Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping
2010; Springer Nature; Volume: 29; Issue: 23 Linguagem: Inglês
10.1038/emboj.2010.267
ISSN1460-2075
Autores Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoArticle2 November 2010Open Access Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping James M Dewar James M Dewar Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle upon Tyne, Tyne-and-Wear, UK Search for more papers by this author David Lydall Corresponding Author David Lydall Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle upon Tyne, Tyne-and-Wear, UK Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, Tyne-and-Wear, UK Search for more papers by this author James M Dewar James M Dewar Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle upon Tyne, Tyne-and-Wear, UK Search for more papers by this author David Lydall Corresponding Author David Lydall Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle upon Tyne, Tyne-and-Wear, UK Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, Tyne-and-Wear, UK Search for more papers by this author Author Information James M Dewar1 and David Lydall 1,2 1Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle upon Tyne, Tyne-and-Wear, UK 2Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, Tyne-and-Wear, UK *Corresponding author. Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, Tyne and Wear NE2 4HH, UK. Tel.: +44 191 222 5318; Fax: +44 191 222 7424; E-mail: [email protected] The EMBO Journal (2010)29:4020-4034https://doi.org/10.1038/emboj.2010.267 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Essential telomere ‘capping’ proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally ‘uncap’ telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA 5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins. Introduction Telomeres consist of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), bound by dsDNA- and ssDNA-binding proteins (Blackburn et al, 2006; Lydall, 2009). This nucleoprotein ‘cap’ has at least two functions: to shield the telomeric DNA from stimulating the DNA damage response (DDR) and to regulate elongation of the telomere by telomerase. In human senescent cells, dysfunctional telomeres induce a sustained DDR (d'Adda di Fagagna et al, 2003). In both budding yeast and mice, nuclease activities that attack dysfunctional telomeres contribute to telomere-driven senescence (Maringele and Lydall, 2004; Schaetzlein et al, 2007). Therefore, understanding the regulation of nuclease activities at dysfunctional telomeres in yeast is likely to be informative about similar processes occurring at mammalian telomeres and the human ageing process. dsDNA-binding proteins and accessory factors are required at both human telomeres (TRF1, TRF2, TIN2, TPP1, RAP1) and budding yeast telomeres (Rap1, Rif1, Rif2) to prevent DDRs (Wotton and Shore, 1997; de Lange, 2005; Celli and de Lange, 2005; Marcand et al, 2008; Bonetti et al, 2010; Vodenicharov et al, 2010). In budding yeast, telomeric ssDNA is bound by Cdc13 with accessory proteins Stn1 and Ten1, whereas in human cells, it is bound by POT1 (de Lange, 2005; Gao et al, 2007). Cdc13–Stn1–Ten1 forms an evolutionarily conserved complex (the CST complex) that has telomeric roles in most organisms studied so far (Miyake et al, 2009; Surovtseva et al, 2009). POT1 binds telomeric ssDNA and is connected to the dsDNA-binding proteins of the telomere cap by TPP1 and TIN2 (de Lange, 2009). Inactivation of POT1 or Cdc13 induces ‘telomere uncapping’ and has similar consequences—initiation of a DDR and resection of the telomeric DNA by nuclease activities (Garvik et al, 1995; Baumann and Cech, 2001; Pitt and Cooper, 2010). The response to telomere uncapping is readily studied in budding yeast by inactivation of Cdc13 using the thermosensitive allele cdc13-1 (Garvik et al, 1995). Following Cdc13 inactivation, a potent DDR is initiated; telomeric DNA is resected by nucleases, which degrade the AC (5′) strand to generate extensive TG (3′) ssDNA that stimulates activation of the DNA damage checkpoint, in a manner analogous to that at DNA double-strand breaks (DSBs) (Figure 1A) (Garvik et al, 1995; Lydall and Weinert, 1995; Vodenicharov and Wellinger, 2006). There is relatively little understanding of the nuclease activities responsible for generating ssDNA at uncapped telomeres (Zubko et al, 2004). In contrast, there has been much recent progress identifying nuclease activities that function at DSBs (Gravel et al, 2008; Mimitou and Symington, 2008; Zhu et al, 2008). Figure 1.Pif1 and Exo1 inhibit growth of cdc13-1 mutants. (A) Inactivation of Cdc13 by use of the temperature-sensitive allele cdc13-1 leads to telomere uncapping. Exo1 and additional nuclease(s) generate ssDNA at uncapped telomeres, which is the stimulus for Mec1-dependent checkpoint activation and cell cycle arrest. (B) Ranked diagram of genes that share genetic interactions with EXO1 and are important in the context of telomeres. (Thicker lines mean more shared genetic interactions). (C) Strains of the genotypes shown were serially diluted across agar plates and grown at the temperatures indicated for 3 days. In this and other figures, strain numbers (DLYs) are shown adjacent. Download figure Download PowerPoint Exo1 is the only nuclease known to generate ssDNA at uncapped telomeres in budding yeast (Maringele and Lydall, 2002). Exo1 is a 5′ to 3′ dsDNA exonuclease involved in DSB resection and in mismatch repair (Tsubouchi and Ogawa, 2000; Gravel et al, 2008; Mimitou and Symington, 2008; Zhu et al, 2008). In the absence of Exo1, ssDNA is still generated following Cdc13 inactivation, demonstrating that other nuclease activities must also function at uncapped telomeres. The determinant(s) of this Exo1-independent ssDNA generation have not so far been identified, but at least two hypothetical nuclease activities have been proposed (ExoX and ExoY) (Zubko et al, 2004). We sought to identify additional nuclease activities functioning at uncapped telomeres following inactivation of Cdc13. Bioinformatic analysis of genetic interactions found the helicase Pif1 to be a candidate for contributing to nuclease activity. Consistent with this hypothesis, we found that Pif1 and Exo1 are required for different nuclease activities that generate ssDNA and activate the DNA damage checkpoint following Cdc13 inactivation. Furthermore, deletion of both PIF1 and EXO1 permits yeast cells to tolerate complete loss of the essential telomere capping protein Cdc13. Results PIF1 and EXO1 define parallel pathways that inhibit growth of cdc13-1 mutants To identify potential nuclease(s) active in cdc13-1 mutants, we reasoned that genes responsible for such activities would interact with similar genes to those that EXO1 interacts with. We used the BioGRID database to create a ranked list of genes that had similar genetic interactions to EXO1 (Figure 1B) (Stark et al, 2006). Of these, 9/19 affected cdc13-1 growth or telomere length. Deletion of EXO1 suppresses cdc13-1 growth defects, so we focussed on those genes that also suppressed cdc13-1 growth defects. By these criteria, two previously characterized checkpoint genes (RAD9 and RAD24) and the helicase-encoding PIF1 behaved similarly to EXO1 (Figure 1B). Rad9 and Rad24 do indeed regulate nuclease activities at uncapped telomeres and are required for checkpoint activation (Garvik et al, 1995; Lydall and Weinert, 1995; Zubko et al, 2004). Pif1 has been shown to inhibit growth of cdc13-1 mutants (Downey et al, 2006), whereas overexpression of Pif1 has been shown to enhance growth defects seen in cdc13-1 mutants, but the contribution of Pif1 to the nuclease activity and checkpoint activation in cdc13-1 mutants had not been assessed (Vega et al, 2007; Chang et al, 2009). Pif1 is a helicase with both mitochondrial and nuclear functions (Van Dyck et al, 1992; Schulz and Zakian, 1994). In the nucleus, Pif1 has been implicated in negative regulation of telomerase, generation of long flaps during Okazaki fragment processing, unwinding of G-quadruplexes and disassembly of stalled replication forks (Zhou et al, 2000; Boule et al, 2005; Budd et al, 2006; Chang et al, 2009; George et al, 2009; Makovets and Blackburn, 2009; Pike et al, 2009; Ribeyre et al, 2009; Zhang and Durocher, 2010). To test the hypothesis that Pif1 contributes to a nuclease activity at uncapped telomeres in cdc13-1 mutants, we compared the effects of Pif1 and Exo1 on cell growth after Cdc13-1 inactivation. At the permissive temperature (23°C), Cdc13-1 is functional and efficiently caps the telomeres, permitting growth of cdc13-1 mutants. At the non-permissive temperature (36°C), Cdc13-1 is completely defective and cdc13-1 mutants are unable to grow (Figure 1A and C). At semi-permissive temperatures (25–29°C), moderate Cdc13-1 inactivation occurs and growth of cdc13-1 mutants is inhibited (Figure 1C). As previously reported, cdc13-1 pif1Δ and cdc13-1 exo1Δ mutants are able to grow at 27°C, whereas cdc13-1 mutants are not (Figure 1C) (Zubko et al, 2004; Downey et al, 2006). These effects on growth are consistent with the hypothesis that Pif1, like Exo1, contributes to nuclease activity at uncapped telomeres. Pif1 and Exo1 inhibit growth of cdc13-1 mutants, possibly by contributing to nuclease activity at uncapped telomeres. To test whether the two proteins worked in the same pathway/complex or in different pathways, we examined the effect Pif1 on growth of cdc13-1 exo1Δ mutants. cdc13-1 exo1Δ mutants were unable to grow at 30°C, whereas cdc13-1 exo1Δ pif1Δ mutants were able to grow at 30 and 36°C (Figure 1C). Remarkably, at 36°C, the growth of cdc13-1 exo1Δ pif1Δ mutants was barely distinguishable from that of CDC13+ exo1Δ pif1Δ mutants (Supplementary Figure S1A). We confirmed that this effect was due to the pif1Δ and exo1Δ mutations and not due to second site suppressors arising in our strains by crossing a cdc13-1 mutant able to grow at 36°C, with a cdc13-1 strain and confirming that all cdc13-1 exo1Δ pif1Δ progeny could all grow at 36°C (Supplementary Figure S1B). We conclude that Pif1 and Exo1 inhibit growth of cdc13-1 mutants through different pathways, and inactivation of these pathways may eliminate the requirement for telomere capping by Cdc13. At DSBs, parallel nuclease activities dependent upon Exo1, the helicase Sgs1 and nuclease Dna2 generate extensive ssDNA (Gravel et al, 2008; Mimitou and Symington, 2008; Zhu et al, 2008). We hypothesized that, as with Exo1, elimination of Sgs1 or Dna2 in cells lacking Pif1 might improve the growth of cdc13-1 mutants and perhaps even permit growth at 36°C. However, we found that cdc13-1 pif1Δ dna2Δ mutants grew less well than cdc13-1 pif1Δ mutants (Supplementary Figure S2A). We were unable to examine the effect of dna2Δ on the growth of cdc13-1 PIF1+ mutants, as DNA2 is an essential gene unless PIF1 is deleted (Budd et al, 2006). We also found that cdc13-1 pif1Δ sgs1Δ mutants grew slightly less well than cdc13-1 pif1Δ mutants and that cdc13-1 sgs1Δ mutants grew slightly less well than cdc13-1 mutants (Supplementary Figure S2B), consistent with other work from our laboratory (Ngo and Lydall, 2010). We conclude that Exo1 inhibits the growth of cdc13-1 mutants with uncapped telomeres, whereas Sgs1 and Dna2 contribute to the vitality of such cells. Therefore, we chose to focus on the roles of Pif1 and Exo1 at uncapped telomeres. Elimination of Pif1 and Exo1 permits telomere maintenance following inactivation of Cdc13 Yeast cells can overcome the requirement for Cdc13 by altering telomere structure, as observed in rare variants, which can be selected for after inactivation of telomerase or after attenuation of nuclease/checkpoint activities at uncapped telomeres (Larrivee and Wellinger, 2006; Zubko and Lydall, 2006). To test whether elimination of Pif1 and Exo1 caused alterations in telomere structure that could explain the growth of cdc13-1 cells at 36°C, we performed Southern blots to examine telomere structure, probing for Y′ sequences (Figure 2B), which are components of the majority of yeast telomeres (Supplementary Figure S3A and B). The Y′ probe contained G-rich sequences and weakly cross-hybridized to telomeres that did not contain Y′ sequences, so we also probed for TG repeat sequences to detect telomeres that lacked Y′ elements (Supplementary Figure S4). Figure 2.Exo1 and nuclear, helicase activity of Pif1 prevent telomere maintenance following inactivation of Cdc13. (A) Cartoon of yeast telomeres, indicating the fragments detected by telomere Southern blots using Y′ probe. Arrows represent XhoI cut sites. (B) Genomic DNA was prepared from two independent CDC13+ (+) or cdc13-1 (TS) strains, grown at 23 or 36°C, digested with XhoI and Southern blotted to detect telomeric Y′ and terminal fragments. Blots were reprobed to detect CDC15 as a loading control. Also see Supplementary Figure S4. (C) cdc13-1 exo1Δ mutants defective in nuclear Pif1 (pif1-m2), mitochondrial Pif1 (pif1-m1) or carrying the helicase-deficient allele of Pif1 (pif1-hd) were serially diluted across agar plates and grown at the temperatures indicated for 3 days. Download figure Download PowerPoint pif1Δ mutants have long telomeres (Schulz and Zakian, 1994) and consistent with this, CDC13+exo1Δ pif1Δ mutants have longer telomeres than CDC13+EXO1+PIF1+ strains (compare lanes 1–2, 3–4 and 7–8; Figure 2B). The telomeres of cdc13-1 exo1Δ pif1Δ mutants grown at 36°C were longer than those of CDC13+exo1Δ pif1Δ mutants grown at 23°C (compare lanes 9–10 with lanes 7–8, Figure 2B) but indistinguishable from those of cdc13-1 exo1Δ pif1Δ mutants grown at 23°C (compare lanes 9–10 with lanes 17–18, Figure 2B). This demonstrates that no gross alterations in telomere structure occur when cdc13-1 exo1Δ pif1Δ mutants are grown at 36°C. Furthermore, cdc13-1 exo1Δ pif1Δ mutants are able to grow at 36°C, whereas cdc13-1 pif1Δ mutants are not, but are indistinguishable in telomere structure (compare lanes 13–14 with lanes 17–18, Figure 2B). We conclude that alterations in telomere structure most likely do not account for the growth of cdc13-1 exo1Δ pif1Δ mutants at 36°C. Pif1 exists as both nuclear and mitochondrial isoforms (Schulz and Zakian, 1994). Therefore, we wanted to know whether the nuclear or mitochondrial function of Pif1 inhibited growth of cdc13-1 exo1Δ mutants at 36°C. The pif1-m2 allele, lacking nuclear Pif1, permitted growth of cdc13-1 exo1Δ mutants at 36°C, whereas the pif1-m1 allele, lacking mitochondrial Pif1, did not (Figure 2C). We note that the growth of cdc13-1 exo1Δ pif1-m2 mutants at 36°C is less than cdc13-1 exo1Δ pif1Δ mutants (Figure 2C). This is consistent with other reports that low levels of nuclear Pif1 activity persist in pif1-m2 mutants (Schulz and Zakian, 1994; Ribeyre et al, 2009). We also confirmed that helicase activity of Pif1 inhibited growth of cdc13-1 exo1Δ mutants because the pif1-hd allele, deficient in helicase activity, also permitted growth at 36°C (Figure 2C) (Zhou et al, 2000; Ribeyre et al, 2009). We conclude that nuclear, helicase-dependent activity of Pif1 inhibits growth of telomere capping-defective cdc13-1 mutants. Pif1- and Exo1-dependent nucleases initiate the DDR following Cdc13 inactivation Upon Cdc13 inactivation, nuclease activities generate ssDNA, which stimulates checkpoint kinase cascades and induces metaphase arrest (Figure 1A) (Garvik et al, 1995). To test the role of Pif1 in cell cycle arrest, cdc13-1 mutants were synchronized in G1 using α factor at 23°C, then released to 36°C to assess metaphase arrest (Figure 3A). All strains also harboured the cdc15-2 mutation so that any cells that overcame cdc13-1-induced metaphase arrest would arrest in late anaphase due to cdc15-2 and be unable to enter another cell cycle (Figure 3A) (Lydall and Weinert, 1995; Zubko et al, 2004). As expected, cdc13-1 mutants accumulated at metaphase and did not pass through to anaphase (Figure 3B and C). cdc13-1 exo1Δ mutants accumulated at metaphase with similar kinetics to cdc13-1 mutants but, as previously reported, a subpopulation of cdc13-1 exo1Δ cells escaped metaphase arrest and accumulated in late anaphase due to the cdc15-2 mutation (Figure 3B and C) (Zubko et al, 2004). cdc13-1 pif1Δ mutants behaved like cdc13-1 mutants and did not pass through to anaphase (Figure 3B and C). Interestingly, cdc13-1 exo1Δ pif1Δ mutants did not accumulate in metaphase at all and passed readily through to anaphase (Figure 3B and C). Taken together, these results show that Pif1 has no effect on metaphase arrest of cdc13-1 mutants at 36°C when Exo1 is present, but it is responsible for the arrest of a subpopulation of cells when Exo1 is absent. Figure 3.Pif1 and Exo1 stimulate cell cycle arrest following inactivation of Cdc13. (A) Synchronous culture experiments to examine the effect of Pif1 and Exo1 on cell cycle arrest following telomere uncapping. cdc15-2 cdc13-1 cells were synchronized in G1 using α-factor then released at 36°C. Cells arrest at metaphase from telomere uncapping (cdc13-1) or at anaphase from Cdc15 inactivation (cdc15-2). Samples taken at the time points indicated were stained with DAPI and >100 cells of each genotype scored for cell cycle position (Zubko et al, 2006). (B) Percentage of cells at metaphase. (C) Percentage of cells at late anaphase. (D) Western blots of Rad53 following shift to 36°C. Upper and lower panels were run in parallel on separate gels, but transferred, detected and imaged simultaneously. (E) Western blots of Rad53 following treatment with bleomycin. Corresponding scoring of cells at metaphase and anaphase given as Supplementary Figure S5. Download figure Download PowerPoint Following inactivation of Cdc13, Mec1-dependent checkpoint activation occurs, leading to activation and hyperphosphorylaiton of the kinase Rad53 (Figure 1A) (Sweeney et al, 2005; Morin et al, 2008). We used the synchronous cultures to examine Rad53 phosphorylation by western blot (Figure 3A). cdc13-1 and cdc13-1 pif1Δ mutants exhibited strong Rad53 phosphorylation, indicated by a marked upward mobility shift of Rad53 (upper panels, Figure 3D). A reduction in Rad53 phosphorylation was seen in cdc13-1 exo1Δ mutants, correlating with the recovery from metaphase arrest displayed by cdc13-1 exo1Δ mutants following telomere uncapping (Figure 3C and D). Interestingly, no discernable change in the mobility of Rad53 could be seen in cdc13-1 exo1Δ pif1Δ mutants, consistent with their complete failure to arrest cell division at 36°C (Figure 3C and D). We conclude that in the absence of Pif1 and Exo1, the checkpoint kinase Rad53 is not activated after telomere uncapping in cdc13-1 mutants. To see whether cdc13-1 exo1Δ pif1Δ strains were defective in the DDR after other types of DNA damage as well as after telomere uncapping, we treated cells with bleomycin to induce DSBs. At both DSBs and uncapped telomeres, ssDNA is an important stimulus for the Mec1-dependent checkpoint. We treated the same set of strains examined in Figure 3B–D, with bleomycin at 23°C after release from G1 arrest. cdc13-1, cdc13-1 pif1Δ, cdc13-1 exo1Δ and cdc13-1 exo1Δ pif1Δ mutants all behaved similarly, phosphorylating Rad53 and arresting at methaphase (Figure 3E; Supplementary Figure S4). We conclude that a functional DDR pathway operates in (cdc13-1) exo1Δ pif1Δ cells but that these cells are specifically defective in responding to telomere uncapping. Telomeric ssDNA stimulates metaphase arrest following telomere uncapping (Garvik et al, 1995). We used synchronous cultures (Figure 3A) and quantitative amplification of ssDNA (QAOS) to measure subtelomeric ssDNA in repetitive Y′ elements (present on Chromosome V and most other chromosome ends) following Cdc13 inactivation (Figure 4A) (Booth et al, 2001). cdc13-1 mutants with uncapped telomeres generated ssDNA at both the Y′600 and Y′5000 loci (Figure 4B and C). cdc13-1 exo1Δ mutants generated less ssDNA following telomere uncapping at the Y′ loci, as previously reported (Maringele and Lydall, 2002; Zubko et al, 2004). Interestingly, cdc13-1 pif1Δ mutants, like cdc13-1 exo1Δ mutants showed reduced ssDNA generation in the Y′600 and Y′5000 loci following telomere uncapping. Furthermore, cdc13-1 exo1Δ pif1Δ mutants generated no detectable ssDNA at Y′600 or Y′5000 loci following telomere uncapping (Figure 4B and C). We confirmed that ssDNA generation occurred on the TG (3′) strand due to degradation of the AC (5′) strand, as we were unable to detect ssDNA on the AC strand (Figure 4D). We conclude that Pif1, like Exo1, is important for ssDNA generation after telomere uncapping in cdc13-1 mutants and appears to regulate a nuclease activity, which functions in parallel to Exo1 at chromosome ends. Figure 4.Pif1 and Exo1 generate ssDNA at uncapped telomeres. (A) Physical map of the right telomere of Chromosome V. Synchronous cultures were subjected to telomere uncapping as in Figure 3A, and samples were taken to measure ssDNA. (B) TG-strand ssDNA 600 bp from the end of the telomere (Y′600) measured by QAOS. (C) TG-strand ssDNA 5000 bp away from the end of the telomere (Y′5000). (D) AC-strand ssDNA 600 bp away from the end of the telomere. (E) TG-strand ssDNA generated at loci indicated in (A), 4 h after telomere uncapping. Dashed line represents 1.56% (1/64), corresponding to the value expected for 1 single-stranded locus per yeast cell in G2. (F) TG-strand ssDNA in the telomeric TG repeats detected by in-gel assay, comparing cdc13-1 mutants (TS) to a yku70Δ control. Loading determined by Southern hybridization with a CDC15 probe. Lanes were run and detected on the same gel and cropped for presentation purposes. (G) Quantification of the ssDNA signal in each lane in (F). ssDNA is measured in KUs (Ku units). In all, 1 Ku Unit is the ssDNA signal in an asynchronously dividing yku70Δ control at 23°C. Download figure Download PowerPoint To examine how Pif1 and Exo1 affect ssDNA accumulation further from chromosome ends, we measured ssDNA at single-copy loci on Chromosome V after Cdc13 inactivation. At 4 h, cdc13-1 mutants generated ssDNA at all loci examined, with the amount of ssDNA decreasing at loci further from the chromosome end, as previously reported (Zubko et al, 2004). cdc13-1 exo1Δ mutants generated less ssDNA in the Y′ repeats and no ssDNA in single-copy loci on Chromosome V, also as previously reported (Figure 4E) (Zubko et al, 2004). cdc13-1 pif1Δ mutants generated similar amounts of ssDNA to cdc13-1 exo1Δ mutants in the Y′ repeats. However, at more distal, single-copy loci, cdc13-1 pif1Δ mutants generated less ssDNA than cdc13-1 mutants but more than exo1Δ cdc13-1 mutants. The higher levels of ssDNA generated further from the chromosome end in cdc13-1 pif1Δ mutants, in comparison with cdc13-1 exo1Δ mutants, most likely accounts for their sustained metaphase arrest following telomere uncapping (Figure 3B and C). Furthermore, the ssDNA generated by cdc13-1 and cdc13-1 pif1Δ mutants 1.6% (1/64) (Figure 4E). Assuming 1 single-stranded telomere per cell is sufficient to stimulate arrest, this suggests that ssDNA extending <10 kb on one of the 64 G2 telomeres in Saccharomyces cerevisiae is sufficient to stimulate metaphase arrest (Figure 4E) (Sandell and Zakian, 1993; Vaze et al, 2002; Zubko et al, 2004). No checkpoint activation was detected in cdc13-1 exo1Δ pif1Δ mutants following telomere uncapping, and no ssDNA was detected in the Y′ elements (Figures 3D and 4B). However, yku70Δ mutants at 23°C have detectable ssDNA in the telomeric TG repeats but do not undergo checkpoint activation (Gravel et al, 1998; Polotnianka et al, 1998; Maringele and Lydall, 2002). Thus, we hypothesized that cdc13-1 exo1Δ pif1Δ mutants might still generate detectable ssDNA in the TG repeats. We used synchronous cultures (Figure 3A) and measured ssDNA by in-gel assay to measure ssDNA in the TG repeats in cdc13-1 mutants (Figure 4F and G). cdc13-1 mutants generated large amounts of TG ssDNA at 2 and 4 h following telomere uncapping (Figure 4F), corresponding to an approximately five-fold increase in signal compared with yku70Δ mutants (Figure 4G). cdc13-1 exo1Δ pif1Δ mutants also generated detectable ssDNA 2 h following telomere uncapping, but at a level approximately equal to that of a yku70Δ mutant (Figure 4F and G). However, the ssDNA generated in cdc13-1 exo1Δ pif1Δ mutants was transient and was no longer detectable 4 h after telomere uncapping (Figure 4F and G). Surprisingly, cdc13-1 pif1Δ mutants displayed only a modest decrease in ssDNA generation in the TG repeats following telomere uncapping, whereas cdc13-1 exo1Δ mutants generated very little ssDNA (Figure 4F and G). We conclude that cdc13-1 exo1Δ pif1Δ mutants generate limited, transient ssDNA that is insufficient to stimulate checkpoint activation and that Exo1 is much more important than Pif1 for ssDNA generation in the TG repeats following telomere uncapping. Pif1 has important functions in cells lacking telomerase It has been suggested that increased levels of telomerase at the telomeres of cdc13-1 pif1Δ cells shields uncapped telomeres from nuclease activities (Vega et al, 2007). However, this is somewhat inconsistent with our observation that Pif1 has relatively little effect on ssDNA generation in the telomeric TG repeats, where telomerase presumably binds (Figure 4G). Therefore, we wanted to know whether the ability of the pif1Δ mutation to improve the growth of cdc13-1 mutants was dependent upon the telomerase template component (TLC1) or catalytic subunit (Est2). Interestingly, we found that cdc13-1 tlc1Δ pif1Δ and cdc13-1 tlc1Δ exo1Δ mutants grew better at 25°C than cdc13-1 tlc1Δ mutants (compare rows 7–8 and 11–12 with 3–4, Figure 5A; Supplementary Figure S6). We also found that cdc13-1 est2Δ pif1Δ and cdc13-1 est2Δ exo1Δ mutants were able to grow at 25°C, whereas cdc13-1 est2Δ mutants were not (Supplementary Figure S7). We conclude that Pif1 has a telomerase (TLC1, Est2) independent effect at uncapped telomeres. However, we note that est2Δ cdc13-1 and tlc1Δ cdc13-1 mutants grow worse than cdc13-1 mutants, demonstrating that telomerase contributes to telomere capping following inactivation of Cdc13. Figure 5.Pif1 has telomerase-independent effects at telomeres. (A) A cdc13-1/CDC13+tlc1Δ/TLC1+exo1Δ/EXO1+pif1Δ/PIF1+ diploid (DLY1628 x DLY5324) was sporulated, dissected and germinated at 23°C to generate strains of the indicated genotype at 23°C. These were taken from the germination plate, grown to saturation, then serially diluted across agar plates and grown at the temperatures indicated for 3 days. (B) Strains of the genotypes indicated were passaged repeatedly by restreaking at 30°C for 3 days along with TLC1+ controls. At the passages indicated, strains were assayed for growth, which was then quantified (Supplementary Figure S9). Growth at each passage is given as a fraction of the growth of the relevant TLC1+ strain and the mean of two independent strains is shown. (C) At passages 1 and 15, strains assayed for growth in (B) had genomic DNA isolated and were Southern blotted with Y′ probe, as in Figure 2B. See Supplementary Figure S10 for detection with TG probe. Download figure Download PowerPoint Pif1 is responsible for the residual checkpoint activation in cdc13-1 exo1Δ mutants (Figure 3D) and inhibits growth of cdc13-1 mutants lacking telomerase (Figure 5A). We hypothesized that Pif1 would contribute to ssDNA generation at uncapped telomeres and subsequent checkpoint activation, even in cdc13-1 mutants lacking telomerase. To test this, we measured Rad53 phosphorylation (Supplementary Figure S8A) and telomeric TG repeat ssDNA (Supplementary Figure S8B and C) in cdc13-1 and cdc13-1 tlc1Δ mutants, before and after telomere uncapping. In cdc13-1 tlc1Δ exo1Δ pif1Δ mutants, there was a decrease in Rad53 phosphorylation compared with cdc13-1 tlc1Δ exo1Δ mutants (Supplementary Figure S8A). We also found that cdc13-1 tlc1Δ pif1Δ and cdc13-1 tlc1Δ exo1Δ pif1Δ mutants generated less ssDNA than cdc13-1 tlc1Δ and cdc13-1 tlc1Δ exo1Δ mutants, respectively, following telomere uncapping (Supplementary Figure S8B and C). We conclude that Pif1 has a contribution to ssDNA generation and checkpoint activation following
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