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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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HR-mediated DSB repair is impaired in GEMIN2-deficient cells. (A) Schematic of the HR-dependent DSB repair assay. Transient expression of the I-SceI restriction enzyme generates a specific DSB in an artificial HR substrate, DR-GFP, inserted into the endogenous OVALBUMIN locus. The SceGFP gene does not produce functional green fluorescent protein (GFP) because of a frame-shift mutation at the I-SceI recognition sequences. Functional GFP is produced only when the recognition sequences are eliminated by HR with the downstream internal GFP fragment (iGFP). (B) Distribution of GEMIN2−/−tetGEMIN2 cells with or without GFP expression. The population of the cells for GFP expression was measured at 12 h after transfection of I-SceI-expression plasmid [I-Sce(+)] or control plasmid [I-Sce(−)] in GEMIN2−/−tetGEMIN2 cells untreated (GEMIN2+) and treated (GEMIN2−) with doxycycline for 3 days. (C) Percentage of cells expressing GFP in the indicated cell line was calculated. Data shown are the mean of three experiments. Error bars indicate standard deviation. (D) Transient transfection efficiency of an intact GFP expression plasmid in the indicated cell lines. Error bars indicate standard deviation.
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Figure 5: HR-mediated DSB repair is impaired in GEMIN2-deficient cells. (A) Schematic of the HR-dependent DSB repair assay. Transient expression of the I-SceI restriction enzyme generates a specific DSB in an artificial HR substrate, DR-GFP, inserted into the endogenous OVALBUMIN locus. The SceGFP gene does not produce functional green fluorescent protein (GFP) because of a frame-shift mutation at the I-SceI recognition sequences. Functional GFP is produced only when the recognition sequences are eliminated by HR with the downstream internal GFP fragment (iGFP). (B) Distribution of GEMIN2−/−tetGEMIN2 cells with or without GFP expression. The population of the cells for GFP expression was measured at 12 h after transfection of I-SceI-expression plasmid [I-Sce(+)] or control plasmid [I-Sce(−)] in GEMIN2−/−tetGEMIN2 cells untreated (GEMIN2+) and treated (GEMIN2−) with doxycycline for 3 days. (C) Percentage of cells expressing GFP in the indicated cell line was calculated. Data shown are the mean of three experiments. Error bars indicate standard deviation. (D) Transient transfection efficiency of an intact GFP expression plasmid in the indicated cell lines. Error bars indicate standard deviation.

Mentions: To directly assess the role of GEMIN2 in HR, we measured the HR-dependent repair of site-specific DSBs generated by the I-SceI nuclease acting on an artificial substrate inserted into the OVALBUMIN locus (60,61). The efficiency of HR-dependent repair can be evaluated by measuring the percentage of green fluorescent protein (GFP)-positive cells (Figure 5A). The percentage of GFP+ cells was three times lower in GEMIN2-deficient than in WT and GEMIN2+GEMIN2−/−tetGEMIN2 cells (Figure 5B and C), though the efficiency of transient transfection in these cells was comparable (Figure 5D). In summary, this observation, as well as the defective repair of DSBs induced by γ-rays and camptothecin, indicates that GEMIN2 indeed facilitates HR-dependent DSB repair.Figure 5.


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)

HR-mediated DSB repair is impaired in GEMIN2-deficient cells. (A) Schematic of the HR-dependent DSB repair assay. Transient expression of the I-SceI restriction enzyme generates a specific DSB in an artificial HR substrate, DR-GFP, inserted into the endogenous OVALBUMIN locus. The SceGFP gene does not produce functional green fluorescent protein (GFP) because of a frame-shift mutation at the I-SceI recognition sequences. Functional GFP is produced only when the recognition sequences are eliminated by HR with the downstream internal GFP fragment (iGFP). (B) Distribution of GEMIN2−/−tetGEMIN2 cells with or without GFP expression. The population of the cells for GFP expression was measured at 12 h after transfection of I-SceI-expression plasmid [I-Sce(+)] or control plasmid [I-Sce(−)] in GEMIN2−/−tetGEMIN2 cells untreated (GEMIN2+) and treated (GEMIN2−) with doxycycline for 3 days. (C) Percentage of cells expressing GFP in the indicated cell line was calculated. Data shown are the mean of three experiments. Error bars indicate standard deviation. (D) Transient transfection efficiency of an intact GFP expression plasmid in the indicated cell lines. Error bars indicate standard deviation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 5: HR-mediated DSB repair is impaired in GEMIN2-deficient cells. (A) Schematic of the HR-dependent DSB repair assay. Transient expression of the I-SceI restriction enzyme generates a specific DSB in an artificial HR substrate, DR-GFP, inserted into the endogenous OVALBUMIN locus. The SceGFP gene does not produce functional green fluorescent protein (GFP) because of a frame-shift mutation at the I-SceI recognition sequences. Functional GFP is produced only when the recognition sequences are eliminated by HR with the downstream internal GFP fragment (iGFP). (B) Distribution of GEMIN2−/−tetGEMIN2 cells with or without GFP expression. The population of the cells for GFP expression was measured at 12 h after transfection of I-SceI-expression plasmid [I-Sce(+)] or control plasmid [I-Sce(−)] in GEMIN2−/−tetGEMIN2 cells untreated (GEMIN2+) and treated (GEMIN2−) with doxycycline for 3 days. (C) Percentage of cells expressing GFP in the indicated cell line was calculated. Data shown are the mean of three experiments. Error bars indicate standard deviation. (D) Transient transfection efficiency of an intact GFP expression plasmid in the indicated cell lines. Error bars indicate standard deviation.
Mentions: To directly assess the role of GEMIN2 in HR, we measured the HR-dependent repair of site-specific DSBs generated by the I-SceI nuclease acting on an artificial substrate inserted into the OVALBUMIN locus (60,61). The efficiency of HR-dependent repair can be evaluated by measuring the percentage of green fluorescent protein (GFP)-positive cells (Figure 5A). The percentage of GFP+ cells was three times lower in GEMIN2-deficient than in WT and GEMIN2+GEMIN2−/−tetGEMIN2 cells (Figure 5B and C), though the efficiency of transient transfection in these cells was comparable (Figure 5D). In summary, this observation, as well as the defective repair of DSBs induced by γ-rays and camptothecin, indicates that GEMIN2 indeed facilitates HR-dependent DSB repair.Figure 5.

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