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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 inhibits homologous pairing by RAD51. (A) The D-loop formation assay. Asterisks indicate the 32P-labeled end of the ssDNA 50-mer. (B) The reactions were conducted with 0.1 μM RAD51 in the presence of increasing amounts of DIDS. Lanes 1 and 6 indicate negative control experiments without and with 10 μM DIDS in the absence of RAD51, respectively. Lane 2 indicates a positive control experiment with RAD51 in the presence of 5% methanol. Lanes 3–5 indicate experiments with RAD51 and DIDS. The DIDS concentrations were 0 μM (lanes 1 and 2), 0.1 μM (lane 3), 1 μM (lane 4) and 10 μM (lanes 5 and 6). (C) Graphic representation of the experiments shown in (B).
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Figure 5: DIDS inhibits homologous pairing by RAD51. (A) The D-loop formation assay. Asterisks indicate the 32P-labeled end of the ssDNA 50-mer. (B) The reactions were conducted with 0.1 μM RAD51 in the presence of increasing amounts of DIDS. Lanes 1 and 6 indicate negative control experiments without and with 10 μM DIDS in the absence of RAD51, respectively. Lane 2 indicates a positive control experiment with RAD51 in the presence of 5% methanol. Lanes 3–5 indicate experiments with RAD51 and DIDS. The DIDS concentrations were 0 μM (lanes 1 and 2), 0.1 μM (lane 3), 1 μM (lane 4) and 10 μM (lanes 5 and 6). (C) Graphic representation of the experiments shown in (B).

Mentions: We then tested whether DIDS inhibits homologous pairing by RAD51, because the homologous-pairing step needs to occur just before strand exchange. To do so, we performed the D-loop formation assay. In this assay, a 32P-labeled ssDNA 50-mer and superhelical dsDNA were used as substrates (Figure 5A), and the reactions were performed in the absence of RPA. As shown in Figure 5B and C, DIDS inhibited the RAD51-mediated D-loop formation in a concentration-dependent manner. These results indicated that DIDS inhibits homologous pairing, as well as strand exchange, by RAD51 in vitro.Figure 5.


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 inhibits homologous pairing by RAD51. (A) The D-loop formation assay. Asterisks indicate the 32P-labeled end of the ssDNA 50-mer. (B) The reactions were conducted with 0.1 μM RAD51 in the presence of increasing amounts of DIDS. Lanes 1 and 6 indicate negative control experiments without and with 10 μM DIDS in the absence of RAD51, respectively. Lane 2 indicates a positive control experiment with RAD51 in the presence of 5% methanol. Lanes 3–5 indicate experiments with RAD51 and DIDS. The DIDS concentrations were 0 μM (lanes 1 and 2), 0.1 μM (lane 3), 1 μM (lane 4) and 10 μM (lanes 5 and 6). (C) Graphic representation of the experiments shown in (B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691838&req=5

Figure 5: DIDS inhibits homologous pairing by RAD51. (A) The D-loop formation assay. Asterisks indicate the 32P-labeled end of the ssDNA 50-mer. (B) The reactions were conducted with 0.1 μM RAD51 in the presence of increasing amounts of DIDS. Lanes 1 and 6 indicate negative control experiments without and with 10 μM DIDS in the absence of RAD51, respectively. Lane 2 indicates a positive control experiment with RAD51 in the presence of 5% methanol. Lanes 3–5 indicate experiments with RAD51 and DIDS. The DIDS concentrations were 0 μM (lanes 1 and 2), 0.1 μM (lane 3), 1 μM (lane 4) and 10 μM (lanes 5 and 6). (C) Graphic representation of the experiments shown in (B).
Mentions: We then tested whether DIDS inhibits homologous pairing by RAD51, because the homologous-pairing step needs to occur just before strand exchange. To do so, we performed the D-loop formation assay. In this assay, a 32P-labeled ssDNA 50-mer and superhelical dsDNA were used as substrates (Figure 5A), and the reactions were performed in the absence of RPA. As shown in Figure 5B and C, DIDS inhibited the RAD51-mediated D-loop formation in a concentration-dependent manner. These results indicated that DIDS inhibits homologous pairing, as well as strand exchange, by RAD51 in vitro.Figure 5.

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