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

Abstract

DNA double-strand breaks (DSBs) and short telomeres are structurally similar, yet they have diametrically opposed fates. Cells must repair DSBs while blocking the action of telomerase on these ends. Short telomeres must avoid recognition by the DNA damage response while promoting telomerase recruitment. In Saccharomyces cerevisiae, the Pif1 helicase, a telomerase inhibitor, lies at the interface of these end-fate decisions. Using Pif1 as a sensor, we uncover a transition point in which 34 bp of telomeric (TG1-3)n repeat sequence renders a DNA end insensitive to Pif1 action, thereby enabling extension by telomerase. A similar transition point exists at natural chromosome ends, where telomeres shorter than ~40 bp are inefficiently extended by telomerase. This phenomenon is not due to known Pif1 modifications and we instead propose that Cdc13 renders TG34+ ends insensitive to Pif1 action. We contend that the observed threshold of Pif1 activity defines a dividing line between DSBs and telomeres.

TheSaccharomyces cerevisiaeHrq1 and Pif1 DNA helicases synergistically modulate telomerase activityin vitro

“…Multiple protein complexes are involved in binding telomeres and protecting them from nuclease-and helicase-controlled resection, thus preventing activation of DNA damage signaling pathways (41,44). These proteins include MRX (Mre11/Rad50/Xrs2), CST (Cdc13/Stn1/Ten1), Ku70/80, and the Rap1/Rif1/Rif2 dsDNA binding complex (reviewed in (45)).…”

“…Pif1 is a central regulator of telomere length and is important for yeast cells to distinguish telomeres from DSBs. Recently, inducible short telomere systems and single telomere extension (STEX) assays have been used to probe the effects on Pif1 binding and activity at telomeres of different lengths (36,41). STEX experiments demonstrate that telomerase processivity and the fraction of telomeres extended in vivo increases in the absence of Pif1 (36).…”

“…Using a similar in vivo telomere length induction system, another group found a transition point where telomeric sequences of 34-125 bp are recognized as short telomeres and are preferentially extended by telomerase (41). Their model suggests that at chromosome ends of ~40 bp, Pif1 blocks resection and telomerase activity, acting as a checkpoint for distinguishing DSBs from telomeres.…”

Repair and Reconstruction of Telomeric and Subtelomeric Regions and Genesis of New Telomeres: Implications for Chromosome Evolution

“…[ 62,63 ] In S. cerevisiae , telomerase does not work effectively when telomere is shorter than ≈40 bp, which is about 1/10 of the average length of a telomere in this species. [ 64 ] This phenomenon involves Pif1 helicase that unwinds RNA–DNA hybrids to inhibit telomerase activity.…”

Telomerase in Space and Time: Regulation of Yeast Telomerase Function at Telomeres and DNA Breaks

“…How does a cell distinguish between these very similar structures? Recent evidence suggests that cooperation between Cdc13 and Pif1 might channel particular substrates into proper repair pathways [145]. It was shown, that if either a natural telomere or a TG-seed flanking a DSB is shorter than 35-40 bp, it is recognized as a break and is protected from telomerase activity by Pif1.…”