Endogenous Hot Spots of De Novo Telomere Addition in the Yeast Genome Contain Proximal Enhancers That Bind Cdc13

Abstract

DNA double-strand breaks (DSBs) pose a threat to genome stability and are repaired through multiple mechanisms. Rarely, telomerase, the enzyme that maintains telomeres, acts upon a DSB in a mutagenic process termed telomere healing. The probability of telomere addition is increased at specific genomic sequences termed sites of repair-associated telomere addition (SiRTAs). By monitoring repair of an induced DSB, we show that SiRTAs on chromosomes V and IX share a bipartite structure in which a core sequence (Core) is directly targeted by telomerase, while a proximal sequence (Stim) enhances the probability of de novo telomere formation. The Stim and Core sequences are sufficient to confer a high frequency of telomere addition to an ectopic site. Cdc13, a single-stranded DNA binding protein that recruits telomerase to endogenous telomeres, is known to stimulate de novo telomere addition when artificially recruited to an induced DSB. Here we show that the ability of the Stim sequence to enhance de novo telomere addition correlates with its ability to bind Cdc13, indicating that natural sites at which telomere addition occurs at high frequency require binding by Cdc13 to a sequence 20 to 100 bp internal from the site at which telomerase acts to initiate de novo telomere addition. Chromosomes in the budding yeast Saccharomyces cerevisiae, as in all eukaryotes, terminate with specialized nucleoprotein structures called telomeres. S. cerevisiae telomeric DNA consists of ϳ250 to 350 bp of TG 1-3 /AC 1-3 repeats and a short (ϳ10-bp) terminal G-rich 3= overhang (1). Because the conventional DNA replication machinery cannot fully replicate chromosome ends, telomeres shorten with each cell division cycle. In most eukaryotes, telomere shortening is counteracted by the enzyme telomerase, a ribonucleoprotein complex that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Associated with telomeric DNA are proteins that protect chromosome ends from nucleolytic resection and prevent chromosome end-to-end fusions by distinguishing natural chromosome ends from ends generated by DNA double-strand breaks (DSBs) (2). These protective functions make telomeres essential for the maintenance of genome integrity and cell viability.In S. cerevisiae, optimal telomere length requires a balance between positive and negative regulatory mechanisms mediated by telomere-binding proteins, including Cdc13 and Rap1 (3). Cdc13, a telomere sequence-specific single-stranded DNA (ssDNA) binding protein, recruits telomerase to telomeres during the S/G 2 phase of the cell cycle through interaction with Est1, a subunit of the telomerase holoenzyme (reviewed in reference 3). Rap1 binds to the double-stranded telomeric repeat, forming a telomere length-regulatory complex through interactions of its C-terminal domain with Rif1 and Rif2 (4,5). Regulation occurs through a counting mechanism in which telomere length is inversely proportional to the number of Rif1 and Rif2 molecules present at a telomere (6, 7).Cells experience insults to t...

Interaction of yeast Rad51 and Rad52 relieves Rad52-mediated inhibition of de novo telomere addition

“…The ability of a sequence to function as a SiRTA is monitored in haploid cells using a previously-described inducible HO endonuclease cleavage assay [20,29]. Briefly, a recognition site for the HO endonuclease is integrated approximately 3 kb distal to the SiRTA, while the gene encoding the HO endonuclease is placed under control of a galactose-inducible promoter.…”

“…While the pathways described above require interaction of the DSB with an intra-or interchromosomal sequence to facilitate repair, terminal deletions can arise through direct addition of a de novo telomere by telomerase to an internal DSB [15,16]. In a haploid strain, GCR events within a non-essential terminal region of yeast chromosome 5 are more likely to involve de novo telomere addition than any other type of rearrangement, whether they occur spontaneously or in response to a single DSB [17][18][19][20]. Sites of de novo telomere addition in yeast typically contain at least a single TG-dinucleotide, likely reflecting a requirement for base pairing between the 3' end of the DSB and the telomerase RNA (which in yeast contains the sequence 5'-CACCACACCCACACAC-3') [18,19,21].…”

“…We have previously characterized two endogenous hotspots of de novo telomere addition on the left arms of yeast chromosomes 5 and 9. These TG-rich sequences, termed SiRTAs (Sites of Repair-associated Telomere Addition), undergo de novo telomere addition at frequen-cies~200-fold higher than neighboring regions [20], even when the initiating chromosome break is located several kilobases distal to the eventual site of telomere addition [20]. The nomenclature for these sites (SiRTAs 5L-35 and 9L-44) reflects the distance each is located (35kb and 44kb, respectively) from the nearest telomere on that chromosome arm.…”

“…The nomenclature for these sites (SiRTAs 5L-35 and 9L-44) reflects the distance each is located (35kb and 44kb, respectively) from the nearest telomere on that chromosome arm. Both sites display a bipartite structure consisting of a Core sequence within which telomerase acts to initiate de novo telomere addition and a Stim sequence that enhances telomere addition at the Core by providing binding site(s) for Cdc13 [20]. The identification of these sequences as hotspots of de novo telomere addition provides a tractable system in which to examine the interplay between alternative non-conservative repair pathways.…”

A Pif1-dependent threshold separates DNA double-strand breaks and telomeres

“…Our investigation into the molecular trigger of the DSB-telomere transition points to a key role for the DNA binding protein Cdc13. This conclusion is supported by work revealing that microsatellite repeats containing Cdc13 binding sites stimulate telomere addition (Piazza et al, 2012), and recently that a hotspot on Chr V also promotes Cdc13 binding and telomere addition (Obodo et al, 2016). Furthermore, the tethering of Cdc13, but not Rap1, to this site was shown to be sufficient for the formation of new telomeres (Obodo et al, 2016).…”