Absence of telomerase increases the shortening of telomere 1L upon 1L TERRA expression.

(A) DNA was extracted from TetO7-1L, and est1Δ/TetO7-1L (four independent clones) strains grown for 25 generations (gen) at 25°C on YPD plates containing 10 µg/ml Dox (lanes 3, 6, 9, 12), before plating them on YPD plates with (+) (lanes 5, 8, 11, 14) or without (−) Dox (lanes 4, 7, 10, 13) for additional 25 generations. Telomere length at 1L was analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp. (B) 1L TERRA expression does not accelerate shortening of telomere 6R in the absence of telomerase. Telomere PCR for telomere 6R performed with DNA from the same strains and growth conditions as in (A). (C) Recruitment of Est2 is not affected by expression of 1L TERRA. Yeast strains were grown for 25 generations at 30°C on YPD plates with (+) or without (−) Dox. Est2-myc associated chromatin was immunoprecipitated after an additional growth to exponential phase in rich medium (−/+Dox) at 30°C (for details see Materials and Methods). The immunoprecipitated telomeres 1L, 7L and 15L were quantified by real-time PCR and expressed as percentage of input. A mock IP lacking the myc-antibody served as negative control. Values of two independent biological replicates with standard deviation are shown.