Author response: A sharp Pif1-dependent threshold separates DNA double-strand breaks from critically short telomeres

Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation inSaccharomyces cerevisiae

“…At wild-type telomeres, telomerase extends only a subset of telomeres in each cell cycle, with a strong preference for the extension of short telomeres (Teixeira et al, 2004;Strecker et al, 2017). At tlc1-tm telomeres, we find that telomere extension frequency is dramatically increased, with nearly all (92%) of telomeres being extended during a single cell cycle ( Figure 3B and 3C).…”

“…To examine telomere length regulation in tlc1-tm strains further, we monitored telomerase-mediated telomere extension events at nucleotide resolution after a single cell cycle using the iSTEX (inducible Single Telomere EXtension) assay (Strecker et al, 2017).…”

“…(C) Telomere VI-R sequences obtained from the iSTEX analysis in B were binned into groups of 10 nt in size according to telomere length before telomerase induction. iSTEX data for the extension of wild-type telomeres were taken from previous studies (Strecker et al, 2017;Stinus et al, 2017) and included for comparison. Groups containing less than four telomeres were excluded from this analysis.…”

“…Logarithmic regression curves for each data set were also included in the plot. Telomeres shorter than 40 nt before telomerase induction, which are not efficiently recognized and extended by telomerase (Strecker et al, 2017), were removed from the regression analysis. All data (except C) are shown as mean SEM (n=3, *** p<10 -3 , **** p<10 -4 ).…”

“…iSTEX was performed essentially as previously described (Strecker et al, 2017), except that the starting PGALL-EST1 strain contained the tlc1-tm allele. This strain was then transformed with a PCR product containing the wild-type TLC1 allele and immediately grown on media containing glucose, which shut off expression of EST1.…”