LETTERS

Telomeres avoid end detection by severing thecheckpoint signal transduction pathwayTiago Carneiro1, Lyne Khair2, Clara C. Reis1, Vanessa Borges1, Bettina A. Moser2, Toru M. Nakamura2

& Miguel Godinho Ferreira1

Telomeres protect the normal ends of chromosomes from beingrecognized as deleterious DNA double-strand breaks. Recent studieshave uncovered an apparent paradox: although DNA repair isprevented, several proteins involved in DNA damage processingand checkpoint responses are recruited to telomeres in every cellcycle and are required for end protection1. It is currently notunderstood how telomeres prevent DNA damage responses fromcausing permanent cell cycle arrest. Here we show that fission yeast(Schizosaccharomyces pombe) cells lacking Taz1, an orthologue ofhuman TRF1 and TRF2 (ref. 2), recruit DNA repair proteins(Rad22RAD52 and Rhp51RAD51, where the superscript indicates thehuman orthologue) and checkpoint sensors (RPA, Rad9, Rad26ATRIP

and Cut5/Rad4TOPBP1) to telomeres. Despite this, telomeres fail toaccumulate the checkpoint mediator Crb253BP1 and, consequently,do not activate Chk1-dependent cell cycle arrest. Artificially recruit-ing Crb253BP1 to taz1D telomeres results in a full checkpointresponse and cell cycle arrest. Stable association of Crb253BP1 toDNA double-strand breaks requires two independent histonemodifications: H4 dimethylation at lysine 20 (H4K20me2) andH2A carboxy-terminal phosphorylation (cH2A)3–5. WhereascH2A can be readily detected, telomeres lack H4K20me2, in con-trast to internal chromosome locations. Blocking checkpoint signaltransduction at telomeres requires Pot1 and Ccq1, and loss of eitherPot1 or Ccq1 from telomeres leads to Crb253BP1 foci formation,Chk1 activation and cell cycle arrest. Thus, telomeres constitute achromatin-privileged region of the chromosomes that lack essen-tial epigenetic markers for DNA damage response amplificationand cell cycle arrest. Because the protein kinases ATM and ATRmust associate with telomeres in each S phase to recruit telomer-ase6, exclusion of Crb253BP1 has a critical role in preventing telo-meres from triggering cell cycle arrest.

Taz1 is required for proper telomere DNA replication and preventsend-joining reactions at chromosome ends that occur in the G1phase of the cell cycle7–9. As fission yeast spend most of the cell cyclein the S and G2 phases, chromosome-end fusions and loss of viabilityare undetectable in taz1D mutants. Despite this, taz1D chromosomeends undergo frequent rearrangements10. To determine the nature ofthese rearrangements, we used a combination of live-cell imaging andchromatin immunoprecipitation (ChIP) to monitor the recruitmentof various DNA repair and checkpoint proteins to telomeres in taz1Dcells.

We endogenously tagged the homologous recombination proteinRad22RAD52 with green fluorescent protein (GFP) and, using Pot1tagged with monomeric red fluorescent protein (mRFP) as a markerfor telomeres (Supplementary Fig. 1), we studied its co-localizationto chromosome ends (Fig. 1a). Few wild-type cells have Rad22–GFPfoci at non-telomeric sites but these become prevalent when exposedto the radiomimetic drug bleomycin. In contrast, taz1D cells had

Rad22–GFP foci at telomeres throughout the cell cycle (Fig. 1a).Using ChIP, we also found that taz1D cells strongly accumulateanother homologous recombination repair protein, Rhp51RAD51, attelomeres (Fig. 1b). Thus, DNA repair is ongoing at taz1D chro-mosome ends.

DNA damage initiates signal transduction pathways, known ascheckpoints, which culminate in cell cycle arrest. Telomeres protectchromosome ends from DNA repair and do not induce cell cyclearrest via checkpoint pathways. Unexpectedly, taz1D telomeresundergo the DNA damage response and yet these cells did not showany cell cycle delay. We were also able to rule out adaptation phe-nomena by analysing newly generated taz1D mutants; they were alsounable to elicit a checkpoint response (Fig. 1c, d).

Next we examined the phosphorylation status of the Chk1 proteinkinase, a well-characterized checkpoint target responsible for inhibitingCdk1 and preventing entry into mitosis in response to DNA damage.Consistent with a lack of checkpoint activation in taz1D cells, phos-phorylated Chk1–Myc was absent in taz1D cell extracts (Fig. 1e). Asexpected, exposure to bleomycin caused strong Chk1 phosphorylationin both wild-type and taz1D cells (Fig. 1e). We performed similarexperiments to investigate the phosphorylation status of Cds1, whichis involved in DNA-replication checkpoints, and failed to detect anyactivation of this branch of the checkpoint pathway unless hydroxyureawas added (Supplementary Fig. 2). Thus, even though taz1D cells arecheckpoint proficient, they are unable to arrest the cell cycle in responseto telomeres undergoing the DNA damage response.

The absence of cell cycle delay prompted us to examine the check-point pathway to identify at which step it was disrupted. Telomeres intaz1D cells are long and accumulate single-stranded DNA through-out the cell cycle11. We started probing the checkpoint pathway(Fig. 2a) at its upstream point by looking for the accumulation ofRPA at telomeres by co-localization of Rad11–GFP, the largest sub-unit of RPA, with Pot1–mRFP. Pot1 protects telomeres from trigger-ing cell cycle arrest via the ATR checkpoint pathway12–14. It has beenproposed that Pot1 accomplishes this function directly by outcom-peting RPA from telomere ends, as both molecules bind to telomericsingle-stranded DNA13. Surprisingly, Pot1–mRFP co-localized withRad11–GFP in taz1D cells (Fig. 2b), indicating that Pot1 does notabrogate checkpoint signalling by exclusion of RPA from dysfunc-tional telomeres. We quantitatively confirmed increased recruitmentof RPA to telomeres in taz1D compared with wild-type cells usingChIP (Fig. 2c). Because telomere-associated RPA greatly increasesduring S phase15, we conclude that we are able to measure proteininteraction with telomeres in S-phase cells using an asynchronouslygrowing population.

Next we analysed Rad26ATRIP, Rad9 (a component of the 9-1-1complex) and Cut5/Rad4TOPBP1. In contrast to wild type, the majorityof taz1D cells had telomeric foci for all three components (Fig. 2b),

1Instituto Gulbenkian de Ciencia, Oeiras 2781-901, Portugal. 2Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607, USA.

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indicating that taz1D dysfunctional telomeres show increased recruit-ment for components of the DNA-damage checkpoint pathway. ChIPanalysis revealed that Cut5TOPBP1 is also strongly recruited to wild-type telomeres (Fig. 2c), consistent with previous observations thattelomeres normally engage DNA damage response components15,16.

Rad3ATR kinase was active in growing taz1D cells, because hyperpho-sphorylation of its known substrates, Rad26ATRIP and Rad9, wasdetected in untreated taz1D extracts as in control cells exposed tobleomycin (Fig. 2d). Further analysis revealed that phosphorylationlevels of haemagglutinin (HA) tagged Rad26ATRIP observed in taz1D

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Figure 1 | taz1D telomeres undergo the DNAdamage response without eliciting a checkpoint-dependent cell cycle arrest. a, Live analysis ofRad22RAD52–GFP and its co-localization withPot1–mRFP used as a telomeric marker. WT,wild type. Bleo, bleomycin. b, Dot-blot ChIPquantification of Rhp51RAD51 bound totelomeres for the indicated strains. rhp51D cellswere used as a control. n $ 3; ** P , 0.01 basedon a two-tailed Student’s t-test to control sample.Error bars represent mean 6 standard deviation(s.d.). c, Strategy to generate taz1D mutants denovo. d, Histograms depict the distribution of cellsizes (mm) after germination over the course ofthe experiment. e, Immunoblot analysis ofChk1–Myc in wild-type and taz1D cells. Theasterisk depicts an antibody cross-reactive bandused as a loading control. The DNA damagingagent bleomycin (Bleo; 5 mU ml21) was added tocultures as a positive control. P indicateshyperphosphorylation.

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Figure 2 | taz1D telomeres initiate aDNA damage checkpoint response.a, Checkpoint pathways in fissionyeast. DNA-damage checkpoint(left) and replication checkpoint(shaded on the right). b, Co-localization of Rad11RPA–GFP,Rad26ATRIP–GFP, Rad9–YFP,Cut5TOPBP1–GFP with Pot1–mRFP(used as a telomere marker) (left)and quantification of cells showingtelomeric and non-telomeric foci(right). c, Telomeric dot-blot ChIPquantification of Rad11RPA–Flagand Cut5TOPBP1–Myc. Untaggedstrains were used as controls. n $ 3;**P , 0.01 and *** P , 0.001 basedon a two-tailed Student’s t-test tocontrol sample. Error bars representmean 6 s.d. d, Immunoblot analysisof Rad9–HA and Rad26ATRIP–HA.Bleomycin (5 mU ml21) was addedto cultures as a positive control.e, Immunoblot analysis ofRad26ATRIP–HA and Chk1–Myc inwild-type cells treated withincreasing concentrations ofbleomycin. Asterisks point to across-reactive band that serves as aloading control. P indicateshyperphosphorylation.

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cells were otherwise sufficient to trigger cell cycle arrest. Exposure toincreasing doses of bleomycin revealed that, at levels of DNA damagewhere phosphorylation of Rad26ATRIP–HA becomes detectable (0.5–1.0 mU bleomycin; Fig. 2e), Chk1–Myc is clearly hyperphosphory-lated. Thus, Rad3ATR activity equivalent to that observed in taz1D cellsis sufficient to activate a full checkpoint response if DNA damageoccurs at non-telomeric regions.

Upon Rad3ATR activation and Cut5TOPBP1 recruitment, Crb253BP1

is brought to sites of DNA damage and, on phosphorylation byRad3ATR, induces activation of the Chk1 kinase and cell cycle arrest17.Unlike other upstream checkpoint proteins, yellow fluorescentprotein (YFP)-tagged Crb2 protein did not co-localize with Pot1–mRFP in most taz1D cells (Fig. 3a). In accordance with this, Crb2 wasnot phosphorylated in taz1D cells (Fig. 3b), even though treatmentwith bleomycin strongly induces Crb2 phosphorylation. We werealso unable to detect telomere-bound tandem affinity purification(TAP)-tagged Crb2 in either wild-type or taz1D cells using ChIP,whereas controls showed clear recruitment of TAP–Crb2 to anHO-endonuclease-induced DNA double-strand break (Fig. 3c).Therefore, unlike other DNA damage response components, Crb2is unable to stably bind to telomeres in either wild-type or taz1D cells,indicating that the checkpoint signalling pathway is blocked at thestep between Cut5TOPBP1 and Crb253BP1.

We next investigated what prevents the stable association ofCrb253BP1 with telomeres. Apart from Cut5, Crb2 requires two his-tone modifications for stable association to double-strand breaks:

C-terminal phosphorylation of cH2A (cH2AX in higher eukaryotes)and H4K20me2 (refs 3–5). These modifications show different res-ponses to DNA damage: whereas cH2A/X is locally phosphorylatedby ATM or ATR at DNA double-strand breaks, H4K20me2 levels donot change on DNA damage18. Instead, H4K20me2 seems to be ubi-quitously distributed even though it participates in the DNA damageresponse and is essential for sustained checkpoint activation19.Consistent with the observed recruitment of Rad3ATR–Rad26ATRIP

to telomeres, we readily detected cH2A by ChIP at both wild-typeand taz1D telomeres (cH2A; Fig. 3c). In contrast, H4K20me2 wasundetectable at telomeres in either wild-type or taz1D cells(H4K20me2 telomere; Fig. 3c and Supplementary Fig. 3). Theabsence of H4K20me2 could not be explained by exclusion of themethyltransferase Set9/Kmt5 from chromosome ends. Telomeresshow Set9-dependent mono- and trimethylated forms of H4K20even though taz1D telomeres lacked the latter (H4K20me1 andH4K20me3; Fig. 3c). Similarly, Clr4 methyltransferase, responsiblefor H3K9 methylation, is not required to maintain the pattern ofH4K20 methylation, despite a marked reduction in H4K20me3(Supplementary Fig. 3). Thus, although H3K9me3 inversely corre-lates with H4K20me2 at fission yeast telomeres, these are likely to beindependently regulated.

Because checkpoint signalling seems to be achieved through theexclusion of Crb2, we reasoned that artificial recruitment of Crb2 totaz1D telomeres might be sufficient to induce cell cycle arrest. Taz1binds to telomeres via its C-terminal Myb domain (MybTaz1);

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Figure 3 | Dysfunctional taz1D telomeres avert cell cycle arrest bypreventing recruitment of Crb253BP1. a, Visualization and quantification ofYFP–Crb2 foci in the indicated strains. b, Immunoblot analysis of YFP–Crb2.Bleomycin (5 mU ml21) was added to cultures where indicated (1).c, Telomeric dot-blot ChIP quantification for Crb253BP1–TAP, cH2A,H4K20me1, H4K20me2 and H4K20me3. Positive controls: quantitativepolymerase chain reaction (qPCR) ChIP was performed for TAP–Crb253BP1

using primers adjacent to an internal double-strand break generated by HOcleavage (HO site) and to wild-type telomeres (Telo) and for H4K20me2

using primers for the ade61 locus (Internal). For negative controls we usedH2A phosphorylation mutants hta1-S129A hta2-S128A (hta1 hta2) for cH2Aand set9D for all forms of H4K20 methylation. n $ 3; * P , 0.05, ** P , 0.01and *** P , 0.001 based on a two-tailed Student’s t-test to controls. Errorbars represent mean 6 s.d. d, Localization of MybTaz1–YFP–Crb2 expressedfrom a low expression vector (pREP81). e, Distribution of sizes (mm) in cellscarrying either MybTaz1–YFP–Crb2 or an empty plasmid control.f, Immunoblot analysis of Chk1–Myc. P indicates hyperphosphorylation.Asterisk indicates a cross-reactive band that serves as a loading control.

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MybTaz1 has been previously used to tether Rap1 directly to taz1Dtelomeres20,21. Likewise, we ectopically expressed a MybTaz1–YFP–Crb2 chimaeric protein in fission yeast. On induction ofMybTaz1–YFP–Crb2, taz1D cells became extremely elongated (Fig. 3d,e), denoting a full checkpoint response confirmed by Chk1 phosphor-ylation (Fig. 3f). None of these phenomena was visible in wild-type cellsexpressing the chimaeric protein (Fig. 3e, f). Also consistent with acheckpoint response, recruitment of MybTaz1–YFP–Crb2 to telomeresin taz1D rad3D cells resulted in neither cell elongation nor Chk1–Mycphosphorylation (Fig. 3e, f). These data demonstrate that Crb2 recruit-ment to telomeres in taz1D cells is sufficient to restore the full check-point pathway.

Next we investigated what marks telomeres different from the rest ofthe genome. Pot1 and its interacting protein Ccq1 are likely candidatesas they bind telomeres independently of Taz1 and prevent checkpointsat telomeres12–14,22. Indeed, in contrast to wild type, both germinatingpot1D and taz1D lig4D pot1D cells derived from heterozygous diploidsbecame elongated and presented Chk1–Myc phosphorylation (Fig. 4a,b). Cell elongation was dependent on Rad3ATR, indicating an activationof this checkpoint pathway (Supplementary Fig. 4). Therefore, Pot1 isrequired to prevent telomeres from eliciting Rad3ATR-dependentcheckpoints not only in wild-type but also in taz1D cells. Ccq1 isrequired for telomerase recruitment and inhibition of Rad3ATR check-points at fission yeast telomeres22,23. Loss of Ccq1 results in progressive

telomere shortening and checkpoint activation before complete telo-mere erosion. Pre-senescent ccq1D and trt1D single mutants have com-parable short telomeres that initiate checkpoints on telomere erosion(Fig. 4c–e). Likewise, ccq1D taz1D and trt1D taz1D double mutantsundergo ALT-like telomere elongation, preserving long and heteroge-nous telomeres (Fig. 4c). However, in contrast to trt1D taz1D doublemutants, ccq1D taz1D mutants had a strong immediate checkpointresponse, becoming extremely elongated with strong Chk1 phosphor-ylation (Fig. 4d, e). As expected, quantification of plasmid-borne YFP–Crb2 revealed that both ccq1D and ccq1D taz1D cells had YFP–Crb2 fociin the majority of observed cells (Fig. 4f). Moreover, ccq1D telomeredeprotection partially requires H4K20 methylation. Preventing H4K20methylation at telomeres by removing Set9 from either ccq1D orccq1D taz1D cells reduces checkpoint activation and YFP–Crb2 fociformation (Supplementary Fig. 5). Thus, absence of the Pot1–Ccq1complex results in prompt Crb2 foci formation, Chk1–Myc phosphor-ylation and cell cycle arrest, indicating that Crb2 telomere exclusionconstitutes a barrier imposed by normal telomeres to a full checkpointresponse. However, loss of Ccq1 in wild-type cells results in a weakerand delayed checkpoint response than in taz1D mutants, establishingthat, in addition to Pot1–Ccq1, the Taz1 complex participates in check-point inhibition at normal telomeres (Fig. 4d, e).

Our work provides new insight into how chromosome ends are pro-tected from DNA damage checkpoints (Fig. 4g). Instead of preventing

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Figure 4 | Pot1 and Ccq1 prevent Crb253BP1-dependent checkpoints attelomeres. a, Distribution of cell sizes over time (mm) after germination ofspores with the indicated genotype. b, Immunoblot analysis of Chk1–Myc.Extracts derived from diploid cells are indicated with a dash. P indicateshyperphosphorylation. Asterisks indicate a cross-reactive band used as aloading control. c, Telomere-length analysis by Southern blotting using atelomere probe. d, Distribution of cell sizes (mm) in the absence of either

Ccq1 or Trt1 in wild-type and taz1D cells. e, Immunoblot analysis ofChk1–Myc. f, Visualization and quantification of plasmid borneYFP–Crb253BP1 foci. g, Model for checkpoint inhibition at fission yeasttelomeres. Pot1 and Ccq1, together with the Taz1 complex, define achromatin-privileged region that excludes H4K20me2 and prevents stableCbr253BP1 association. As a result, the full checkpoint response is severed attelomeres and cell cycle arrest is averted.

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the detection of DNA damage, fission yeast telomeres constitute achromatin-privileged region on the chromosome that blocks transduc-tion of an active checkpoint signal. This is achieved by preventing stableassociation of Crb2 with telomeres, a checkpoint adaptor proteinrequired for the delivery and activation of Chk1. Studies in buddingyeast using a short telomere seed adjacent to a HO break postulated ananticheckpoint activity at telomeres24. Following studies showed thatcheckpoint repression in budding yeast is primarily regulated by theinhibition of Mec1ATR recruitment to telomeres25–27. Fission yeast telo-meres initiate a checkpoint response that fails to phosphorylate Crb2and Chk1. However, rather than preventing recruitment and activationof Rad3ATR, exclusion of Crb253BP1 from telomeres represents the criticalstep in the inhibition of the checkpoint pathway.

The inability of telomeres to stably recruit Crb253BP1 is probablydue to the lack of H4K20me2 epigenetic marks at chromosome ends.Studies in both mammalian cells and in fission yeast demonstratethat H4K20me2 is the relevant histone modification for Crb2 bindingand is required for the stable recruitment of Crb2 to sites of DNAdamage3,4,19,28. Although H4K20me2 is the most abundant form ofH4K20me18, how it is regulated or localized is not understood. Ourfinding that telomeres have low levels of H4K20me2 provides arationale for its ubiquitous nature throughout the genome.Conceivably, H4K20me2 marks regions of the genome required forchromosome contiguity, and cell division should not be attemptedunless the DNA damage response is disengaged. In contrast, theabsence of H4K20me2 marks regions where DNA perceived asdamaged, such as chromosome ends, would not interfere with genomestability thus precluding a full checkpoint response.

Normal telomeres undergoing DNA replication temporarily loseDNA protection. Studies in both yeast model systems and mam-malian cells show that during S phase, concomitant with the replica-tion fork passage, chromosome ends engage ATR and ATM15,16,29,30,which are redundantly required for telomerase recruitment6.However, the periodic activation of ATM and ATR at telomeres doesnot result in either Chk1 or Chk2 phosphorylation or in cell cycledelay. As dividing cells use cell-cycle-staged telomere exposure toprocess chromosome ends and engage telomerase, it is likely thatthese mechanisms have evolved to prevent cell cycle arrest in res-ponse to normal replicating telomeres.

METHODS SUMMARY

All experiments were performed using the Schizosaccharomyces pombe strains

listed in Supplementary Information. Checkpoint studies were performed using

a combination of microscopy, ChIP and western blotting. Telomere length was

analysed by Southern blotting using telomere probes. For details of the methods

used, see Methods.

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 12 August 2009; accepted 14 July 2010.

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18. Sanders, S. L. et al. Methylation of histone H4 lysine 20 controls recruitment ofCrb2 to sites of DNA damage. Cell 119, 603–614 (2004).

19. Wang, Y. & Jia, S. Degrees make all the difference: the multifunctionality ofhistone H4 lysine 20 methylation. Epigenetics 4, 273–276 (2009).

20. Chikashige, Y. & Hiraoka, Y. Telomere binding of the Rap1 protein is required formeiosis in fission yeast. Curr. Biol. 11, 1618–1623 (2001).

21. Miller, K. M., Ferreira, M. G. & Cooper, J. P. Taz1, Rap1 and Rif1 act bothinterdependently and independently to maintain telomeres. EMBO J. 24,3128–3135 (2005).

22. Tomita, K. & Cooper, J. P. Fission yeast Ccq1 is telomerase recruiter and localcheckpoint controller. Genes Dev. 22, 3461–3474 (2008).

23. Miyoshi, T., Kanoh, J., Saito, M. & Ishikawa, F. Fission yeast Pot1-Tpp1 protectstelomeres and regulates telomere length. Science 320, 1341–1344 (2008).

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25. Abdallah, P. et al. A two-step model for senescence triggered by a single criticallyshort telomere. Nature Cell Biol. 11, 988–993 (2009).

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28. Botuyan, M. V. et al. Structural basis for the methylation state-specific recognitionof histone H4-K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361–1373 (2006).

29. Goudsouzian, L. K., Tuzon, C. T. & Zakian, V. A. S. cerevisiae Tel1p and Mre11p arerequired for normal levels of Est1p and Est2p telomere association. Mol. Cell 24,603–610 (2006).

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank J. Cooper, K. Tomita and the rest of the TelomereBiology Laboratory (Cancer Research UK, London) for support at the start of thisproject. We thank J. Cooper, T. Wolkow, P. Russell and A. M. Carr for strains andplasmids. We thank W. Kaufman for her initial effort in the establishment of HOstrains. We are grateful to S. Grewal for sharing unpublished results and to D. Lydallfor insights on the quantitative analysis of checkpoint activation. We thank K. Labib,L. Jansen, K. Xavier, R. Martinho and S. Lopes for critically reading the manuscript.T.C. and C.C.R. are supported by Fundacao para a Ciencia e a Tecnologia (FCT)postdoctoral fellowships. T.M.N. was supported by the Sidney Kimmel ScholarProgram and his laboratory is supported by NIH grant GM078253. This work wassupported by the FCT (PTDC/BIA-BCM/67261/2006) and the Association forInternational Cancer Research (06-396).

Author Contributions T.C. helped with the design and executed most experiments.L.K. performed ChIP experiments. C.C.R. performed western and Southern blottingexperiments. V.B. performed live cell analysis. B.A.M. established the ChIP and HOassays. T.M.N. contributed to the design of the ChIP and HO assays. All authorscontributed with strains and data analysis. M.G.F. conceived the study, performedlive cell analysis and wrote the paper.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of this article atwww.nature.com/nature. Correspondence and requests for materials should beaddressed to M.G.F. (mgferreira@igc.gulbenkian.pt).

LETTERS NATURE | Vol 467 | 9 September 2010

232Macmillan Publishers Limited. All rights reserved©2010

METHODSStrains and media. The strains used in this work are listed in Supplementary

Table 1. Strains were constructed using standard techniques31. Standard media

and growth conditions were used throughout this work32. Bleomycin sulphate

(5 mU ml21; Sigma-Aldrich) or hydroxyurea (20 mM; Sigma-Aldrich) was added

to exponentially growing cultures where indicated and incubated for 1 h for

bleomycin or 4 h for hydroxurea before cells were processed for immunoblot or

microscopy analysis. For the strains containing the HO system or the pJR plas-

mids, cultures were grown overnight in media containing 4 mM thiamine to

repress the HO endonuclease (or the protein cloned in the pJR plasmid). Cellswere then washed twice to remove thiamine and incubated for 20 h to induce

expression of HO endonuclease (or the protein encoded in the pJR plasmid).

Southern blot analysis. Genomic DNA was obtained from exponentially grow-

ing cells and digested with ApaI. Southern blot analysis was performed as

described10. A telomere probe labelled with 32P using the Prime-it II random

primer labelling kit (Stratagene) was used for Southern blotting.

ChIP. ChIP was performed as described15 with minor modifications. For TAP–

Crb2 ChIP, lysis buffer containing 0.1% SDS and pan-mouse IgG Dynabeads

(#110.41 Invitrogen) was added to 1.5 mg whole-cell extracts. For all the other

ChIPs, Dynabeads Protein G (#100.04D Invitrogen) was added to 1.5 mg whole-

cell extract pre-incubated with anti-Myc (9B11; Cell Signaling), anti-Flag (M2-

F4802; Sigma), anti-Rad51 (Sc-8349; Santa Cruz), anti-histone H2A (phospho

S129; ab15083; Abcam), anti-histone H4 (mono methyl K20; ab9051; Abcam),

anti-histone H4 (di-methyl K20; ab9052; Abcam) and anti-histone H4 (tri-methyl

K20; ab9053; Abcam). Recovered DNA was either analysed in triplicate by SYBR-

Green-based real-time PCR (Bio-Rad) using the primers listed in Supplementary

Table 2 or by dot-blot Southern blotting as described33. Per cent precipitated DNA

values were then calculated based onDCT between input and immunoprecipitatedsamples from RT–PCR analysis or based on the difference in signal intensity

between input and immunoprecipitated samples from the dot-blot analysis

(Supplementary Fig. 6).

Immunoblot analysis. Whole-cell extracts prepared using exponentially grow-

ing cells were processed for western blot as described10. For detection of Chk1–

Myc, we used a 1:2,000 dilution of mouse anti-Myc monoclonal antibody (9E10;

Santa Cruz). For detection of Cds1–HA, Rad9–HA and Rad26–HA, we used a

1:1,000 dilution mouse anti-HA monoclonal antibody (16B12; Covance). Cds1–

HA was separated in 10% polyacrylamide gels containing 25 mM Phos-tag

(AAL-107; NARD Institute). For detection of YFP–Crb2, we used a 1:200 dilu-

tion mouse anti-GFP monoclonal antibody (11814460001; Roche).

Checkpoint studies. Diploids were sporulated in Edinburgh minimal media

(EMM) complete plates directly from 280 uC glycerol stocks and incubated

for 2 days at 32 uC. Cells were then treated with 10 ml of helix pomatia juice

(Pall Biosepra) in 1 ml water overnight with shaking. Spores were germinated in

yeast extract with supplements (YES) containing 50 mg ml21 ampicillin for 7 h

before changing into appropriate selective medium (time zero). Samples were

then collected every 3 h up to 12 h and a final sample was collected at 24 h. Cells

were fixed in 70% ethanol and processed for microscopy. Samples were collected

for immunoblot analysis and processed as described10.

Microscopy. Cells were grown at 25 uC in EMM with all supplements added. For

each individual experiment, at least 100 cells were analysed. For GFP, YFP and

mRFP visualization, live cells were imaged using a Delta Vision Core System

(Applied Precision) using a 1003 1.4 numerical aperture UplanSApo objective

and a cascade2 EMCCD camera (Photometrics). Images were acquired at 14

z-sections separated by 0.2-mm intervals. Deconvolution was performed using

the enhanced ratio method in softWoRx software. Co-localization experiments

were performed using maximum intensity projections of deconvoluted images.

To measure cell size in checkpoint studies, images were acquired in a Leica

DMIRE2 using a 1003 1.4 numerical aperture HCX PlaAPO objective and a

Hamamatsu EM-CCD camara. Cell size was measured using ImageJ software

(National Institutes of Health) calibrated with a reference image.

31. Bahler, J. et al. Heterologous modules for efficient and versatile PCR-based genetargeting in Schizosaccharomyces pombe. Yeast 14, 943–951 (1998).

32. Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeastSchizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).

33. Khair, L., Subramanian, L., Moser, B. A. & Nakamura, T. M. Roles ofheterochromatin and telomere proteins in regulation of fission yeast telomererecombination and telomerase recruitment. J. Biol. Chem. 285, 5327–5337(2010).

doi:10.1038/nature09353

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