Abstract
Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Here we demonstrated that the budding yeast Saccharomyces cerevisiae type I survivors derived from telomerase-deficient cells were hypersensitive to DNA damaging agents. Assays to track telomere lengths and drug sensitivity of telomerase-deficient cells from spore colonies to survivors suggested a correlation between telomere shortening and bleomycin sensitivity. Our genetic studies demonstrated that this sensitivity depends on Mec1, which signals checkpoint activation, leading to prolonged cell-cycle arrest in senescent budding yeasts. Moreover, we also observed that when cells equipped with short telomeres, recruitments of homologous recombination proteins, Rad51 and Rad52, were reduced at an HO-endonuclease-catalyzed double-strand break (DSB), while their associations were increased at chromosome ends. These results suggested that the sensitive phenotype may be attributed to the sequestration of repair proteins to compromised telomeres, thus limiting the repair capacity at bona fide DSB sites.
Highlights
Telomeres are the specialized DNA-proteins complexes at the ends of eukaryotic chromosomes that allow intact ends to be distinguished from broken chromosomes, prevent chromosomes from end-to-end fusion, degradation, genomic instability and the associated risk of cancer
In order to confirm that growth inhibition induced by bleomycin was not restricted to a single source of type I survivors, we further tested the sensitivity of 448 type I survivors derived from nine independent tlc1 spore colonies to bleomycin
The bindings of both Rad proteins at VII-L reduced to the wild-type level in type I cells after treated with bleomycin (Figure 5B). Both wild-type and pre-senescent tlc1 mutant strains displayed indistinguishable bindings of Rad proteins at VII-L before and after bleomycin treatment. These results demonstrate that sequestration of Rad51 and Rad52 at short telomeres reduces the efficiency of their recruitments to an induced double-strand break (DSB) site and that the mere onset of telomerase deprivation in pre-senescent tlc1 mutant does not lead to the same level of biased distribution of Rad proteins as shown in type I survivors
Summary
Telomeres are the specialized DNA-proteins complexes at the ends of eukaryotic chromosomes that allow intact ends to be distinguished from broken chromosomes, prevent chromosomes from end-to-end fusion, degradation, genomic instability and the associated risk of cancer. Telomeric DNA is composed of a tandem array of short sequences. These repeats in S. cerevisiae consist of ,350675 bp of duplex TG1–3/ C1–3A tracts. The majority of telomerase-minus S. cerevisiae cells enter cell cycle arrest eventually following the senescence stage [10,11], survivors arise through RAD52-dependent homologous recombination (HR). Most survivors are type I, which have multiple tandem copies of the subtelomeric Y’ element followed by very short terminal tracts of TG1–3/CA1–3 DNA [10,12]. The generation of type I survivors depends on the presence of helicase Rad, Rad that acts in strand invasion, Rad55-Rad complex that stabilizes recombination filament [13], and 59-to-39 exonuclease Exo1 [14]. The formation of type II survivors, on the other hand, requires MRX complex (Mre, Rad and Xrs2), Rad for strand invasion, and the Sgs1-Top complex, which is involved in the process of HR [15,16,17,18,19]
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