Limits...
Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ.

Hsiao SJ, Smith S - J. Cell Biol. (2009)

Bottom Line: Holtgreve-Grez, A.Curr.These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Program, The Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462-2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635-1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV-mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.

Show MeSH

Related in: MedlinePlus

Tankyrase 1 depletion leads to DNA damage and a senescence-like phenotype in HTC75 tumor cells. (A) Immunoblot analysis of extracts from HTC75 cells harvested 48 h after infection with GFP, TNKS1-7, TNKS1-11, TNKS1-12, or TNKS1-13 lentiviral shRNAs. (B) Graphical representation of the effect of tankyrase 1 depletion on HTC75 cell growth. Growth was monitored from 48 h after infection (day 0) to day 6. Cell growth was plotted as PDs versus days in culture. (C) Phase contrast microscopic images of HTC75 cells stained for SA β-galactosidase activity 48 h after infection with the indicated lentiviral shRNAs. (D) Quantification of SA β-galactosidase–positive cells. Approximately 500 cells were scored for each sample. (E) Immunofluorescence analysis of HTC75 cells infected with GFP or TNKS1 lentiviral shRNA. 48 h after infection, cells were incubated with BrdU for 1 h, ethanol fixed, and probed with anti-BrdU antibody (red); DNA was stained with DAPI (blue). (F) Quantification of BrdU-positive cells. Approximately 1,000 cells were scored for each sample. (G) Immunoblot analysis of extracts from HTC75 cells harvested 48 h after infection with GFP or TNKS1 lentiviral shRNA shows up-regulation of p21 expression and induction of γ-H2AX in tankyrase 1–depleted cells. (H) Immunofluorescence analysis of HTC75 cells infected with GFP or TNKS1 lentiviral shRNA, fixed with paraformaldehyde 48 h after infection, and stained with antibodies against p53BP1 (green) and γ-H2AX (red); DNA was stained with DAPI (blue). (I) Quantification of DNA damage foci. Cells with >10 foci that costained with 53BP1 and γ-H2AX were scored. Approximately 1,000 cells were scored for each sample. Bars: (C and E) 20 µm; (H) 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2654126&req=5

fig1: Tankyrase 1 depletion leads to DNA damage and a senescence-like phenotype in HTC75 tumor cells. (A) Immunoblot analysis of extracts from HTC75 cells harvested 48 h after infection with GFP, TNKS1-7, TNKS1-11, TNKS1-12, or TNKS1-13 lentiviral shRNAs. (B) Graphical representation of the effect of tankyrase 1 depletion on HTC75 cell growth. Growth was monitored from 48 h after infection (day 0) to day 6. Cell growth was plotted as PDs versus days in culture. (C) Phase contrast microscopic images of HTC75 cells stained for SA β-galactosidase activity 48 h after infection with the indicated lentiviral shRNAs. (D) Quantification of SA β-galactosidase–positive cells. Approximately 500 cells were scored for each sample. (E) Immunofluorescence analysis of HTC75 cells infected with GFP or TNKS1 lentiviral shRNA. 48 h after infection, cells were incubated with BrdU for 1 h, ethanol fixed, and probed with anti-BrdU antibody (red); DNA was stained with DAPI (blue). (F) Quantification of BrdU-positive cells. Approximately 1,000 cells were scored for each sample. (G) Immunoblot analysis of extracts from HTC75 cells harvested 48 h after infection with GFP or TNKS1 lentiviral shRNA shows up-regulation of p21 expression and induction of γ-H2AX in tankyrase 1–depleted cells. (H) Immunofluorescence analysis of HTC75 cells infected with GFP or TNKS1 lentiviral shRNA, fixed with paraformaldehyde 48 h after infection, and stained with antibodies against p53BP1 (green) and γ-H2AX (red); DNA was stained with DAPI (blue). (I) Quantification of DNA damage foci. Cells with >10 foci that costained with 53BP1 and γ-H2AX were scored. Approximately 1,000 cells were scored for each sample. Bars: (C and E) 20 µm; (H) 5 µm.

Mentions: Our previous study showed that depletion of tankyrase 1 using transient transfection with siRNA oligonucleotides resulted in persistent sister telomere associations and mitotic arrest in HeLa cells (Dynek and Smith, 2004). We sought to determine whether tankyrase 1 depletion would lead to a similar phenotype in other human cell lines. Unexpectedly, we found that treatment of a different human tumor cell line, HTC75 (a derivative of HT1080), with tankyrase 1 siRNA oligonucleotides did not lead to a mitotic arrest despite efficient knockdown of tankyrase 1 protein (unpublished data). To determine whether long-term (rather than transient) depletion of tankyrase 1 would influence HTC75 cell growth, we generated stable cell lines lacking tankyrase 1 using lentiviral short hairpin RNAs (shRNAs). HTC75 cells were infected with four different tankyrase 1 shRNAs and a GFP shRNA as a control, and stable lines were selected with puromycin and harvested for analysis 48 h after infection. Immunoblot analysis shows efficient depletion of tankyrase 1 protein (Fig. 1 A). To determine the effect of tankyrase 1 depletion on cell growth, stable cell lines were passaged from day 0 (48 h after infection) to day 6, and the population doublings (PDs) were determined. As predicted from transient siRNA experiments, HTC75 cells lacking tankyrase 1 did not arrest in mitosis. However, they did have a striking phenotype; cell proliferation was inhibited (Fig. 1 B). Moreover, analysis of the cells on day 0 revealed hallmarks of cellular senescence: large vacuolated, multinuclear cells that stained positive for the senescence-associated (SA) β-galactosidase marker that is active at pH 6.0 (Fig. 1, C and D; Dimri et al., 1995). These effects were observed with four distinct lentiviral shRNAs (Fig. 1, A–D). For all subsequent experiments, the TNKS1-13 lentivirus was used, and the analysis was performed at 48 h after infection (day 0). Consistent with a senescence-like G1 arrest, tankyrase 1–depleted cells show a block in BrdU incorporation (Fig. 1, E and F) and an up-regulation of p21 (Fig. 1 G; Brown et al., 1997).


Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ.

Hsiao SJ, Smith S - J. Cell Biol. (2009)

Tankyrase 1 depletion leads to DNA damage and a senescence-like phenotype in HTC75 tumor cells. (A) Immunoblot analysis of extracts from HTC75 cells harvested 48 h after infection with GFP, TNKS1-7, TNKS1-11, TNKS1-12, or TNKS1-13 lentiviral shRNAs. (B) Graphical representation of the effect of tankyrase 1 depletion on HTC75 cell growth. Growth was monitored from 48 h after infection (day 0) to day 6. Cell growth was plotted as PDs versus days in culture. (C) Phase contrast microscopic images of HTC75 cells stained for SA β-galactosidase activity 48 h after infection with the indicated lentiviral shRNAs. (D) Quantification of SA β-galactosidase–positive cells. Approximately 500 cells were scored for each sample. (E) Immunofluorescence analysis of HTC75 cells infected with GFP or TNKS1 lentiviral shRNA. 48 h after infection, cells were incubated with BrdU for 1 h, ethanol fixed, and probed with anti-BrdU antibody (red); DNA was stained with DAPI (blue). (F) Quantification of BrdU-positive cells. Approximately 1,000 cells were scored for each sample. (G) Immunoblot analysis of extracts from HTC75 cells harvested 48 h after infection with GFP or TNKS1 lentiviral shRNA shows up-regulation of p21 expression and induction of γ-H2AX in tankyrase 1–depleted cells. (H) Immunofluorescence analysis of HTC75 cells infected with GFP or TNKS1 lentiviral shRNA, fixed with paraformaldehyde 48 h after infection, and stained with antibodies against p53BP1 (green) and γ-H2AX (red); DNA was stained with DAPI (blue). (I) Quantification of DNA damage foci. Cells with >10 foci that costained with 53BP1 and γ-H2AX were scored. Approximately 1,000 cells were scored for each sample. Bars: (C and E) 20 µm; (H) 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654126&req=5

fig1: Tankyrase 1 depletion leads to DNA damage and a senescence-like phenotype in HTC75 tumor cells. (A) Immunoblot analysis of extracts from HTC75 cells harvested 48 h after infection with GFP, TNKS1-7, TNKS1-11, TNKS1-12, or TNKS1-13 lentiviral shRNAs. (B) Graphical representation of the effect of tankyrase 1 depletion on HTC75 cell growth. Growth was monitored from 48 h after infection (day 0) to day 6. Cell growth was plotted as PDs versus days in culture. (C) Phase contrast microscopic images of HTC75 cells stained for SA β-galactosidase activity 48 h after infection with the indicated lentiviral shRNAs. (D) Quantification of SA β-galactosidase–positive cells. Approximately 500 cells were scored for each sample. (E) Immunofluorescence analysis of HTC75 cells infected with GFP or TNKS1 lentiviral shRNA. 48 h after infection, cells were incubated with BrdU for 1 h, ethanol fixed, and probed with anti-BrdU antibody (red); DNA was stained with DAPI (blue). (F) Quantification of BrdU-positive cells. Approximately 1,000 cells were scored for each sample. (G) Immunoblot analysis of extracts from HTC75 cells harvested 48 h after infection with GFP or TNKS1 lentiviral shRNA shows up-regulation of p21 expression and induction of γ-H2AX in tankyrase 1–depleted cells. (H) Immunofluorescence analysis of HTC75 cells infected with GFP or TNKS1 lentiviral shRNA, fixed with paraformaldehyde 48 h after infection, and stained with antibodies against p53BP1 (green) and γ-H2AX (red); DNA was stained with DAPI (blue). (I) Quantification of DNA damage foci. Cells with >10 foci that costained with 53BP1 and γ-H2AX were scored. Approximately 1,000 cells were scored for each sample. Bars: (C and E) 20 µm; (H) 5 µm.
Mentions: Our previous study showed that depletion of tankyrase 1 using transient transfection with siRNA oligonucleotides resulted in persistent sister telomere associations and mitotic arrest in HeLa cells (Dynek and Smith, 2004). We sought to determine whether tankyrase 1 depletion would lead to a similar phenotype in other human cell lines. Unexpectedly, we found that treatment of a different human tumor cell line, HTC75 (a derivative of HT1080), with tankyrase 1 siRNA oligonucleotides did not lead to a mitotic arrest despite efficient knockdown of tankyrase 1 protein (unpublished data). To determine whether long-term (rather than transient) depletion of tankyrase 1 would influence HTC75 cell growth, we generated stable cell lines lacking tankyrase 1 using lentiviral short hairpin RNAs (shRNAs). HTC75 cells were infected with four different tankyrase 1 shRNAs and a GFP shRNA as a control, and stable lines were selected with puromycin and harvested for analysis 48 h after infection. Immunoblot analysis shows efficient depletion of tankyrase 1 protein (Fig. 1 A). To determine the effect of tankyrase 1 depletion on cell growth, stable cell lines were passaged from day 0 (48 h after infection) to day 6, and the population doublings (PDs) were determined. As predicted from transient siRNA experiments, HTC75 cells lacking tankyrase 1 did not arrest in mitosis. However, they did have a striking phenotype; cell proliferation was inhibited (Fig. 1 B). Moreover, analysis of the cells on day 0 revealed hallmarks of cellular senescence: large vacuolated, multinuclear cells that stained positive for the senescence-associated (SA) β-galactosidase marker that is active at pH 6.0 (Fig. 1, C and D; Dimri et al., 1995). These effects were observed with four distinct lentiviral shRNAs (Fig. 1, A–D). For all subsequent experiments, the TNKS1-13 lentivirus was used, and the analysis was performed at 48 h after infection (day 0). Consistent with a senescence-like G1 arrest, tankyrase 1–depleted cells show a block in BrdU incorporation (Fig. 1, E and F) and an up-regulation of p21 (Fig. 1 G; Brown et al., 1997).

Bottom Line: Holtgreve-Grez, A.Curr.These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Program, The Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462-2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635-1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV-mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.

Show MeSH
Related in: MedlinePlus