Length-dependent processing of telomeres in the absence of telomerase

Abstract

In the absence of telomerase, telomeres progressively shorten with every round of DNA replication, leading to replicative senescence. In telomerase-deficient Saccharomyces cerevisiae, the shortest telomere triggers the onset of senescence by activating the DNA damage checkpoint and recruiting homologous recombination (HR) factors. Yet, the molecular structures that trigger this checkpoint and the mechanisms of repair have remained elusive. By tracking individual telomeres, we show that telomeres are subjected to different pathways depending on their length. We first demonstrate a progressive accumulation of subtelomeric single-stranded DNA (ssDNA) through 5'-3' resection as telomeres shorten. Thus, exposure of subtelomeric ssDNA could be the signal for cell cycle arrest in senescence. Strikingly, early after loss of telomerase, HR counteracts subtelomeric ssDNA accumulation rather than elongates telomeres. We then asked whether replication repair pathways contribute to this mechanism. We uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the DNA damage tolerance (DDT) pathway, associates with native telomeres and cooperates with HR in senescent cells. We propose that DDT acts in a length-independent manner, whereas an HR-based repair using the sister chromatid as a template buffers precocious 5'-3' resection at the shortest telomeres.

Figure 1.

5′-3′ resection factors promote senescence. (A and B) Experimental setting used in this study. (A) Two sets of yeast strains deleted for telomerase activity (tlc1Δ) and containing a modified version of the telomere VIIL were used [adapted from (33,75)]. In Control cells the VIIL telomere is of wild-type length, and in VST cells the VIIL telomere is very short. The proliferation capacity of several independent clones of such strains, obtained through the procedure described in Supplementary Figure S1, was compared. (B) Telomeric constructs. The VST strain contains an artificial VIIL telomere, such that a URA3 selection marker and 600 nt of TG_1-3 repeats were inserted between two flipase recognition target (FRT) sequences close to the chromosome end. These internal repeats negatively regulated telomerase activity via the protein-counting mechanism (76), resulting in a short array of terminal telomeric repeats after the second FRT. The Control strain did not display internal telomeric sequences, resulting in normal homeostasis of the number of distal telomeric repeats. The induction of Flp1, a site-directed recombinase, resulted in the excision of the fragment between the two FRTs, leaving only the terminal telomeric repeats. (C) Quantitative analysis of senescence by serial spot assays in the presence (VST) or absence (Control) of a very short VIIL telomere was performed on 8 MRE11 tlc1Δ (dotted lines) and 8 mre11Δ tlc1Δ (full lines) independent spores derived from yT235 (Control cells) and yT236 (VST cells; see Supplementary Figure S1 and ‘Materials and Methods’ section). Adjusted P-values were obtained by Wilcoxon rank-sum test with a false discovery rate correction. *P < 0.1; **P < 0.05. See Supplementary Table S2 for detailed P-values. (D) Same as in (C) for 16 SAE2 tlc1Δ and sae2Δ tlc1Δ independent spores derived from yT231 and yT232. (E) Same as in (C) for 16 EXO1 tlc1Δ and 16 exo1Δ tlc1Δ independent spores derived from yT403 and yT404.

Figure 2.

The accumulation of subtelomeric ssDNA by end resection contributes to senescence. (A) A mixture of tlc1Δ colonies derived from yT136 or yT138 was grown in liquid-rich medium with daily dilutions. DNA was prepared every day, and for each culture the number of PDs grown in liquid culture was estimated. Genomic DNA was prepared and ssDNA quantity was monitored by QAOS using a probe located 56 nt away from the telomeric repeats of telomere VIR (open circles) and 139 nt away from the telomeric repeats of telomere VIIL (full circles) in Control (blue circles) and VST (red circles) tlc1Δ cells and plotted according to the PDs since the loss of telomerase. (B) DNA samples from (A) were used to determine the telomere length of VIR and VIIL by telomere-PCR. ssDNA determined as above was plotted according to telomere length. Circle colors indicate whether DNA was from Control cells (blue circles) or VST cells (red-brown circles). Full or open circles indicate whether data correspond to VIR (open circles) or VIIL (full circles) telomeres. PD indicates the number of estimated PDs in liquid cultures after mass sporulation and colony formation. (C) MRE11 tlc1Δ and mre11Δ tlc1Δ colonies derived from yT235 or yT236 were grown in liquid-rich medium for ∼25 population doublings. Genomic DNA was prepared from Control (blue) and VST (red) cells and ssDNA quantity was monitored by QAOS as in (A) (n = 3–4 clones per genotype). (D) Same as in (C) with EXO1 tlc1Δ and exo1Δ tlc1Δ colonies derived from yT403 or yT404 grown for ∼27 population doublings (n = 3–4 clones per genotype).

Figure 3.

HR counteracts senescence. (A) The deletion of RAD51 accelerates senescence. Quantitative analysis of senescence as in Figure 1C on 8 RAD51 tlc1Δ and rad51Δ tlc1Δ colonies derived from yT347 and yT348. Adjusted P-values were obtained by Wilcoxon rank-sum test with a false discovery rate correction. *P < 0.1; **P < 0.05. See Supplementary Table S2 for detailed P-values. (B) The deletion of POL32 accelerates senescence. Quantitative analysis of senescence as in Figure 1C on 8 POL32 tlc1Δ and 8 pol32Δ tlc1Δ colonies derived from yT225 and yT226. (C) Rad52 and Rad51 associate preferentially with short telomeres in the absence of telomerase. Cultures from independent tlc1Δ spores derived from yT136 or yT138 were grown in liquid YPD for ∼30 generations after sporulation in the absence (Control cells) or presence (VST cells) of a short VIIL telomere. Chromatin was immunoprecipitated using primary antibodies directed against Rad52 (left panel) or Rad51 (right panel). The association of each protein with the VIR or VIIL telomeres or with an internal locus (ARO1) was quantified by qPCR, and the fold increase in telomere enrichment over ARO1 is indicated. Error bars indicate the SEM from three independent spores. (D) Rad52 association with the short telomere depends on Tel1. ChIP of Rad52 was performed as in (B) in tlc1Δ independent spores derived yT174 or yT176 with the indicated genotypes. Association of each protein with the Y′ telomeres or VIIL telomeres or to an internal locus (ARO1) was quantified by qPCR, and fold increase of telomere enrichment over ARO1 is indicated. Error bars indicate SEM from at least three independent spores.

Figure 4.

Rad52 counteracts the accumulation of subtelomeric ssDNA. (A) Rad52 counteracts ssDNA accumulation at subtelomeres of the very short telomere. ssDNA at VIIL (left panel) and VIR (right panel) subtelomeres from Control or VST cells was measured by QAOS as described for Figure 2A ∼9 PDs after the loss of TLC1 in RAD52 (derived from yT136 and yT138) or rad52Δ (derived from yT143 and yT145) cells. Mean ± SEM for three independent experiments. (B) Lengths of Y′ (left panel) and VIIL (right panel) telomeres in Control and VST cells were measured by Southern blot at the indicated PD after the loss of TLC1. Full arrow: internal ura3 locus; open arrow: fragment of the VIIL when excision did not occur. (C) Mean shortening rate per PD was determined from Southern blot as in (B) between 9 and 33–35 PDs for Y′ and VIIL telomeres from three independent cultures. Mean ± SEM.

Figure 5.

DDT and HR act independently to sustain the viability of senescent cells. (A and B) DDT factors sustain the viability of senescent cells. Quantitative analysis of senescence as in Figure 1C on 16 rad5-535 tlc1Δ and rad5Δ tlc1Δ derivatives of yT299 and yT300 (A) and 16 MMS2 tlc1Δ and mms2Δ tlc1Δ derivatives of yT303 and yT304 (B). Adjusted P-values were obtained by Wilcoxon rank-sum test with a false discovery rate correction. *P < 0.1; **P < 0.05. See Supplementary Table S2 for detailed P-values. (C and D) rad5Δ and rad52Δ display synthetic slow growth phenotypes in TLC1 or tlc1Δ background. TLC1/tlc1Δ RAD52/rad52Δ rad5-535/rad5Δ diploids (yT567) were sporulated and meiosis products dissected and analysed after 2 days of growth on rich medium, photographed and genotyped. (C) Representative tetrads are shown, and genotypes of the tlc1Δ colonies are indicated. (D) Spore colonies of each genotype were given an index according to their size. −: non-growing spore or microcolony, +: small colony, ++: medium colony, +++: normal size colony. n = 15 tetrads/60 spore colonies were analysed.

Figure 6.

Rad5 localizes to a subset of telomeres in wild-type cells. (A) Rad5-535 and Rad5 are enriched at telomeres. Telomerase-positive cells expressing Rad5-535 (yT580), a myc-tagged version of Rad5-535 (yT581), Rad5 (yT583) or a myc-tagged version of Rad5 (yT582) and a wild-type length telomere VIIL (Control construct after excision of the URA3-containing circle) were submitted to ChIP using an anti-myc antibody. The association of Rad5-535-myc and Rad5-myc with the VIIL telomere, the VIR telomere or the internal ARO1 locus was quantified by qPCR. The mean fraction of input ± SEM was calculated from four independent cultures. (B) ChIP of Rad5-535-myc was performed as in (A) in tlc1Δ independent spores derived yT382 or yT383 with the indicated genotypes grown ∼30 PDs after sporulation. Association with the Y′ telomeres or VIIL telomeres or to an internal locus (ARO1) was quantified by qPCR. Error bars indicate SEM from three independent spores. (C-E) Rad5 colocalizes with Rap1 in telomerase-positive cells. Rad5 foci were examined by fluorescence microscopy in wild-type cells expressing Rad5-YFP, Rad52-RFP and Rap1-CFP (ML690-16A) and in est2Δ mutant cells (ML691-12C) 1, 2 or 3 streaks after the loss of a plasmid-borne EST2. (C) Representative images of wild-type (left) and est2Δ (right) cells are shown. Scale bar: 3 µm. (D) Percentage of S/G2 cells displaying Rad5 foci. Blue: percentage of S/G2 cells displaying a Rad5 focus co-localizing with Rap1; red: percentage of S/G2 cells displaying a Rad5 focus co-localizing with Rad52. In EST2 cells, ∼30% of Rad5 foci colocalize with Rap1 and 10% colocalize with a Rad52 focus. Rad5 foci are observed exclusively in budded cells (S/G2). A total of 60–120 cells were counted per genotype. Error bars represent 95% confidence intervals. (E) Quantification of Rad52 foci in S/G2 cells in the experiment shown in (B and C). Error bars represent 95% confidence intervals.

Figure 7.

Distinct effects of Mus81 and Sgs1 in senescence triggered by a very short telomere. Quantitative analysis of senescence as in Figure 1C of 8 MUS81 tlc1Δ and mus81Δ tlc1Δ derivatives of yT355 and yT356 (A) and 16 SGS1 tlc1Δ and sgs1Δ tlc1Δ derivatives of yT257 and yT258 (B). Adjusted P-values were obtained by Wilcoxon rank-sum test with a false discovery rate correction. *P < 0.1; **P < 0.05. See Supplementary Table S2 for detailed P-values.

Figure 8.

Model for telomere maintenance in the absence of telomerase. Factors studied in this work are indicated. In the presence of telomerase or early after the loss of telomerase, a Rad5-dependent mechanism could contribute to the semi-conservative DNA replication of telomeres. As they become short in the absence of telomerase, telomeres are subject to progressive increased resection and/or incomplete replication, increasing telomeric overhang length and subsequently increasing the asymmetry in length of replication products. ssDNA may then trigger the recruitment of HR and error-free DDT factors. A recombination-based mechanism, possibly combined with DDT, would limit the increased resection by allowing re-elongation of the shortened strand using the sister chromatid as the template. This would counteract the generation of long overhangs and diminish the activation of Mec1 in a feedback loop. Note that in all these events, a minimal DNA end replication should remain, causing a minimal shortening rate. Permanent cell cycle arrest is thus inexorably expected when telomeric repeats become exhausted.