Abstract
In yeast telomerase mutants, the Sgs1 RecQ helicase slows the rate of senescence and also facilitates the appearance of certain types of survivors of critical telomere shortening via mechanisms dependent on Rad52-dependent homologous recombination (HR). Here we describe a third function for Sgs1 in telomerase-deficient cells, inhibition of survivors that grow independent of Rad52. Unlike tlc1 rad52 double mutants, which do not form survivors of telomere dysfunction, tlc1 rad52 sgs1 triple mutants readily generated survivors. After emerging from growth crisis, the triple mutants progressively lost telomeric and subtelomeric sequences, yet grew for more than 1 year. Analysis of cloned chromosome termini and of copy number changes of loci genome-wide using tiling arrays revealed terminal deletions extending up to 57 kb, as well as changes in Ty retrotransposon copy numbers. Amplification of the remaining terminal sequences generated large palindromes at some chromosome termini. Sgs1 helicase activity but not checkpoint function was essential for inhibiting the appearance of the survivors, and the continued absence of Sgs1 was required for the growth of the established survivors. Thus, in addition to facilitating the maintenance of telomere repeat sequences via HR-dependent mechanisms, a RecQ helicase can prevent the adoption of HR-independent mechanisms that stabilize chromosome termini without the use of natural telomere sequences. This provides a novel mechanism by which RecQ helicases may help maintain genome integrity and thus prevent age-related diseases and cancer.
Highlights
The linear chromosomes of eukaryotic cells require specialized structures to protect their ends from exonucleolytic degradation and to prevent them from engaging the checkpoint and repair activities that normally respond to DNA double strand breaks
By investigating the mechanism by which sgs1 deletion accelerates the senescence of telomerase mutants, we demonstrated previously that tlc1 rad52 mutants senesced about as rapidly as tlc1 sgs1 rad52 mutants, indicating that Sgs1 functions via an homologous recombination (HR)-dependent pathway to slow the senescence of telomerase mutants [27, 28]
Survivors have a slow population doubling rate, attributable primarily to slowed progression through the cell cycle rather than an increased frequency of permanent cycle arrest, as indicated by near normal colony-forming efficiency in spot assays (Fig. 1B). These findings indicate that the absence of Sgs1 somehow enables the formation or maintenance of Rad52-independent survivors of telomerase inactivation
Summary
Yeast Strains and Senescence Assays—All yeast strains were derived from JKM111 [34] (Table 1) and were cultured in standard YPAD media at 30 °C. Liquid senescence assays were performed as described [21], inoculating 2 million cells per 5 ml of culture at the start of each day of growth. XhoI-digested DNA was separated on 1% agarose gels, blotted onto Hybond-XL membrane, and hybridized to a 784-bp PCR-amplified YЈ telomere fragment [21]. Fragmented chromosome ends with poly(C) tails were amplified by vectorettespecific and poly(G) primers, separated by agarose gel electrophoresis, and fragments between 0.1 and 1 kb were isolated and purified by a QIAquick gel extraction kit (Qiagen), and cloned into the pCR4-TOPO vector (Invitrogen). A two-sample comparison was performed using genomic DNA from est sgs rad triple mutants (JL76-10B) at days 5 (pre-survivors) and 504 (survivors) after the loss of telomerase. Affymetrix CEL file data were normalized using RMA, and for each probe the log2(intensity in survivors) Ϫ log2(intensity in presurvivors) was plotted
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