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ATR contributes to telomere maintenance in human cells.

Pennarun G, Hoffschir F, Revaud D, Granotier C, Gauthier LR, Mailliet P, Biard DS, Boussin FD - Nucleic Acids Res. (2010)

Bottom Line: The telomere aberrations resulting from ATR deficiency (i.e. sister telomere fusions and chromatid-type telomere aberrations) are mainly generated during and/or after telomere replication, and involve both leading and lagging strand telomeres as shown by chromosome orientation-FISH (CO-FISH).Moreover, we show that ATR deficiency strongly sensitizes cells to the G-quadruplex ligand 360A, enhancing sister telomere fusions and chromatid-type telomere aberrations involving specifically the lagging strand telomeres.Altogether, these data reveal that ATR plays a critical role in telomere maintenance during and/or after telomere replication in human cells.

View Article: PubMed Central - PubMed

Affiliation: CEA/DSV/iRCM/SCSR, Laboratoire de Radiopathologie, INSERM-Université Paris VII U967, 92265 Fontenay-aux-Roses, France.

ABSTRACT
Telomere maintenance is essential to preserve genomic stability and involves several telomere-specific proteins as well as DNA replication and repair proteins. The kinase ATR, which has a crucial function in maintaining genome integrity from yeast to human, has been shown to be involved in telomere maintenance in several eukaryotic organisms, including yeast, Arabidopsis and Drosophila. However, its role in telomere maintenance in mammals remains poorly explored. Here, we report by using telomere-fluorescence in situ hybridization (Telo-FISH) on metaphase chromosomes that ATR deficiency causes telomere instability both in primary human fibroblasts from Seckel syndrome patients and in HeLa cells. The telomere aberrations resulting from ATR deficiency (i.e. sister telomere fusions and chromatid-type telomere aberrations) are mainly generated during and/or after telomere replication, and involve both leading and lagging strand telomeres as shown by chromosome orientation-FISH (CO-FISH). Moreover, we show that ATR deficiency strongly sensitizes cells to the G-quadruplex ligand 360A, enhancing sister telomere fusions and chromatid-type telomere aberrations involving specifically the lagging strand telomeres. Altogether, these data reveal that ATR plays a critical role in telomere maintenance during and/or after telomere replication in human cells.

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ATR inhibition destabilizes both lagging and leading strand telomeres. (A) Histograms show the percentages of sister telomere losses affecting the lagging or the leading strand in ATRKD and CtKD cells detected by CO-FISH on metaphase spreads. ‘n’ represents the total number of telomere losses analyzed. No significant difference in the repartition of missing lagging or leading strand telomeres between the two cell lines was detected by chi-square analysis. Representative images of chromosomes missing lagging or leading strand telomeres are shown on the right. Lagging strand telomeres are labeled in red by hybridization of the parental G strands with Cy3-PNA probe and the leading telomeres in green by hybridization of the parental C strand with FITC-PNA probe. (B) Histograms show the respective percentages of telomere doublets containing two parental C-strand telomeres (C–C doublets) or two parental G-strand telomeres (G–G doublets) or both parental strand telomeres (C–G or G–C doublets) in ATRKD and CtKD cells. ‘n’ represents the total number of telomere doublets analyzed. No significant difference in the repartition of the three classes of telomere doublets between the two cell lines was detected by chi-square analysis. Representative images of telomere doublets detected by CO-FISH are shown on the right.
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Figure 3: ATR inhibition destabilizes both lagging and leading strand telomeres. (A) Histograms show the percentages of sister telomere losses affecting the lagging or the leading strand in ATRKD and CtKD cells detected by CO-FISH on metaphase spreads. ‘n’ represents the total number of telomere losses analyzed. No significant difference in the repartition of missing lagging or leading strand telomeres between the two cell lines was detected by chi-square analysis. Representative images of chromosomes missing lagging or leading strand telomeres are shown on the right. Lagging strand telomeres are labeled in red by hybridization of the parental G strands with Cy3-PNA probe and the leading telomeres in green by hybridization of the parental C strand with FITC-PNA probe. (B) Histograms show the respective percentages of telomere doublets containing two parental C-strand telomeres (C–C doublets) or two parental G-strand telomeres (G–G doublets) or both parental strand telomeres (C–G or G–C doublets) in ATRKD and CtKD cells. ‘n’ represents the total number of telomere doublets analyzed. No significant difference in the repartition of the three classes of telomere doublets between the two cell lines was detected by chi-square analysis. Representative images of telomere doublets detected by CO-FISH are shown on the right.

Mentions: In order to better characterize telomere instability induced by ATR deficiency, we performed CO-FISH, which enables to identify parental telomeric C and G strands on metaphase chromosomes after degradation of newly synthesized strands (35) (Figure 3). This allows to distinguish telomeres replicated by either leading or lagging strand synthesis. Results showed that almost all fused sister chromatids in ATRKD cells had both parental telomeric C and G strand sequences at the fusion points (Supplementary Figure S6), confirming the involvement of both sister telomeres in sister chromatid fusions and thus that sister chromatid fusions resulted directly from telomere destabilization in ATRKD cells.Figure 3.


ATR contributes to telomere maintenance in human cells.

Pennarun G, Hoffschir F, Revaud D, Granotier C, Gauthier LR, Mailliet P, Biard DS, Boussin FD - Nucleic Acids Res. (2010)

ATR inhibition destabilizes both lagging and leading strand telomeres. (A) Histograms show the percentages of sister telomere losses affecting the lagging or the leading strand in ATRKD and CtKD cells detected by CO-FISH on metaphase spreads. ‘n’ represents the total number of telomere losses analyzed. No significant difference in the repartition of missing lagging or leading strand telomeres between the two cell lines was detected by chi-square analysis. Representative images of chromosomes missing lagging or leading strand telomeres are shown on the right. Lagging strand telomeres are labeled in red by hybridization of the parental G strands with Cy3-PNA probe and the leading telomeres in green by hybridization of the parental C strand with FITC-PNA probe. (B) Histograms show the respective percentages of telomere doublets containing two parental C-strand telomeres (C–C doublets) or two parental G-strand telomeres (G–G doublets) or both parental strand telomeres (C–G or G–C doublets) in ATRKD and CtKD cells. ‘n’ represents the total number of telomere doublets analyzed. No significant difference in the repartition of the three classes of telomere doublets between the two cell lines was detected by chi-square analysis. Representative images of telomere doublets detected by CO-FISH are shown on the right.
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Figure 3: ATR inhibition destabilizes both lagging and leading strand telomeres. (A) Histograms show the percentages of sister telomere losses affecting the lagging or the leading strand in ATRKD and CtKD cells detected by CO-FISH on metaphase spreads. ‘n’ represents the total number of telomere losses analyzed. No significant difference in the repartition of missing lagging or leading strand telomeres between the two cell lines was detected by chi-square analysis. Representative images of chromosomes missing lagging or leading strand telomeres are shown on the right. Lagging strand telomeres are labeled in red by hybridization of the parental G strands with Cy3-PNA probe and the leading telomeres in green by hybridization of the parental C strand with FITC-PNA probe. (B) Histograms show the respective percentages of telomere doublets containing two parental C-strand telomeres (C–C doublets) or two parental G-strand telomeres (G–G doublets) or both parental strand telomeres (C–G or G–C doublets) in ATRKD and CtKD cells. ‘n’ represents the total number of telomere doublets analyzed. No significant difference in the repartition of the three classes of telomere doublets between the two cell lines was detected by chi-square analysis. Representative images of telomere doublets detected by CO-FISH are shown on the right.
Mentions: In order to better characterize telomere instability induced by ATR deficiency, we performed CO-FISH, which enables to identify parental telomeric C and G strands on metaphase chromosomes after degradation of newly synthesized strands (35) (Figure 3). This allows to distinguish telomeres replicated by either leading or lagging strand synthesis. Results showed that almost all fused sister chromatids in ATRKD cells had both parental telomeric C and G strand sequences at the fusion points (Supplementary Figure S6), confirming the involvement of both sister telomeres in sister chromatid fusions and thus that sister chromatid fusions resulted directly from telomere destabilization in ATRKD cells.Figure 3.

Bottom Line: The telomere aberrations resulting from ATR deficiency (i.e. sister telomere fusions and chromatid-type telomere aberrations) are mainly generated during and/or after telomere replication, and involve both leading and lagging strand telomeres as shown by chromosome orientation-FISH (CO-FISH).Moreover, we show that ATR deficiency strongly sensitizes cells to the G-quadruplex ligand 360A, enhancing sister telomere fusions and chromatid-type telomere aberrations involving specifically the lagging strand telomeres.Altogether, these data reveal that ATR plays a critical role in telomere maintenance during and/or after telomere replication in human cells.

View Article: PubMed Central - PubMed

Affiliation: CEA/DSV/iRCM/SCSR, Laboratoire de Radiopathologie, INSERM-Université Paris VII U967, 92265 Fontenay-aux-Roses, France.

ABSTRACT
Telomere maintenance is essential to preserve genomic stability and involves several telomere-specific proteins as well as DNA replication and repair proteins. The kinase ATR, which has a crucial function in maintaining genome integrity from yeast to human, has been shown to be involved in telomere maintenance in several eukaryotic organisms, including yeast, Arabidopsis and Drosophila. However, its role in telomere maintenance in mammals remains poorly explored. Here, we report by using telomere-fluorescence in situ hybridization (Telo-FISH) on metaphase chromosomes that ATR deficiency causes telomere instability both in primary human fibroblasts from Seckel syndrome patients and in HeLa cells. The telomere aberrations resulting from ATR deficiency (i.e. sister telomere fusions and chromatid-type telomere aberrations) are mainly generated during and/or after telomere replication, and involve both leading and lagging strand telomeres as shown by chromosome orientation-FISH (CO-FISH). Moreover, we show that ATR deficiency strongly sensitizes cells to the G-quadruplex ligand 360A, enhancing sister telomere fusions and chromatid-type telomere aberrations involving specifically the lagging strand telomeres. Altogether, these data reveal that ATR plays a critical role in telomere maintenance during and/or after telomere replication in human cells.

Show MeSH
Related in: MedlinePlus