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Telomere stability and development of ctc1 mutants are rescued by inhibition of EJ recombination pathways in a telomerase-dependent manner.

Amiard S, Olivier M, Allain E, Choi K, Smith-Unna R, Henderson IR, White CI, Gallego ME - Nucleic Acids Res. (2014)

Bottom Line: In this work, we set out to specifically test this hypothesis in the plant, Arabidopsis.It is thus the chromosomal fusions, per se, which are the underlying cause of the severe developmental defects.This rescue is mediated by telomerase-dependent telomere extension, revealing a competition between telomerase and end-joining recombination proteins for access to deprotected telomeres.

View Article: PubMed Central - PubMed

Affiliation: Génétique, Reproduction et Développement, UMR CNRS 6293, Clermont Université, INSERM U1103, Aubière, France megalleg@univ-bpclermont.fr.

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Short telomeres in ctc1 ku80 xrcc1 xpf mutants are deprotected. (A) γ-H2AX immunostaining and subtelomeric (upper panel) or telomeric (lower panel) FISH labelling of root tip nuclei of the ctc1 ku80 xrcc1 xpf mutants. Nuclei were stained with DAPI (blue), FISH signals are coloured in magenta and γ-H2AX foci are coloured in green. Images are collapsed Z-stack projections of a deconvolved three-dimensional image stack. The arrow (upper right image) indicates a focus with colocalized γ-H2AX and subtelomeric probe signals. Bar in (A) = 2 μm. (B) Table with mean number of foci per nucleus with standard error (s.e.m.), the percentages of γ-H2AX foci colocalizing with the subtelomeric and the telomeric probes. The numbers (n) of telomeric or non-telomeric foci counted are indicated in each case. n.d. means ‘not determined’.
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Figure 3: Short telomeres in ctc1 ku80 xrcc1 xpf mutants are deprotected. (A) γ-H2AX immunostaining and subtelomeric (upper panel) or telomeric (lower panel) FISH labelling of root tip nuclei of the ctc1 ku80 xrcc1 xpf mutants. Nuclei were stained with DAPI (blue), FISH signals are coloured in magenta and γ-H2AX foci are coloured in green. Images are collapsed Z-stack projections of a deconvolved three-dimensional image stack. The arrow (upper right image) indicates a focus with colocalized γ-H2AX and subtelomeric probe signals. Bar in (A) = 2 μm. (B) Table with mean number of foci per nucleus with standard error (s.e.m.), the percentages of γ-H2AX foci colocalizing with the subtelomeric and the telomeric probes. The numbers (n) of telomeric or non-telomeric foci counted are indicated in each case. n.d. means ‘not determined’.

Mentions: Unprotected telomeres generated in the absence of the CST complex activate a DNA damage response, as visualized by the detection of Telomere Induced Foci (TIF), phosphorylated H2AX (γ-H2AX) foci that colocalize with telomeres (30). Given the absence of chromosomal fusions and normal growth of ctc1 plants lacking the three EJ pathways, it is clearly of interest to determine whether or not the DDR is activated in these plants. We thus monitored the presence of γ-H2AX foci in nuclei of ctc1 ku80 xrcc1 xpf mutants. As shown in Figure 3A and B, γ-H2AX foci were visible in the nuclei of ctc1 cells lacking the EJ pathway proteins, although in a slightly lower number of nuclei than the ctc1 mutant plants (mean number of foci/nucleus of 0.73 versus 1.04 in ctc1 single mutant). FISH was performed on the same slides using nine subtelomeric specific BACs corresponding to nine of the 10 Arabidopsis chromosome ends. As previously reported for the ctc1 single mutant (30), 68.1% of the γ-H2AX foci colocalize with the subtelomere-specific probes revealing that ctc1 ku80 xrcc1 xpf telomeres are recognized as DNA damage in the mutant plants. Colocalization of γ-H2AX foci with telomeres using a probe against the telomeric repeats (TTTAGGG) however showed that only 17.2% of γ-H2AX foci colocalize with the telomeric repeat sequences. The remaining (50.9%) of TIFs defined by the subtelomeric BAC probes are thus not associated with sequences detectable by the telomeric repeat probe. The DDR is thus being induced at only a subset of dysfunctional telomeres in these plants—mostly those with telomeric sequences too short to be detected by the repeat probe. Our observations suggest that only chromosomes ends with short telomeric repeats activate a DNA damage response.


Telomere stability and development of ctc1 mutants are rescued by inhibition of EJ recombination pathways in a telomerase-dependent manner.

Amiard S, Olivier M, Allain E, Choi K, Smith-Unna R, Henderson IR, White CI, Gallego ME - Nucleic Acids Res. (2014)

Short telomeres in ctc1 ku80 xrcc1 xpf mutants are deprotected. (A) γ-H2AX immunostaining and subtelomeric (upper panel) or telomeric (lower panel) FISH labelling of root tip nuclei of the ctc1 ku80 xrcc1 xpf mutants. Nuclei were stained with DAPI (blue), FISH signals are coloured in magenta and γ-H2AX foci are coloured in green. Images are collapsed Z-stack projections of a deconvolved three-dimensional image stack. The arrow (upper right image) indicates a focus with colocalized γ-H2AX and subtelomeric probe signals. Bar in (A) = 2 μm. (B) Table with mean number of foci per nucleus with standard error (s.e.m.), the percentages of γ-H2AX foci colocalizing with the subtelomeric and the telomeric probes. The numbers (n) of telomeric or non-telomeric foci counted are indicated in each case. n.d. means ‘not determined’.
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Related In: Results  -  Collection

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Figure 3: Short telomeres in ctc1 ku80 xrcc1 xpf mutants are deprotected. (A) γ-H2AX immunostaining and subtelomeric (upper panel) or telomeric (lower panel) FISH labelling of root tip nuclei of the ctc1 ku80 xrcc1 xpf mutants. Nuclei were stained with DAPI (blue), FISH signals are coloured in magenta and γ-H2AX foci are coloured in green. Images are collapsed Z-stack projections of a deconvolved three-dimensional image stack. The arrow (upper right image) indicates a focus with colocalized γ-H2AX and subtelomeric probe signals. Bar in (A) = 2 μm. (B) Table with mean number of foci per nucleus with standard error (s.e.m.), the percentages of γ-H2AX foci colocalizing with the subtelomeric and the telomeric probes. The numbers (n) of telomeric or non-telomeric foci counted are indicated in each case. n.d. means ‘not determined’.
Mentions: Unprotected telomeres generated in the absence of the CST complex activate a DNA damage response, as visualized by the detection of Telomere Induced Foci (TIF), phosphorylated H2AX (γ-H2AX) foci that colocalize with telomeres (30). Given the absence of chromosomal fusions and normal growth of ctc1 plants lacking the three EJ pathways, it is clearly of interest to determine whether or not the DDR is activated in these plants. We thus monitored the presence of γ-H2AX foci in nuclei of ctc1 ku80 xrcc1 xpf mutants. As shown in Figure 3A and B, γ-H2AX foci were visible in the nuclei of ctc1 cells lacking the EJ pathway proteins, although in a slightly lower number of nuclei than the ctc1 mutant plants (mean number of foci/nucleus of 0.73 versus 1.04 in ctc1 single mutant). FISH was performed on the same slides using nine subtelomeric specific BACs corresponding to nine of the 10 Arabidopsis chromosome ends. As previously reported for the ctc1 single mutant (30), 68.1% of the γ-H2AX foci colocalize with the subtelomere-specific probes revealing that ctc1 ku80 xrcc1 xpf telomeres are recognized as DNA damage in the mutant plants. Colocalization of γ-H2AX foci with telomeres using a probe against the telomeric repeats (TTTAGGG) however showed that only 17.2% of γ-H2AX foci colocalize with the telomeric repeat sequences. The remaining (50.9%) of TIFs defined by the subtelomeric BAC probes are thus not associated with sequences detectable by the telomeric repeat probe. The DDR is thus being induced at only a subset of dysfunctional telomeres in these plants—mostly those with telomeric sequences too short to be detected by the repeat probe. Our observations suggest that only chromosomes ends with short telomeric repeats activate a DNA damage response.

Bottom Line: In this work, we set out to specifically test this hypothesis in the plant, Arabidopsis.It is thus the chromosomal fusions, per se, which are the underlying cause of the severe developmental defects.This rescue is mediated by telomerase-dependent telomere extension, revealing a competition between telomerase and end-joining recombination proteins for access to deprotected telomeres.

View Article: PubMed Central - PubMed

Affiliation: Génétique, Reproduction et Développement, UMR CNRS 6293, Clermont Université, INSERM U1103, Aubière, France megalleg@univ-bpclermont.fr.

Show MeSH
Related in: MedlinePlus