Survival and growth of yeast without telomere capping by Cdc13 in the absence of Sgs1, Exo1, and Rad9

Abstract

Maintenance of telomere capping is absolutely essential to the survival of eukaryotic cells. Telomere capping proteins, such as Cdc13 and POT1, are essential for the viability of budding yeast and mammalian cells, respectively. Here we identify, for the first time, three genetic modifications that allow budding yeast cells to survive without telomere capping by Cdc13. We found that simultaneous inactivation of Sgs1, Exo1, and Rad9, three DNA damage response (DDR) proteins, is sufficient to allow cell division in the absence of Cdc13. Quantitative amplification of ssDNA (QAOS) was used to show that the RecQ helicase Sgs1 plays an important role in the resection of uncapped telomeres, especially in the absence of checkpoint protein Rad9. Strikingly, simultaneous deletion of SGS1 and the nuclease EXO1, further reduces resection at uncapped telomeres and together with deletion of RAD9 permits cell survival without CDC13. Pulsed-field gel electrophoresis studies show that cdc13-1 rad9Delta sgs1Delta exo1Delta strains can maintain linear chromosomes despite the absence of telomere capping by Cdc13. However, with continued passage, the telomeres of such strains eventually become short and are maintained by recombination-based mechanisms. Remarkably, cdc13Delta rad9Delta sgs1Delta exo1Delta strains, lacking any Cdc13 gene product, are viable and can grow indefinitely. Our work has uncovered a critical role for RecQ helicases in limiting the division of cells with uncapped telomeres, and this may provide one explanation for increased tumorigenesis in human diseases associated with mutations of RecQ helicases. Our results reveal the plasticity of the telomere cap and indicate that the essential role of telomere capping is to counteract specific aspects of the DDR.

Figure 1. Sgs1, Exo1, and Rad9 affect cell proliferation of cdc13-1 strains.

(A) Serial dilutions of yeast strains with the indicated genotypes and growing at 23°C were spotted onto YPD agar plates and incubated at the indicated temperatures for four days before being photographed. (B) Schematic representation of the end of a Y' repeat-containing yeast chromosome with position of XhoI sites and Southern blot hybridisation probe (black bar) indicated. (C) DNA was purified from the indicated strains following incubation in liquid culture overnight at 23°C or 36°C. The DNA was cut with XhoI and hybridised with a Y'+TG probe as described previously . The membrane was stripped and reprobed with a CDC15 probe as described previously . (D) Yeast strains with the indicated genotypes were grown in liquid cultures at 23°C or 36°C overnight before subjected to pulsed-field gel electrophoresis as described previously .

Figure 2. Checkpoint response to telomere uncapping in the absence of Sgs1, Exo1, and Rad9.

(A) Yeast strains of the indicated genotypes (all with cdc13-1 cdc15-2 bar1 mutations) were arrested in G1 at 23°C with α factor and released into 36°C, cells were collected at the indicated time points, and scored for the percentage of cells arrested in medial nuclear division using DAPI staining. (B) As in (A) but the percentage of cells arrested in late nuclear division were quantified. (C) Yeast strains of the indicated genotypes (all with cdc13-1 cdc15-2 bar1 mutations) were arrested in G1 at 23°C and released into 36°C for up to two hours. Western blot were first probed with anti-Rad53 antibody, membranes were stripped and reprobed with anti-tubulin antibody.

Figure 3. Resection of uncapped telomeres in the absence of Sgs1, Exo1, and Rad9.

(A–H) A series of yeast strains with the indicated genotypes (all with cdc13-1 cdc15-2 bar1 mutations) were arrested in G1 at 23°C and released into 36°C to induce telomere uncapping, the amount of ssDNA at the TG strands at two repetitive telomeric locus, Y'600 and Y'5000 and two single copy loci, YER188W and PDA1 were measured by QAOS as described previously . The values plotted are the mean value ± SD.

Figure 4. Cellular response to long term absence of Cdc13.

(A) Yeast strains of the indicated genotype were streaked onto YPD agar plates and the plates were incubated at 23°C or 36°C for three days. The strains were then photographed and restreaked onto another YPD plate. This cycle is repeated for nine times (p = passage). (B) DNA was purified from the strains in (A) or a wild-type strain following further incubation in liquid culture for 48 hours at 36°C. Southern blots were performed as in Figure 1C.

Figure 5. Cdc13 is dispensable for cell viability in the absence of Sgs1, Exo1, and Rad9.

(A) Spores from a yeast strain heterozygous for cdc13Δ, sgs1Δ, exo1Δ and rad9Δ were dissected onto YPD agar plate and the plates were incubated at 23°C for five days before being photographed. Four tetrads (labelled 1–4) are shown (see Figure S10 for full genotype). (B) DNA was purified from fresh cdc13Δ sgs1Δ exo1Δ rad9Δ strains or control strains following further incubation in liquid culture for 48 hours at 23°C. Southern blots were performed as in Figure 1C.