Limits...
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.

Show MeSH

Related in: MedlinePlus

The strand-exchange assay with human RAD51. (A) A schematic diagram of the strand-exchange assay. (B) The strand exchange activity of RAD51 under the three different conditions. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) 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 molecules and nicked circular DNA are indicated by jm and nc, respectively. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate experiments with RAD51 in the absence and presence of 0.2 M KCl, respectively. Lane 4 indicates an experiment with 5% methanol in the absence of 0.2 M KCl.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2691838&req=5

Figure 1: The strand-exchange assay with human RAD51. (A) A schematic diagram of the strand-exchange assay. (B) The strand exchange activity of RAD51 under the three different conditions. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) 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 molecules and nicked circular DNA are indicated by jm and nc, respectively. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate experiments with RAD51 in the absence and presence of 0.2 M KCl, respectively. Lane 4 indicates an experiment with 5% methanol in the absence of 0.2 M KCl.

Mentions: To identify chemical compounds that affect the recombinase activity of RAD51, we employed the strand-exchange assay. In this assay, ϕX174 phage circular ssDNA (5386 bases) and linearized ϕX174 dsDNA (5386 base pairs) were used as DNA substrates, and the reactions were conducted in the presence of RPA. In this combination of DNA substrates, both intermediate (joint molecule; JM) and complete strand-exchange products (nicked circular; NC) were potentially detectable (Figure 1A). However, under the reaction conditions used in this study, only the JM product was detected, because 5% methanol (as a solvent for chemical compounds) and low concentrations of salt, which are not optimal for the reaction, were present in the reaction mixture (Figure 1B).Figure 1.


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)

The strand-exchange assay with human RAD51. (A) A schematic diagram of the strand-exchange assay. (B) The strand exchange activity of RAD51 under the three different conditions. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) 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 molecules and nicked circular DNA are indicated by jm and nc, respectively. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate experiments with RAD51 in the absence and presence of 0.2 M KCl, respectively. Lane 4 indicates an experiment with 5% methanol in the absence of 0.2 M KCl.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: The strand-exchange assay with human RAD51. (A) A schematic diagram of the strand-exchange assay. (B) The strand exchange activity of RAD51 under the three different conditions. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) 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 molecules and nicked circular DNA are indicated by jm and nc, respectively. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate experiments with RAD51 in the absence and presence of 0.2 M KCl, respectively. Lane 4 indicates an experiment with 5% methanol in the absence of 0.2 M KCl.
Mentions: To identify chemical compounds that affect the recombinase activity of RAD51, we employed the strand-exchange assay. In this assay, ϕX174 phage circular ssDNA (5386 bases) and linearized ϕX174 dsDNA (5386 base pairs) were used as DNA substrates, and the reactions were conducted in the presence of RPA. In this combination of DNA substrates, both intermediate (joint molecule; JM) and complete strand-exchange products (nicked circular; NC) were potentially detectable (Figure 1A). However, under the reaction conditions used in this study, only the JM product was detected, because 5% methanol (as a solvent for chemical compounds) and low concentrations of salt, which are not optimal for the reaction, were present in the reaction mixture (Figure 1B).Figure 1.

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