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. 2003 Mar;14(3):987-1001.
doi: 10.1091/mbc.02-04-0057.

Short telomeres induce a DNA damage response in Saccharomyces cerevisiae

Arne S IJpma  1 , Carol W Greider
Affiliations

Short telomeres induce a DNA damage response in Saccharomyces cerevisiae

Arne S IJpma et al. Mol Biol Cell. 2003 Mar.

Abstract

Telomerase-deficient Saccharomyces cerevisiae cells show a progressive decrease in telomere length. When grown for several days in log phase, the tlc1Delta cells initially display wild-type growth kinetics with subsequent loss of growth potential after which survivors are generated via RAD52-dependent homologous recombination. We found that chromosome loss in these telomerase-deficient cells only increased after a significant decline in growth potential of the culture. At earlier stages of growth, as the telomerase-deficient cells began to show loss of growth potential, the cells arrested in G2/M and showed RNR3 induction and Rad53p phosphorylation. These responses were dependent on RAD24 and MEC1, suggesting that short telomeres are recognized as DNA damage and signal G2/M arrest.

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Figures

Figure 1
Figure 1
Chromosome loss and plating efficiency of wild-type, est2Δ and rad52Δ cells. (A) The plating efficiency of all cells was measured and was normalized to the wild-type plating efficiency. For plating efficiency, wild-type is denoted as blue diamonds, est2Δ as red circles and rad52Δ as green triangles. (B) Chromosome loss was measured in the wild-type, est2Δ, and rad52Δ cultures. Wild-type is denoted as blue filled bars, est2Δ as red horizontally striped bars, and rad52Δ as green hatched bars. The rad52Δ cells were only grown until generation 75 because of a loss of function mutation in the SUP11 gene after this point. No chromosome loss data was generated for wild-type cells at generation 35 and for rad52Δ cells at generation 45.
Figure 2
Figure 2
Cell viability of wild-type and tlc1Δ cells in the growth potential assay. (A) Wild-type (black lines) and tlc1Δ (gray lines) cells were grown in log-phase for 11 d. Every day the growth potential of the cultures was measured. For each genotype four independent isolates were analyzed. (B) The percent of phloxin positive cells was plotted for wild-type (black line) and tlc1Δ cells (gray line) derived from the corresponding days of the growth potential assay in A.
Figure 3
Figure 3
Individual telomerase mutant cells show decreased growth potential. (A) The growth potential assay was carried out for wild-type (⋄) and tlc1Δ (●) cultures. (B) Wild-type and tlc1Δ cells from day 5 of the growth potential assay were plated out and incubated at 30°C for 6 h. Pictures were taken at the beginning (0 h) and the end (6 h) of the experiment. (C) Quantitation of the growth potential of individual wild-type (□) and tlc1Δ (▪) cells. The increase in cell number in the microcolonies in the 6-h time span was counted and converted to total number of divisions. All images were captured at 400× magnification.
Figure 4
Figure 4
Telomerase-deficient cells accumulate in the G2/M phase of the cell cycle. (A) A growth potential assay was carried out for wild-type (⋄) and tlc1Δ (●) and tlc1Δ rad24Δ (▪). (B) DAPI (red) stained wild-type, tlc1Δ and tlc1Δ rad24Δ cells from days 1, 3, and 5 of the growth potential assay. All images were captured at the same (×1000) magnification.
Figure 5
Figure 5
Accumulation of G2/M-arrested cells in tlc1Δ cells depends on RAD24 and to a lesser extent on RAD9. (A) Growth potential assay for wild-type (gray diamonds), tlc1Δ (black circles), tlc1Δ rad9Δ cells (red X), tlc1Δ rad24Δ cells (blue squares) and tlc1Δ rad9 Δrad24Δ (green triangles) (B) Quantitation of cell cycle distribution of DAPI-stained cells. Wild-type (gray bars), tlc1Δ (black bars), tlc1Δ rad9Δ (horizontally red striped bars), tlc1Δ rad24Δ (hatched blue bars) and tlc1Δ rad9Δ rad24Δ (vertically striped green bars) cells were divided into different cell cycle stages as described in the materials and methods and as depicted at the bottom of the graphs. Wild-type cells are only shown for first and last day of experiment but showed similar profiles for days 4, 5, and 6. The data for each mutant is derived from the analysis of one mutant spore that is representative for the multiple isolates analyzed. For each mutant three independent isolates were analyzed; for the tlc1Δ rad9Δ rad24Δ mutant two independent isolates were analyzed.
Figure 6
Figure 6
Accumulation of large G2/M-arrested cells in tlc1Δ cells depends on MEC1. (A) Growth potential assay for wild-type (⋄), tlc1Δ (●) and tlc1Δ mec1Δ sml1Δ cells (▴). (B) Quantitation of cell cycle distribution of DAPI-stained cells. Wild-type (white bars), tlc1Δ (black bars) and tlc1Δ mec1Δ sml1Δ (dotted bars) cells. Cells were divided into different cell cycle stages as depicted at the bottom of the graphs. SML1 was also deleted in these cells to rescue the lethality of a MEC1 deletion. Deletion of SML1 in the background of tlc1Δ did not change the G2/M cell cycle arrest. Wild-type cells are only shown for first and last day of experiment but showed similar profiles for days 4, 6, and 7. The data shown for each mutant is derived from the analysis of one mutant spore. Another isolate that was analyzed for each mutant gave similar results.
Figure 7
Figure 7
Accumulation of G2/M-arrested cells in tlc1Δ cells does not depend on TEL1 and MAD2. (A) Growth potential assay for wild-type (⋄), tlc1Δ (●) and tlc1Δ tel1Δ cells (filled triangles). (B) Growth potential assay for wild-type (⋄), tlc1Δ (●) and tlc1Δ mad2Δ cells (♦). (C) Quantitation of cell cycle distribution of DAPI-stained cells. Wild-type (white bars), tlc1Δ (black bars) and tlc1Δ tel1Δ (hatched bars) cells. Cells were divided into different cell cycle stages as depicted at the bottom of the graphs. (D) Quantitation of cell cycle distribution of DAPI-stained cells. Wild-type (white bars), tlc1Δ (black bars) and tlc1Δ mad2Δ (waved bars) cells. Cells were divided into different cell cycle stages as depicted at the bottom of the graphs. Wild-type cells are only shown for first and last day of both experiments but showed similar profiles for the other days that were tested. The data shown for each mutant is derived from the analysis of one mutant spore. Another isolate that was analyzed for each mutant gave similar results.
Figure 8
Figure 8
RNR3 mRNA induction and Rad53p phosphorylation in tlc1Δ cells. (A) Northern blot analysis was performed for the induction of RNR3 mRNA in tlc1Δ and (B) tlc1Δ rad24Δ cells at different days of the growth potential assay (see Figure 4A). The ACT1 mRNA signal was used to normalize the RNR3 mRNA signal. As a positive control for RNR3 induction wild-type cells were treated with 0.04% MMS for 2 h. Quantitation of the signal is plotted below the blot. (C) Rad53p phosphorylation was assayed by Western blot. Rad53p is indicated by an arrow and phosphorylated species of Rad53p are indicated by the bracket. Cells from day 1 and day 5 of the growth potential assay (Figures 5A and 6A) were analyzed. As a positive control for Rad53p phosphorylation wild-type cells were treated with 0.04% MMS for 2 h with 1 h recovery.
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