiments without DIDS with RAD51 in the absence and presence of 5% methanol, respectively. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. (D) Graphic representation of the experiments shown in (C). The band intensities of the jm products were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (C, lane 3) were plotted against the DIDS concentration.
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Figure 3: DIDS efficiently inhibits RAD51-mediated strand exchange. (A) DIDS titration experiments in the absence of 0.2 M KCl. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) in the presence of DIDS at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecule is indicated by jm. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 and 5% methanol in the absence of DIDS. DIDS concentrations were 0.01 μM (lane 3), 0.1 μM (lane 4), 1 μM (lane 5) and 10 μM (lane 6). Lane 7 indicates an experiment with 10 μM DIDS in the absence of RAD51. (B) Graphic representation of the experiments shown in (A). The band intensities of the jm product were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (A, lane 2) were plotted against the DIDS concentration. (C) The strand-exchange assay in the presence of 0.2 M KCl. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate control experiments without DIDS with RAD51 in the absence and presence of 5% methanol, respectively. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. (D) Graphic representation of the experiments shown in (C). The band intensities of the jm products were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (C, lane 3) were plotted against the DIDS concentration.

Mentions: We then performed the RAD51-mediated strand exchange in the presence of each of 185 chemical compounds, which were selected as potential antitumor chemical compounds by the program of Scientific Research on Priority Areas, Cancer, Japan (Supplementary Table 1). As shown in Figure 2A, most of the chemical compounds did not affect the RAD51-mediated strand exchange; however, we found that DIDS (Figure 2A, #137 and B) was a potential inhibitor for the RAD51-mediated strand exchange. Careful titration experiments confirmed that DIDS actually inhibited the RAD51-mediated strand exchange (Figure 3A and B). DIDS also inhibited the RAD51-mediated strand exchange under the conditions with 0.2 M KCl, which significantly stimulates the strand exchange (Figure 3C and D).Figure 2.


DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange.

Ishida T, Takizawa Y, Kainuma T, Inoue J, Mikawa T, Shibata T, Suzuki H, Tashiro S, Kurumizaka H - Nucleic Acids Res. (2009)

DIDS efficiently inhibits RAD51-mediated strand exchange. (A) DIDS titration experiments in the absence of 0.2 M KCl. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) in the presence of DIDS at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecule is indicated by jm. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 and 5% methanol in the absence of DIDS. DIDS concentrations were 0.01 μM (lane 3), 0.1 μM (lane 4), 1 μM (lane 5) and 10 μM (lane 6). Lane 7 indicates an experiment with 10 μM DIDS in the absence of RAD51. (B) Graphic representation of the experiments shown in (A). The band intensities of the jm product were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (A, lane 2) were plotted against the DIDS concentration. (C) The strand-exchange assay in the presence of 0.2 M KCl. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate control experiments without DIDS with RAD51 in the absence and presence of 5% methanol, respectively. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. (D) Graphic representation of the experiments shown in (C). The band intensities of the jm products were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (C, lane 3) were plotted against the DIDS concentration.
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Figure 3: DIDS efficiently inhibits RAD51-mediated strand exchange. (A) DIDS titration experiments in the absence of 0.2 M KCl. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) in the presence of DIDS at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecule is indicated by jm. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 and 5% methanol in the absence of DIDS. DIDS concentrations were 0.01 μM (lane 3), 0.1 μM (lane 4), 1 μM (lane 5) and 10 μM (lane 6). Lane 7 indicates an experiment with 10 μM DIDS in the absence of RAD51. (B) Graphic representation of the experiments shown in (A). The band intensities of the jm product were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (A, lane 2) were plotted against the DIDS concentration. (C) The strand-exchange assay in the presence of 0.2 M KCl. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate control experiments without DIDS with RAD51 in the absence and presence of 5% methanol, respectively. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. (D) Graphic representation of the experiments shown in (C). The band intensities of the jm products were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (C, lane 3) were plotted against the DIDS concentration.
Mentions: We then performed the RAD51-mediated strand exchange in the presence of each of 185 chemical compounds, which were selected as potential antitumor chemical compounds by the program of Scientific Research on Priority Areas, Cancer, Japan (Supplementary Table 1). As shown in Figure 2A, most of the chemical compounds did not affect the RAD51-mediated strand exchange; however, we found that DIDS (Figure 2A, #137 and B) was a potential inhibitor for the RAD51-mediated strand exchange. Careful titration experiments confirmed that DIDS actually inhibited the RAD51-mediated strand exchange (Figure 3A and B). DIDS also inhibited the RAD51-mediated strand exchange under the conditions with 0.2 M KCl, which significantly stimulates the strand exchange (Figure 3C and D).Figure 2.

Bottom Line: We found that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) efficiently inhibited the RAD51-mediated strand exchange.A gel mobility shift assay showed that DIDS significantly inhibited the DNA-binding activity of RAD51.Therefore, DIDS may bind near the DNA binding site(s) of RAD51 and compete with DNA for RAD51 binding.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.

ABSTRACT
RAD51, an essential eukaryotic DNA recombinase, promotes homologous pairing and strand exchange during homologous recombination and the recombinational repair of double strand breaks. Mutations that up- or down-regulate RAD51 gene expression have been identified in several tumors, suggesting that inappropriate expression of the RAD51 activity may cause tumorigenesis. To identify chemical compounds that affect the RAD51 activity, in the present study, we performed the RAD51-mediated strand exchange assay in the presence of 185 chemical compounds. We found that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) efficiently inhibited the RAD51-mediated strand exchange. DIDS also inhibited the RAD51-mediated homologous pairing in the absence of RPA. A surface plasmon resonance analysis revealed that DIDS directly binds to RAD51. A gel mobility shift assay showed that DIDS significantly inhibited the DNA-binding activity of RAD51. Therefore, DIDS may bind near the DNA binding site(s) of RAD51 and compete with DNA for RAD51 binding.

Show MeSH
Related in: MedlinePlus