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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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Defective repair of DSBs induced by γ-rays and camptothecin in GEMIN2-deficient cells. (A) Time line of γ-H2AX foci per cell at the indicated time after treatment with 4Gy γ-rays. Results for WT cells and for GEMIN2−/−tetGEMIN2 untreated (GEMIN2+) and treated (GEMIN2−) with doxycycline for 4 days are shown. Error bars represent standard deviation. Statistical analysis was performed using the t test. (B) Ionizing-radiation-induced chromosome aberrations. Mitotic cells were harvested at 6 h after exposure of an asynchronous population of cells to 4Gy γ-rays. Irradiated cells were treated with colcemid for the last 3 h before harvest to enrich the mitotic cells. Results from WT and GEMIN2−/−tetGEMIN2 cells untreated with doxycycline (GEMIN2+) and treated (GEMIN2−) with doxycycline for 4 days are shown. The number of chromosomal aberrations per 50 mitotic cells is shown on the Y-axis. Error bars represent standard deviation. (C) Time course of the formation of γ-H2AX foci per cell at the indicated times after treatment with camptothecin (CPT, 100 ng). Results for WT cells and for GEMIN2−/−tetGEMIN2 untreated (GEMIN2+) and treated (2 days, GEMIN2− 2D; 3 days, GEMIN2− 3D; 4 days, GEMIN2− 4D) with doxycycline are shown. CPT is supposed to induce DSBs only in cells at the S phase, and the number of γ-H2AX foci was counted only in γ-H2AX foci positive cells. Error bars represent standard deviation.
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Figure 4: Defective repair of DSBs induced by γ-rays and camptothecin in GEMIN2-deficient cells. (A) Time line of γ-H2AX foci per cell at the indicated time after treatment with 4Gy γ-rays. Results for WT cells and for GEMIN2−/−tetGEMIN2 untreated (GEMIN2+) and treated (GEMIN2−) with doxycycline for 4 days are shown. Error bars represent standard deviation. Statistical analysis was performed using the t test. (B) Ionizing-radiation-induced chromosome aberrations. Mitotic cells were harvested at 6 h after exposure of an asynchronous population of cells to 4Gy γ-rays. Irradiated cells were treated with colcemid for the last 3 h before harvest to enrich the mitotic cells. Results from WT and GEMIN2−/−tetGEMIN2 cells untreated with doxycycline (GEMIN2+) and treated (GEMIN2−) with doxycycline for 4 days are shown. The number of chromosomal aberrations per 50 mitotic cells is shown on the Y-axis. Error bars represent standard deviation. (C) Time course of the formation of γ-H2AX foci per cell at the indicated times after treatment with camptothecin (CPT, 100 ng). Results for WT cells and for GEMIN2−/−tetGEMIN2 untreated (GEMIN2+) and treated (2 days, GEMIN2− 2D; 3 days, GEMIN2− 3D; 4 days, GEMIN2− 4D) with doxycycline are shown. CPT is supposed to induce DSBs only in cells at the S phase, and the number of γ-H2AX foci was counted only in γ-H2AX foci positive cells. Error bars represent standard deviation.

Mentions: To assess the role of GEMIN2 in DSB repair, we exposed GEMIN2−/−tetGEMIN2 cells to γ-rays, and measured the kinetics of DSB repair by counting the number of phosphorylated histone H2AX (γ-H2AX) foci (Figure 4A) as well as by counting chromosomal breaks in the subsequent M phase (Figure 4B). To this end, 4 days after adding the doxycycline, we exposed an asynchronous population of cells to 4Gy γ-rays. The numbers of induced γ-H2AX foci were comparable irrespective of GEMIN2 expression (Figure 4A), indicating that GEMIN2 does not affect the kinetics of γ-induced DSB formation. However, at 6 and 12 h after irradiation, larger numbers of DSBs were left unrepaired in the GEMIN2-deficient cells than in the GEMIN2+ cells, indicating that GEMIN2 promotes DSB repair. We also harvested mitotic cells after 6 h and measured the number of chromosomal breaks (Figure 4B). Note that cells irradiated in the S to G2 phases can enter the M phase within 6 h after γ-irradiation (54). GEMIN2-deficient cells exhibited a ∼3-fold increase in the number of induced chromosome breaks in comparison with WT and GEMIN2+GEMIN2−/−tetGEMIN2 cells. Since DSB repair is primarily carried out by HR in DT40 cells (55), the compromised DSB repair in the S to G2 phases raises the possibility that GEMIN2 is required for HR-mediated DSB repair.Figure 4.


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)

Defective repair of DSBs induced by γ-rays and camptothecin in GEMIN2-deficient cells. (A) Time line of γ-H2AX foci per cell at the indicated time after treatment with 4Gy γ-rays. Results for WT cells and for GEMIN2−/−tetGEMIN2 untreated (GEMIN2+) and treated (GEMIN2−) with doxycycline for 4 days are shown. Error bars represent standard deviation. Statistical analysis was performed using the t test. (B) Ionizing-radiation-induced chromosome aberrations. Mitotic cells were harvested at 6 h after exposure of an asynchronous population of cells to 4Gy γ-rays. Irradiated cells were treated with colcemid for the last 3 h before harvest to enrich the mitotic cells. Results from WT and GEMIN2−/−tetGEMIN2 cells untreated with doxycycline (GEMIN2+) and treated (GEMIN2−) with doxycycline for 4 days are shown. The number of chromosomal aberrations per 50 mitotic cells is shown on the Y-axis. Error bars represent standard deviation. (C) Time course of the formation of γ-H2AX foci per cell at the indicated times after treatment with camptothecin (CPT, 100 ng). Results for WT cells and for GEMIN2−/−tetGEMIN2 untreated (GEMIN2+) and treated (2 days, GEMIN2− 2D; 3 days, GEMIN2− 3D; 4 days, GEMIN2− 4D) with doxycycline are shown. CPT is supposed to induce DSBs only in cells at the S phase, and the number of γ-H2AX foci was counted only in γ-H2AX foci positive cells. Error bars represent standard deviation.
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Figure 4: Defective repair of DSBs induced by γ-rays and camptothecin in GEMIN2-deficient cells. (A) Time line of γ-H2AX foci per cell at the indicated time after treatment with 4Gy γ-rays. Results for WT cells and for GEMIN2−/−tetGEMIN2 untreated (GEMIN2+) and treated (GEMIN2−) with doxycycline for 4 days are shown. Error bars represent standard deviation. Statistical analysis was performed using the t test. (B) Ionizing-radiation-induced chromosome aberrations. Mitotic cells were harvested at 6 h after exposure of an asynchronous population of cells to 4Gy γ-rays. Irradiated cells were treated with colcemid for the last 3 h before harvest to enrich the mitotic cells. Results from WT and GEMIN2−/−tetGEMIN2 cells untreated with doxycycline (GEMIN2+) and treated (GEMIN2−) with doxycycline for 4 days are shown. The number of chromosomal aberrations per 50 mitotic cells is shown on the Y-axis. Error bars represent standard deviation. (C) Time course of the formation of γ-H2AX foci per cell at the indicated times after treatment with camptothecin (CPT, 100 ng). Results for WT cells and for GEMIN2−/−tetGEMIN2 untreated (GEMIN2+) and treated (2 days, GEMIN2− 2D; 3 days, GEMIN2− 3D; 4 days, GEMIN2− 4D) with doxycycline are shown. CPT is supposed to induce DSBs only in cells at the S phase, and the number of γ-H2AX foci was counted only in γ-H2AX foci positive cells. Error bars represent standard deviation.
Mentions: To assess the role of GEMIN2 in DSB repair, we exposed GEMIN2−/−tetGEMIN2 cells to γ-rays, and measured the kinetics of DSB repair by counting the number of phosphorylated histone H2AX (γ-H2AX) foci (Figure 4A) as well as by counting chromosomal breaks in the subsequent M phase (Figure 4B). To this end, 4 days after adding the doxycycline, we exposed an asynchronous population of cells to 4Gy γ-rays. The numbers of induced γ-H2AX foci were comparable irrespective of GEMIN2 expression (Figure 4A), indicating that GEMIN2 does not affect the kinetics of γ-induced DSB formation. However, at 6 and 12 h after irradiation, larger numbers of DSBs were left unrepaired in the GEMIN2-deficient cells than in the GEMIN2+ cells, indicating that GEMIN2 promotes DSB repair. We also harvested mitotic cells after 6 h and measured the number of chromosomal breaks (Figure 4B). Note that cells irradiated in the S to G2 phases can enter the M phase within 6 h after γ-irradiation (54). GEMIN2-deficient cells exhibited a ∼3-fold increase in the number of induced chromosome breaks in comparison with WT and GEMIN2+GEMIN2−/−tetGEMIN2 cells. Since DSB repair is primarily carried out by HR in DT40 cells (55), the compromised DSB repair in the S to G2 phases raises the possibility that GEMIN2 is required for HR-mediated DSB repair.Figure 4.

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