DNA damage signalling prevents deleterious telomere addition at DNA breaks

Abstract

The response to DNA damage involves regulation of multiple essential processes to maximize the accuracy of DNA damage repair and cell survival 1. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage, to increase the accuracy of repair. Here we report that telomerase action is regulated as a part of the cellular response to a DNA double-strand break (DSB). Using yeast, we show that the major ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. Upon DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Utilizing a separation of function PIF1 mutation, we show that this phosphorylation is required for the Pif1-mediated telomerase inhibition that takes place specifically at DNA breaks, but not telomeres. Hence DNA damage signalling down-modulates telomerase action at a DNA break via Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a novel regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity.

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

“…De novo telomere addition was assayed in SRS2 and srs2 Δ using a previously described genetic test (Makovets & Blackburn, 2009) involving a single galactose‐inducible DSB (Fig 2A). Because de novo telomere addition normally occurs with a very low frequency due to telomerase inhibition by Pif1 (Schulz & Zakian, 1994), the pif1‐m2 background was used in the genetic assay.…”

In Vitro Biological Characterization of DCUN1D5 in DNA Damage Response

“…Additionally, there was a putative Rad53 binding site. Recently, researchers uncovered a novel MEC1-RAD53-DCN1-dependent signaling pathway that prevented deleterious DSB-specific telomerase additions at DNA breaks, thus preserving genomic integrity (Makovets et al, 2009;Kurz et al, 2008). Taken together, these findings suggested that the DCUN1D5 might play a role in DNA damage response to genotoxic stress and probably subsequent DNA repair.…”

Suppression of Chromosome Healing and Anticheckpoint Pathways in Yeast Postsenescence Survivors

“…In contrast to prolonged Rad53 activation in postsenescent est2Δ and tlc1Δ survivors, checkpoint activation in presenescent telomerase-deficient est2Δ cells was as short-lived as in wild-type cells ( Figure 2C). Thus, these data indicate that it was the establishment of the ALT pathway, rather than just the loss of telomerase, that was responsible for prolonged checkpoint activation in our survivors.Abolition of the checkpoint response does not restore de novo telomere addition in survivors As indicated above, DNA damage checkpoints inhibit de novo telomere addition at DSBs by telomerase to prevent genome instability (Makovets and Blackburn 2009;Zhang and Durocher 2009). We thus investigated if prolonged checkpoint activation also contributes to impaired de novo telomere addition in postsenescent survivors.…”

“…Abolition of the checkpoint response does not restore de novo telomere addition in survivors As indicated above, DNA damage checkpoints inhibit de novo telomere addition at DSBs by telomerase to prevent genome instability (Makovets and Blackburn 2009;Zhang and Durocher 2009). We thus investigated if prolonged checkpoint activation also contributes to impaired de novo telomere addition in postsenescent survivors.…”

“…However, telomerase can sometimes also be a source of genome instability when it acts on internal telomere repeat-like sequences adjacent to DSBs and converts them to de novo telomeres in a mutagenic repair process called chromosome healing (Kramer and Haber 1993). To prevent such genome rearrangements, de novo telomere addition at DSBs is normally suppressed by DNA damage checkpoint-signaling pathways (Makovets and Blackburn 2009;Zhang and Durocher 2009). However, checkpoint signals at telomere seed-containing DSBs turn off more quickly than at non-seed-containing DSBs (2-3 hr vs. .10 hr); and, strikingly, a seed-containing end also rapidly turns off the checkpoint signal generated from the other seed-free end of the same DSB, a phenomenon referred to as the anticheckpoint function of telomeres (Michelson et al 2005).…”