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.

Pif1 Activity is Modulated by DNA Sequence and Structure

“…cerevisiae telomeres may adopt an alternate higher order structure that Pif1 is better adapted to and that is different from the G4 structures formed by human telomeric ssDNA. Regardless of the higher-order structure, these data support a model where Pif1 will preferentially bind to telomeres, rDNA, and G-rich DSBs due to its high affinity for G-rich ssDNA …”

“…Regardless of the higher-order structure, these data support a model where Pif1 will preferentially bind to telomeres, rDNA, and G-rich DSBs due to its high affinity for G-rich ssDNA. 61 A critical focus of this work was the role of the length of ssDNA loading strands of potential Pif1 substrates. Efficient unwinding of immobilized gapped forks by Pif1 requires a ssDNA gap size of >5 nt in a unique set of single-molecule experiments using Brownian motion for detection of unwinding.…”

Hotspot ofde novotelomere addition stabilizes linear amplicons in yeast grown in sulfate-limiting conditions

“…The observation that many of the telomere addition events associated with extrachromosomal SUL1 fragments cluster within a small (∼100 bp) region is reminiscent of sequences in the yeast genome that are hotspots of de novo telomere addition (termed SiRTAs or Sites of Repair-associated Telomere Addition) (Stellwagen et al 2003; Obodo et al 2016; Hoerr et al 2021). SiRTAs contain strand-specific, TG-rich sequence motifs (Obodo et al 2016; Ngo et al 2020).…”

A proto-telomere is elongated by telomerase in a shelterin-dependent manner in quiescent fission yeast cells

“…Like in cycling cells, the presence of the telomeric seed (48 bp) near the break promotes de novo telomere addition despite the fact that the I-SceI 3′-overhang does not exhibit telomeric sequences. This is reminiscent to HO-cleavage adjacent to a telomeric seed sequence which has been extensively exploited to study telomere elongation in budding yeast ( 64 ). We further report that telomerase can extend this short proto-telomere despite that telomere elongation was thought to depend on replication fork passage in most organisms studied ( 18 , 22 , 23 ).…”