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.

Dysfunctional telomere length regulation is detrimental to human health, and both activation and inhibition of telomerase have been proposed in potential therapies to treat human diseases. The Saccharomyces cerevisiae Pif1 protein is an evolutionarily conserved helicase that inhibits telomerase activity at DNA ends. Recent studies have indicated that Pif1 is specifically important for inhibiting telomerase at DNA ends with very little or no telomeric sequence and at long telomeres. At the former, Pif1 prevents the inappropriate addition of a telomere at DNA double-strand breaks. For the latter, Pif1 has been shown to bind long telomeres to presumably promote the extension of the short ones. These observations leave the impression that Pif1 does not act at DNA ends with telomeric sequence of intermediate length. Here, we provide in vivo evidence that Pif1 inhibits telomerase activity at DNA ends regardless of telomere sequence length.

In Saccharomyces cerevisiae, recruitment of telomerase to telomeres requires an interaction between Cdc13, which binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. A second pathway involving an interaction between the yKu complex and telomerase RNA (TLC1) contributes to telomerase recruitment, but cannot sufficiently recruit telomerase on its own to prevent replicative senescence when the primary Cdc13-Est1 pathway is abolished—for example, in the cdc13-2 mutant. In this study, we find that mutation of PIF1, which encodes a helicase that inhibits telomerase, suppresses the replicative senescence of cdc13-2 by increasing reliance on the yKu-TLC1 pathway for telomerase recruitment. Our findings reveal new insight into telomerase-mediated telomere maintenance.