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A role for the arginine methylation of Rad9 in checkpoint control and cellular sensitivity to DNA damage.

He W, Ma X, Yang X, Zhao Y, Qiu J, Hang H - Nucleic Acids Res. (2011)

Bottom Line: In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5).The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated.In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules and Center for Computational and Systems Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
The genome stability is maintained by coordinated action of DNA repairs and checkpoints, which delay progression through the cell cycle in response to DNA damage. Rad9 is conserved from yeast to human and functions in cell cycle checkpoint controls. Here, a regulatory mechanism for Rad9 function is reported. In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5). Arginine methylation of Rad9 plays a critical role in S/M and G2/M cell cycle checkpoints. The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated. Additionally, Rad9 methylation is also required for cellular resistance to DNA damaging stresses. In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

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Deficiency of hRad9 methylation leads to S/M and G2/M checkpoint control defects. (A) The lack of hRad9 methylation affects ionizing radiation-induced G2 arrest. The four types of mouse ES cells (mRad9+/+, mRad9−/−, mRad9−/− cells expressing wild-type hRad9 and mRad9−/− cells expressing hRad9-3RA, respectively) were mock-treated or irradiated with 6 Gy of γ rays in the absence or presence of colcemid. Regions of the profiles corresponding to G1, S or G2/M are delineated above the first row of graphs, and the ratio of cells in G1, S or G2/M phase were shown. (B) Statistics analysis of the relative cell number in G1 phase out of three independent experiments in (A). Double asterisks indicate extremely significant difference (P < 0.01) and asterisk indicates significant (P < 0.05). (C) Lack of hRad9-methylation results in the S/M checkpoint control defect. The four types of cells were treated or mock-treated with 1 mM HU for 8 h. Cells were collected and labeled with the mitotic marker phosphor-histone H3 antibody, stained with propidium iodide, and analyzed by flow cytometry. Staining intensity for PI (x-axis) is plotted versus that for phosphor-histone H3 (y-axis). The cells in the boxed region are premature mitotic cells. Numbers above the box are the percentage of the cells boxed in the total cells. (D) Statistics analysis of relative premature mitotic cells derived from three independent experiments descried in (C). In (B) and (D), Double asterisks indicate extremely significant difference (P < 0.01) and asterisk indicates significance (P < 0.05).
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Figure 5: Deficiency of hRad9 methylation leads to S/M and G2/M checkpoint control defects. (A) The lack of hRad9 methylation affects ionizing radiation-induced G2 arrest. The four types of mouse ES cells (mRad9+/+, mRad9−/−, mRad9−/− cells expressing wild-type hRad9 and mRad9−/− cells expressing hRad9-3RA, respectively) were mock-treated or irradiated with 6 Gy of γ rays in the absence or presence of colcemid. Regions of the profiles corresponding to G1, S or G2/M are delineated above the first row of graphs, and the ratio of cells in G1, S or G2/M phase were shown. (B) Statistics analysis of the relative cell number in G1 phase out of three independent experiments in (A). Double asterisks indicate extremely significant difference (P < 0.01) and asterisk indicates significant (P < 0.05). (C) Lack of hRad9-methylation results in the S/M checkpoint control defect. The four types of cells were treated or mock-treated with 1 mM HU for 8 h. Cells were collected and labeled with the mitotic marker phosphor-histone H3 antibody, stained with propidium iodide, and analyzed by flow cytometry. Staining intensity for PI (x-axis) is plotted versus that for phosphor-histone H3 (y-axis). The cells in the boxed region are premature mitotic cells. Numbers above the box are the percentage of the cells boxed in the total cells. (D) Statistics analysis of relative premature mitotic cells derived from three independent experiments descried in (C). In (B) and (D), Double asterisks indicate extremely significant difference (P < 0.01) and asterisk indicates significance (P < 0.05).

Mentions: It has been reported that hRad9 is critical in S/M and G2/M checkpoint controls (7,25). Here, we tested whether hRad9 methylation plays roles in these checkpoints. The classical cell cycle checkpoint analysis was introduced to test S/M and G2/M checkpoint controls (7,13). To test the G2/M cell cycle checkpoint, four types of cells (wild-type, mRad9−/−, mRad9−/− cells expressing wild-type hRad9 and mRad9−/− cells expressing hRad9-3RA) were mock irradiated or exposed to 6 Gy of γ rays. At various post-irradiation times, the cells were fixed and examined with flow cytometry. Another set of cells was treated with colcemid immediately after radiation exposure and harvested 12 h after irradiation. As shown in Figure 5A and B, at 8 h and 12 h after exposure to 6-Gy γ rays, more cells expressing hRad9-3RA accumulated in the G1 and S phase (arrow) than cells expressing wild-type hRad9, and difference between the two types of cells above in G1 phase is statistically significant (Figure 5B). mRad9−/− and mRad9+/+ cells were used as negative and positive controls, respectively. A similar G2/M checkpoint deficient result of mRad9 knockout was reported previously by Hopkins et al. (7,26). As all the cells treated with colcemid were blocked in G2/M at 12 h after radiation exposure, these results suggest that unmethylatable hRad9-3RA leads to G2/M checkpoint deficiency.Figure 5.


A role for the arginine methylation of Rad9 in checkpoint control and cellular sensitivity to DNA damage.

He W, Ma X, Yang X, Zhao Y, Qiu J, Hang H - Nucleic Acids Res. (2011)

Deficiency of hRad9 methylation leads to S/M and G2/M checkpoint control defects. (A) The lack of hRad9 methylation affects ionizing radiation-induced G2 arrest. The four types of mouse ES cells (mRad9+/+, mRad9−/−, mRad9−/− cells expressing wild-type hRad9 and mRad9−/− cells expressing hRad9-3RA, respectively) were mock-treated or irradiated with 6 Gy of γ rays in the absence or presence of colcemid. Regions of the profiles corresponding to G1, S or G2/M are delineated above the first row of graphs, and the ratio of cells in G1, S or G2/M phase were shown. (B) Statistics analysis of the relative cell number in G1 phase out of three independent experiments in (A). Double asterisks indicate extremely significant difference (P < 0.01) and asterisk indicates significant (P < 0.05). (C) Lack of hRad9-methylation results in the S/M checkpoint control defect. The four types of cells were treated or mock-treated with 1 mM HU for 8 h. Cells were collected and labeled with the mitotic marker phosphor-histone H3 antibody, stained with propidium iodide, and analyzed by flow cytometry. Staining intensity for PI (x-axis) is plotted versus that for phosphor-histone H3 (y-axis). The cells in the boxed region are premature mitotic cells. Numbers above the box are the percentage of the cells boxed in the total cells. (D) Statistics analysis of relative premature mitotic cells derived from three independent experiments descried in (C). In (B) and (D), Double asterisks indicate extremely significant difference (P < 0.01) and asterisk indicates significance (P < 0.05).
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Figure 5: Deficiency of hRad9 methylation leads to S/M and G2/M checkpoint control defects. (A) The lack of hRad9 methylation affects ionizing radiation-induced G2 arrest. The four types of mouse ES cells (mRad9+/+, mRad9−/−, mRad9−/− cells expressing wild-type hRad9 and mRad9−/− cells expressing hRad9-3RA, respectively) were mock-treated or irradiated with 6 Gy of γ rays in the absence or presence of colcemid. Regions of the profiles corresponding to G1, S or G2/M are delineated above the first row of graphs, and the ratio of cells in G1, S or G2/M phase were shown. (B) Statistics analysis of the relative cell number in G1 phase out of three independent experiments in (A). Double asterisks indicate extremely significant difference (P < 0.01) and asterisk indicates significant (P < 0.05). (C) Lack of hRad9-methylation results in the S/M checkpoint control defect. The four types of cells were treated or mock-treated with 1 mM HU for 8 h. Cells were collected and labeled with the mitotic marker phosphor-histone H3 antibody, stained with propidium iodide, and analyzed by flow cytometry. Staining intensity for PI (x-axis) is plotted versus that for phosphor-histone H3 (y-axis). The cells in the boxed region are premature mitotic cells. Numbers above the box are the percentage of the cells boxed in the total cells. (D) Statistics analysis of relative premature mitotic cells derived from three independent experiments descried in (C). In (B) and (D), Double asterisks indicate extremely significant difference (P < 0.01) and asterisk indicates significance (P < 0.05).
Mentions: It has been reported that hRad9 is critical in S/M and G2/M checkpoint controls (7,25). Here, we tested whether hRad9 methylation plays roles in these checkpoints. The classical cell cycle checkpoint analysis was introduced to test S/M and G2/M checkpoint controls (7,13). To test the G2/M cell cycle checkpoint, four types of cells (wild-type, mRad9−/−, mRad9−/− cells expressing wild-type hRad9 and mRad9−/− cells expressing hRad9-3RA) were mock irradiated or exposed to 6 Gy of γ rays. At various post-irradiation times, the cells were fixed and examined with flow cytometry. Another set of cells was treated with colcemid immediately after radiation exposure and harvested 12 h after irradiation. As shown in Figure 5A and B, at 8 h and 12 h after exposure to 6-Gy γ rays, more cells expressing hRad9-3RA accumulated in the G1 and S phase (arrow) than cells expressing wild-type hRad9, and difference between the two types of cells above in G1 phase is statistically significant (Figure 5B). mRad9−/− and mRad9+/+ cells were used as negative and positive controls, respectively. A similar G2/M checkpoint deficient result of mRad9 knockout was reported previously by Hopkins et al. (7,26). As all the cells treated with colcemid were blocked in G2/M at 12 h after radiation exposure, these results suggest that unmethylatable hRad9-3RA leads to G2/M checkpoint deficiency.Figure 5.

Bottom Line: In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5).The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated.In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules and Center for Computational and Systems Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
The genome stability is maintained by coordinated action of DNA repairs and checkpoints, which delay progression through the cell cycle in response to DNA damage. Rad9 is conserved from yeast to human and functions in cell cycle checkpoint controls. Here, a regulatory mechanism for Rad9 function is reported. In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5). Arginine methylation of Rad9 plays a critical role in S/M and G2/M cell cycle checkpoints. The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated. Additionally, Rad9 methylation is also required for cellular resistance to DNA damaging stresses. In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

Show MeSH
Related in: MedlinePlus