Telomere stability and development of ctc1 mutants are rescued by inhibition of EJ recombination pathways in a telomerase-dependent manner.
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.
Affiliation: Génétique, Reproduction et Développement, UMR CNRS 6293, Clermont Université, INSERM U1103, Aubière, France firstname.lastname@example.org.Show MeSH
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Mentions: Arabidopsis ku80-deficient plants have longer telomeres than wild-type plants and this telomere elongation has been shown to be telomerase dependent (38,39). The new telomeric addition we observe ctc1 ku80 xrcc1 xpf mutant plants being dependent on the absence of KU, it seemed likely that it would be also the result of direct addition by telomerase. To test this hypothesis, we mutated the telomerase catalytic subunit (TERT) in plants lacking both the CST complex and the EJ proteins. Arabidopsis ctc1-/- ku80 xrcc1 xpf plants were crossed with plants mutated for tert ku80 xrcc1 genes (see Supplementary Figure S7). Homozygous mutant lines were identified in the F2 generation and TRF analysis realized in G1 ctc1 tert ku80 xrcc1 and ctc1 tert ku80 xrcc1 xpf mutant plants by Southern analysis of MboI-digested genomic DNA using the telomeric repeat probe. Results in Figure 4 clearly show a dramatic loss of telomeric repeats caused by absence of telomerase in both ctc1 tert ku80 xrcc1 xpf (Figure 4A) and ctc1 tert ku80 xrcc1 (Figure 4B). The loss of repeats was faster and more heterogeneous than in sibling CTC1 control plants (wild type for the CST complex) (lanes 5 and 2 in Figure 4A and lanes 4 and 2 in Figure 4B). This was further confirmed by examining dynamics of one particular telomere by reprobing the same Southern blot with the subtelomeric probe specific for the long arm of chromosome 2 (Figure 4C). With this probe a sharp-defined band was detected in CTC1 plants lacking telomerase, while in the absence of the CTC1 protein a shorter and heterogeneous band was observed (lanes 4 and 2 in Figure 4C). Absence of CTC1 thus accelerates the telomere shortening of telomerase mutant plants. As expected from their loss of telomeric repeats, ku80 xrcc1 plants lacking both CST and telomerase proteins show a 3-fold increase of γ-H2AX foci per nuclei as compared to the control plants (Figure 4D and E). This telomere shortening is accompanied by equally dramatic effects on growth, with first generation ctc1 tert ku80 xrcc1 xpf plants showing severe developmental defects and being completely sterile. First generation ctc1 tert ku80 xrcc1 mutant were phenotypically wild type, however only 34% of their seeds germinated and the resulting progeny show severe developmental defects (Figure 5A and B). This contrasts clearly with second-generation plants expressing the CST complex, which present a wild-type phenotype irrespective of the presence or absence of telomerase.
Affiliation: Génétique, Reproduction et Développement, UMR CNRS 6293, Clermont Université, INSERM U1103, Aubière, France email@example.com.