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Role of ATM in the telomere response to the G-quadruplex ligand 360A.

Pennarun G, Granotier C, Hoffschir F, Mandine E, Biard D, Gauthier LR, Boussin FD - Nucleic Acids Res. (2008)

Bottom Line: To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM.We demonstrate that ATM reduced telomere instability independently of apoptosis induction.Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres.

View Article: PubMed Central - PubMed

Affiliation: CEA, DSV, iRCM, Laboratoire de Radiopathologie-IPSC, 92265 Fontenay-aux-Roses, France.

ABSTRACT
Telomeres are known to prevent chromosome ends from being recognized as DNA double-strand breaks. Conversely, many DNA damage response proteins, including ATM, are thought to participate to telomere maintenance. However, the precise roles of ATM at telomeres remain unclear due to its multiple functions in cell checkpoints and apoptosis. To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM. We showed, by using Fluorescence in situ hybridization (FISH) and Chromosome Orientation-FISH using telomere PNA probes, that 360A induced specific telomere aberrations occurring during or after replication, mainly consisting in sister telomere fusions and also recombinations that involved preferentially the lagging strand telomeres. We demonstrate that ATM reduced telomere instability independently of apoptosis induction. Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres.

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360A induces DNA damage signaling. (A–C) 53BP1 and phosphorylated forms (P-) of ATM and SMC1 form foci that co-localized with γ-H2AX following 360A treatment in HeLa cells. HeLa cells were treated with 5 µM 360A (or 0.05% DMSO as control) for 7 days, fixed and costained with anti-γ-H2AX and specific antibodies of 53BP1, P-ATM and P-SMC1. Typical images of 360A-treated HeLa cells exhibited colocalizations between foci of γ-H2AX and DNA repair factors are shown in (A). Histograms (B) show the numbers of P-ATM, γ-H2AX foci and of their colocalization per cell calculated from at least 50 randomly chosen cells for each condition ± SE (***indicate a t-test P-value <0.0001 and **P < 0.005). (C) Colocalizations were quantified by scoring the number of 53BP1 and P-SMC1 foci colocalized or not with γ-H2AX foci in at least 50 randomly chosen cells per condition (***P < 0.0001 and **P < 0.005). (D–F) Colocalization of telomeric-PNA signals (green, in D) and 53BP1 foci (red, in D) in HeLa cells treated with 5 µM 360A (or 0.05% DMSO) for 7 days or exposed to 2 Gy-irradiation (IR) and collected 1 h after irradiation. (D) Colocalization was appreciated on merge images shown on the left for the three conditions. Each image was obtained from a maximum projection of a Z-stack of 15 images, which were previously subjected to 2D deconvolution using the Metamorph software (Universal imaging corp.). Nuclei stained with DAPI (blue) and enlarged views of colocalized foci from the merged image are shown on the right. Percentage of 53BP1 signal colocalized with telomeric PNA signal (E) and percentages of nucleus surfaces occupied by 53BP1 foci (F) in the different conditions were calculated with colocalization module of the Metamorph software (**P < 0.001; ***P < 0.0001).
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Figure 5: 360A induces DNA damage signaling. (A–C) 53BP1 and phosphorylated forms (P-) of ATM and SMC1 form foci that co-localized with γ-H2AX following 360A treatment in HeLa cells. HeLa cells were treated with 5 µM 360A (or 0.05% DMSO as control) for 7 days, fixed and costained with anti-γ-H2AX and specific antibodies of 53BP1, P-ATM and P-SMC1. Typical images of 360A-treated HeLa cells exhibited colocalizations between foci of γ-H2AX and DNA repair factors are shown in (A). Histograms (B) show the numbers of P-ATM, γ-H2AX foci and of their colocalization per cell calculated from at least 50 randomly chosen cells for each condition ± SE (***indicate a t-test P-value <0.0001 and **P < 0.005). (C) Colocalizations were quantified by scoring the number of 53BP1 and P-SMC1 foci colocalized or not with γ-H2AX foci in at least 50 randomly chosen cells per condition (***P < 0.0001 and **P < 0.005). (D–F) Colocalization of telomeric-PNA signals (green, in D) and 53BP1 foci (red, in D) in HeLa cells treated with 5 µM 360A (or 0.05% DMSO) for 7 days or exposed to 2 Gy-irradiation (IR) and collected 1 h after irradiation. (D) Colocalization was appreciated on merge images shown on the left for the three conditions. Each image was obtained from a maximum projection of a Z-stack of 15 images, which were previously subjected to 2D deconvolution using the Metamorph software (Universal imaging corp.). Nuclei stained with DAPI (blue) and enlarged views of colocalized foci from the merged image are shown on the right. Percentage of 53BP1 signal colocalized with telomeric PNA signal (E) and percentages of nucleus surfaces occupied by 53BP1 foci (F) in the different conditions were calculated with colocalization module of the Metamorph software (**P < 0.001; ***P < 0.0001).

Mentions: In order to further investigate the role of ATM in response to 360A, we then studied ATM signaling in Hela cells. We found that 360A significantly increased nuclear foci of activated ATM by performing immunofluorescence staining with an antibody against its phosphorylated form (serine 1981) (Figure 5A and B). These foci colocalized with that of phosphorylated H2AX, a member of the nucleosome core histone H2A family, which is phosphorylated on serine 139 (referred to as γ-H2AX) mainly by ATM, at chromatin adjacent to DNA DSBs (40) and at dysfunctional telomeres (10,41). 360A induced a significant increase in the number of γ-H2AX nuclear foci in HeLa cells compared to DMSO controls (Figure 5A and B). 360A also induced the formation of nuclear foci of 53BP1 and of the phosphorylated forms of SMC1, which for the most colocalized with that of γ-H2AX (Figure 5A and C), consistently with the well-known role of γ-H2AX in the recruitment of DNA repair and checkpoint proteins at DNA damage sites (42).Figure 5.


Role of ATM in the telomere response to the G-quadruplex ligand 360A.

Pennarun G, Granotier C, Hoffschir F, Mandine E, Biard D, Gauthier LR, Boussin FD - Nucleic Acids Res. (2008)

360A induces DNA damage signaling. (A–C) 53BP1 and phosphorylated forms (P-) of ATM and SMC1 form foci that co-localized with γ-H2AX following 360A treatment in HeLa cells. HeLa cells were treated with 5 µM 360A (or 0.05% DMSO as control) for 7 days, fixed and costained with anti-γ-H2AX and specific antibodies of 53BP1, P-ATM and P-SMC1. Typical images of 360A-treated HeLa cells exhibited colocalizations between foci of γ-H2AX and DNA repair factors are shown in (A). Histograms (B) show the numbers of P-ATM, γ-H2AX foci and of their colocalization per cell calculated from at least 50 randomly chosen cells for each condition ± SE (***indicate a t-test P-value <0.0001 and **P < 0.005). (C) Colocalizations were quantified by scoring the number of 53BP1 and P-SMC1 foci colocalized or not with γ-H2AX foci in at least 50 randomly chosen cells per condition (***P < 0.0001 and **P < 0.005). (D–F) Colocalization of telomeric-PNA signals (green, in D) and 53BP1 foci (red, in D) in HeLa cells treated with 5 µM 360A (or 0.05% DMSO) for 7 days or exposed to 2 Gy-irradiation (IR) and collected 1 h after irradiation. (D) Colocalization was appreciated on merge images shown on the left for the three conditions. Each image was obtained from a maximum projection of a Z-stack of 15 images, which were previously subjected to 2D deconvolution using the Metamorph software (Universal imaging corp.). Nuclei stained with DAPI (blue) and enlarged views of colocalized foci from the merged image are shown on the right. Percentage of 53BP1 signal colocalized with telomeric PNA signal (E) and percentages of nucleus surfaces occupied by 53BP1 foci (F) in the different conditions were calculated with colocalization module of the Metamorph software (**P < 0.001; ***P < 0.0001).
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Related In: Results  -  Collection

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Figure 5: 360A induces DNA damage signaling. (A–C) 53BP1 and phosphorylated forms (P-) of ATM and SMC1 form foci that co-localized with γ-H2AX following 360A treatment in HeLa cells. HeLa cells were treated with 5 µM 360A (or 0.05% DMSO as control) for 7 days, fixed and costained with anti-γ-H2AX and specific antibodies of 53BP1, P-ATM and P-SMC1. Typical images of 360A-treated HeLa cells exhibited colocalizations between foci of γ-H2AX and DNA repair factors are shown in (A). Histograms (B) show the numbers of P-ATM, γ-H2AX foci and of their colocalization per cell calculated from at least 50 randomly chosen cells for each condition ± SE (***indicate a t-test P-value <0.0001 and **P < 0.005). (C) Colocalizations were quantified by scoring the number of 53BP1 and P-SMC1 foci colocalized or not with γ-H2AX foci in at least 50 randomly chosen cells per condition (***P < 0.0001 and **P < 0.005). (D–F) Colocalization of telomeric-PNA signals (green, in D) and 53BP1 foci (red, in D) in HeLa cells treated with 5 µM 360A (or 0.05% DMSO) for 7 days or exposed to 2 Gy-irradiation (IR) and collected 1 h after irradiation. (D) Colocalization was appreciated on merge images shown on the left for the three conditions. Each image was obtained from a maximum projection of a Z-stack of 15 images, which were previously subjected to 2D deconvolution using the Metamorph software (Universal imaging corp.). Nuclei stained with DAPI (blue) and enlarged views of colocalized foci from the merged image are shown on the right. Percentage of 53BP1 signal colocalized with telomeric PNA signal (E) and percentages of nucleus surfaces occupied by 53BP1 foci (F) in the different conditions were calculated with colocalization module of the Metamorph software (**P < 0.001; ***P < 0.0001).
Mentions: In order to further investigate the role of ATM in response to 360A, we then studied ATM signaling in Hela cells. We found that 360A significantly increased nuclear foci of activated ATM by performing immunofluorescence staining with an antibody against its phosphorylated form (serine 1981) (Figure 5A and B). These foci colocalized with that of phosphorylated H2AX, a member of the nucleosome core histone H2A family, which is phosphorylated on serine 139 (referred to as γ-H2AX) mainly by ATM, at chromatin adjacent to DNA DSBs (40) and at dysfunctional telomeres (10,41). 360A induced a significant increase in the number of γ-H2AX nuclear foci in HeLa cells compared to DMSO controls (Figure 5A and B). 360A also induced the formation of nuclear foci of 53BP1 and of the phosphorylated forms of SMC1, which for the most colocalized with that of γ-H2AX (Figure 5A and C), consistently with the well-known role of γ-H2AX in the recruitment of DNA repair and checkpoint proteins at DNA damage sites (42).Figure 5.

Bottom Line: To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM.We demonstrate that ATM reduced telomere instability independently of apoptosis induction.Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres.

View Article: PubMed Central - PubMed

Affiliation: CEA, DSV, iRCM, Laboratoire de Radiopathologie-IPSC, 92265 Fontenay-aux-Roses, France.

ABSTRACT
Telomeres are known to prevent chromosome ends from being recognized as DNA double-strand breaks. Conversely, many DNA damage response proteins, including ATM, are thought to participate to telomere maintenance. However, the precise roles of ATM at telomeres remain unclear due to its multiple functions in cell checkpoints and apoptosis. To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM. We showed, by using Fluorescence in situ hybridization (FISH) and Chromosome Orientation-FISH using telomere PNA probes, that 360A induced specific telomere aberrations occurring during or after replication, mainly consisting in sister telomere fusions and also recombinations that involved preferentially the lagging strand telomeres. We demonstrate that ATM reduced telomere instability independently of apoptosis induction. Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres.

Show MeSH
Related in: MedlinePlus