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GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination.

Takizawa Y, Qing Y, Takaku M, Ishida T, Morozumi Y, Tsujita T, Kogame T, Hirota K, Takahashi M, Shibata T, Kurumizaka H, Takeda S - Nucleic Acids Res. (2010)

Bottom Line: We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis.The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2.These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan.

ABSTRACT
RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51-DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.

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GEMIN2 stabilizes the RAD51–DNA filament. (A) Complex formation of RAD51 and dsDNA was evaluated by electrophoresis of unbound free DNA in agarose gel. Increased concentrations of competitor DNA were incubated with 2 µM of RAD51 in the presence or absence of 4 µM of GEMIN2, prior to the addition of ϕX174 dsDNA. (B) Quantification of results from panel A. The relative amounts of RAD51-unbound DNA are shown. Closed and open circles indicate experiments with and without GEMIN2. Average values and standard deviation were calculated from three independent experiments. (C) Complex formation of RAD51 and dsDNA in the presence of the BRC4 polypeptide. The experiments were done as described for panel A. (D) Quantification of the data from panel C. (E) Surface plasmon resonance analysis. The RAD51- or GEMIN2-conjugated sensor chips were used. Sensorgrams of RAD51-BRC4 and GEMIN2-BRC4 interactions are presented. The BRC4 polypeptide concentration was 10 µM. Time 0 of the horizontal axis indicates the initiation time of the peptide injection.
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Figure 2: GEMIN2 stabilizes the RAD51–DNA filament. (A) Complex formation of RAD51 and dsDNA was evaluated by electrophoresis of unbound free DNA in agarose gel. Increased concentrations of competitor DNA were incubated with 2 µM of RAD51 in the presence or absence of 4 µM of GEMIN2, prior to the addition of ϕX174 dsDNA. (B) Quantification of results from panel A. The relative amounts of RAD51-unbound DNA are shown. Closed and open circles indicate experiments with and without GEMIN2. Average values and standard deviation were calculated from three independent experiments. (C) Complex formation of RAD51 and dsDNA in the presence of the BRC4 polypeptide. The experiments were done as described for panel A. (D) Quantification of the data from panel C. (E) Surface plasmon resonance analysis. The RAD51- or GEMIN2-conjugated sensor chips were used. Sensorgrams of RAD51-BRC4 and GEMIN2-BRC4 interactions are presented. The BRC4 polypeptide concentration was 10 µM. Time 0 of the horizontal axis indicates the initiation time of the peptide injection.

Mentions: To test whether GEMIN2 inhibited the dissociation of RAD51 from DNA, we generated the RAD51–DNA complex and subsequently measured the stability of this complex in the presence of excess amounts of either competitor DNA (975–1246 bp) (Figure 2A and B) or the BRCA2-derived BRC4 polypeptide (Figure 2C and D), which is known to disassemble the RAD51–DNA filament (43–46). In these assays, we used ϕX174 linear dsDNA (5386 bp) associated with RAD51 as the RAD51–DNA complex. We evaluated the stability by measuring the amount of free dsDNA using agarose gel electrophoresis. Excess amounts of competitor DNA disrupted the RAD51–DNA complex, as evidenced by the fact that substantial amounts of free ϕX174 linear dsDNA were detected, as the amount of competitor DNA was increased (Figure 2A, lanes 2–5). The disruption of the RAD51–DNA complex was significantly suppressed by the addition of GEMIN2 (Figure 2A, lanes 6–9 and Figure 2B), indicating that GEMIN2 may inhibit the RAD51–DNA dissociation. We also analyzed the impact of the BRC4 polypeptide on the RAD51–DNA complex with and without GEMIN2. The BRC4 polypeptide dissociated free RAD51 from ϕX174 linear dsDNA. This inhibitory effect of BRC4 on the RAD51–DNA complex was significantly reversed by the addition of GEMIN2 (Figure 2C and D). SPR analysis revealed that the BRC4 polypeptide did not bind to GEMIN2 (Figure 2E), eliminating the possibility that GEMIN2 masked the BRC4 polypeptide from the RAD51–BRC4 interaction. We therefore conclude that GEMIN2 facilitates the formation of the RAD51–DNA complex, probably by inhibiting the dissociation of RAD51 from damaged DNA sites.Figure 2.


GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination.

Takizawa Y, Qing Y, Takaku M, Ishida T, Morozumi Y, Tsujita T, Kogame T, Hirota K, Takahashi M, Shibata T, Kurumizaka H, Takeda S - Nucleic Acids Res. (2010)

GEMIN2 stabilizes the RAD51–DNA filament. (A) Complex formation of RAD51 and dsDNA was evaluated by electrophoresis of unbound free DNA in agarose gel. Increased concentrations of competitor DNA were incubated with 2 µM of RAD51 in the presence or absence of 4 µM of GEMIN2, prior to the addition of ϕX174 dsDNA. (B) Quantification of results from panel A. The relative amounts of RAD51-unbound DNA are shown. Closed and open circles indicate experiments with and without GEMIN2. Average values and standard deviation were calculated from three independent experiments. (C) Complex formation of RAD51 and dsDNA in the presence of the BRC4 polypeptide. The experiments were done as described for panel A. (D) Quantification of the data from panel C. (E) Surface plasmon resonance analysis. The RAD51- or GEMIN2-conjugated sensor chips were used. Sensorgrams of RAD51-BRC4 and GEMIN2-BRC4 interactions are presented. The BRC4 polypeptide concentration was 10 µM. Time 0 of the horizontal axis indicates the initiation time of the peptide injection.
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Related In: Results  -  Collection

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Figure 2: GEMIN2 stabilizes the RAD51–DNA filament. (A) Complex formation of RAD51 and dsDNA was evaluated by electrophoresis of unbound free DNA in agarose gel. Increased concentrations of competitor DNA were incubated with 2 µM of RAD51 in the presence or absence of 4 µM of GEMIN2, prior to the addition of ϕX174 dsDNA. (B) Quantification of results from panel A. The relative amounts of RAD51-unbound DNA are shown. Closed and open circles indicate experiments with and without GEMIN2. Average values and standard deviation were calculated from three independent experiments. (C) Complex formation of RAD51 and dsDNA in the presence of the BRC4 polypeptide. The experiments were done as described for panel A. (D) Quantification of the data from panel C. (E) Surface plasmon resonance analysis. The RAD51- or GEMIN2-conjugated sensor chips were used. Sensorgrams of RAD51-BRC4 and GEMIN2-BRC4 interactions are presented. The BRC4 polypeptide concentration was 10 µM. Time 0 of the horizontal axis indicates the initiation time of the peptide injection.
Mentions: To test whether GEMIN2 inhibited the dissociation of RAD51 from DNA, we generated the RAD51–DNA complex and subsequently measured the stability of this complex in the presence of excess amounts of either competitor DNA (975–1246 bp) (Figure 2A and B) or the BRCA2-derived BRC4 polypeptide (Figure 2C and D), which is known to disassemble the RAD51–DNA filament (43–46). In these assays, we used ϕX174 linear dsDNA (5386 bp) associated with RAD51 as the RAD51–DNA complex. We evaluated the stability by measuring the amount of free dsDNA using agarose gel electrophoresis. Excess amounts of competitor DNA disrupted the RAD51–DNA complex, as evidenced by the fact that substantial amounts of free ϕX174 linear dsDNA were detected, as the amount of competitor DNA was increased (Figure 2A, lanes 2–5). The disruption of the RAD51–DNA complex was significantly suppressed by the addition of GEMIN2 (Figure 2A, lanes 6–9 and Figure 2B), indicating that GEMIN2 may inhibit the RAD51–DNA dissociation. We also analyzed the impact of the BRC4 polypeptide on the RAD51–DNA complex with and without GEMIN2. The BRC4 polypeptide dissociated free RAD51 from ϕX174 linear dsDNA. This inhibitory effect of BRC4 on the RAD51–DNA complex was significantly reversed by the addition of GEMIN2 (Figure 2C and D). SPR analysis revealed that the BRC4 polypeptide did not bind to GEMIN2 (Figure 2E), eliminating the possibility that GEMIN2 masked the BRC4 polypeptide from the RAD51–BRC4 interaction. We therefore conclude that GEMIN2 facilitates the formation of the RAD51–DNA complex, probably by inhibiting the dissociation of RAD51 from damaged DNA sites.Figure 2.

Bottom Line: We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis.The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2.These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan.

ABSTRACT
RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51-DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.

Show MeSH
Related in: MedlinePlus