When the Ends Justify the Means: Regulation of Telomere Addition at Double-Strand Breaks in Yeast

Abstract

Telomeres, repetitive sequences located at the ends of most eukaryotic chromosomes, provide a mechanism to replenish terminal sequences lost during DNA replication, limit nucleolytic resection, and protect chromosome ends from engaging in double-strand break (DSB) repair. The ribonucleoprotein telomerase contains an RNA subunit that serves as the template for the synthesis of telomeric DNA. While telomere elongation is typically primed by a 3' overhang at existing chromosome ends, telomerase can act upon internal non-telomeric sequences. Such de novo telomere addition can be programmed (for example, during chromosome fragmentation in ciliated protozoa) or can occur spontaneously in response to a chromosome break. Telomerase action at a DSB can interfere with conservative mechanisms of DNA repair and results in loss of distal sequences but may prevent additional nucleolytic resection and/or chromosome rearrangement through formation of a functional telomere (termed "chromosome healing"). Here, we review studies of spontaneous and induced DSBs in the yeast Saccharomyces cerevisiae that shed light on mechanisms that negatively regulate de novo telomere addition, in particular how the cell prevents telomerase action at DSBs while facilitating elongation of critically short telomeres. Much of our understanding comes from the use of perfect artificial telomeric tracts to "seed" de novo telomere addition. However, endogenous sequences that are enriched in thymine and guanine nucleotides on one strand (TG-rich) but do not perfectly match the telomere consensus sequence can also stimulate unusually high frequencies of telomere formation following a DSB. These observations suggest that some internal sites may fully or partially escape mechanisms that normally negatively regulate de novo telomere addition.

Keywords: DNA repair; Pif1; de novo telomere addition; telomerase; telomere.

FIGURE 1

Models of telomerase regulation at a resecting break in the presence and absence of telomere-like sequences. (A) Regulation of telomerase at endogenous hotspots of de novo telomere addition [Sites of Repair-associated Telomere Addition (SiRTAs)]. Following induction of a double-strand break (DSB), the MRX complex (Mre11–Xrs2–Rad50) along with Sae2 initiates 5’ end resection. Multiple nucleases act at DSBs, but extensive resection requires the exonuclease Exo1 and helicase Sgs1 (Gravel et al., 2008; Mimitou and Symington, 2008; Zhu et al., 2008). The resulting generation of single-stranded DNA (ssDNA) triggers a checkpoint kinase cascade and cell cycle arrest (Villa et al., 2016). Following resection through the TG-rich sequences, Cdc13 binds to a “Core” sequence and recruits telomerase through interactions with Est1. Cdc13, in complex with Stn1 and Ten1 [likely as a hexamer (Ge et al., 2020)], also binds to a proximal “Stim” sequence to prevent further 5′ resection. The limited generation of ssDNA inhibits Pif1 loading and removal of telomerase (see text). While both the Stim and Core sequences are necessary to stimulate de novo telomere addition, it is unclear whether Cdc13 complexes bound to each are functionally distinct (as depicted here). Telomerase must access a 3′ terminus, which is generated through an unknown mechanism to prime telomere synthesis (depicted by a red *). Following de novo telomere addition by telomerase, the CST complex recruits the lagging strand machinery for C-strand fill-in (see text). If the site of telomere addition is oriented correctly relative to the centromere, the resulting product is a stable truncated chromosome. (B) Regulation of telomerase at sequences lacking extensive TG-rich sequences. In the absence of DSB repair, 5’ resection proceeds unimpeded. Phosphorylation of Cdc13 at serine 306 by Mec1 inhibits Cdc13 accumulation at TG_1–3 sequences less than 11 bases (Zhang and Durocher, 2010). Pph3 phosphatase (in a manner requiring the activator Rrd1) counteracts Cdc13 phosphorylation (Zhang and Durocher, 2010), but Pif1 binds and inhibits telomerase action to strongly repress de novo telomere addition (Schulz and Zakian, 1994; Boulé et al., 2005; Li et al., 2014).