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• W2034949406 abstract "Article17 April 2008Open Access Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres Federico Lazzaro Federico Lazzaro Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy Search for more papers by this author Vasileia Sapountzi Vasileia Sapountzi Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Henry Wellcome Laboratory for Biogerontology Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Magda Granata Magda Granata Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy Search for more papers by this author Achille Pellicioli Achille Pellicioli Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy Search for more papers by this author Moreshwar Vaze Moreshwar Vaze Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USAPresent address: Boston Biomedicals Inc., Norwood, MA 0206, USA Search for more papers by this author James E Haber James E Haber Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA Search for more papers by this author Paolo Plevani Corresponding Author Paolo Plevani Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy 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, Henry Wellcome Laboratory for Biogerontology Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Marco Muzi-Falconi Corresponding Author Marco Muzi-Falconi Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy Search for more papers by this author Federico Lazzaro Federico Lazzaro Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy Search for more papers by this author Vasileia Sapountzi Vasileia Sapountzi Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Henry Wellcome Laboratory for Biogerontology Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Magda Granata Magda Granata Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy Search for more papers by this author Achille Pellicioli Achille Pellicioli Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy Search for more papers by this author Moreshwar Vaze Moreshwar Vaze Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USAPresent address: Boston Biomedicals Inc., Norwood, MA 0206, USA Search for more papers by this author James E Haber James E Haber Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA Search for more papers by this author Paolo Plevani Corresponding Author Paolo Plevani Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy 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, Henry Wellcome Laboratory for Biogerontology Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Marco Muzi-Falconi Corresponding Author Marco Muzi-Falconi Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy Search for more papers by this author Author Information Federico Lazzaro1,‡, Vasileia Sapountzi2,‡, Magda Granata1, Achille Pellicioli1, Moreshwar Vaze3, James E Haber3, Paolo Plevani 1, David Lydall 2 and Marco Muzi-Falconi 1 1Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Milano, Italy 2Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Henry Wellcome Laboratory for Biogerontology Research, Newcastle University, Newcastle upon Tyne, UK 3Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA ‡These authors contributed equally to this work *Corresponding authors: Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Via Celoria 26, Milano 20133, Italy. Tel.: +39 02 50315034; Fax: +39 02 50315044; E-mail: [email protected] Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Henry Wellcome Laboratory for Biogerontology Research, Newcastle University, Newcastle upon Tyne NE4 5PL, UK. Tel.: +44 191 256 3449; Fax: +44 191 256 3445; E-mail: [email protected] The EMBO Journal (2008)27:1502-1512https://doi.org/10.1038/emboj.2008.81 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage. Introduction Eukaryotic cells evolved a complex system to protect the genome from spontaneous and exogenous DNA damage. A central role is played by DNA damage checkpoint pathways, signal transduction cascades coordinating DNA replication, repair and recombination with cell cycle progression. The defining feature of an active checkpoint is the arrest of cell proliferation at the G1/S or G2/M transitions, or the slowing down of DNA replication (Elledge, 1996; Nyberg et al, 2002). Many details of the DNA damage checkpoint have been established using genetic and biochemical approaches in budding and fission yeast; the basic checkpoint response has been shown to be conserved in other eukaryotes (Longhese et al, 1998; Melo and Toczyski, 2002; Rouse and Jackson, 2002; Lydall and Whitehall, 2005). In budding yeast, it has been useful to examine the roles of checkpoint proteins at uncapped telomeres and unrepaired double-strand breaks (DSBs). Several repair, recombination and checkpoint factors are recruited at a DSB site, according to a well-established order (Lisby et al, 2004). DSB ends are initially processed to generate long 3′ single-stranded DNA (ssDNA) tails. The Mre11, Rad50, Xrs2 (MRX) complex is involved in this process, as mutations in the corresponding genes reduce the resection rate (White and Haber, 1990; Ivanov et al, 1994; Lee et al, 1998). However, MRX is not likely to be the nuclease itself: point mutations within Mre11 catalytic site do not affect resection of DSB ends (Moreau et al, 1999; Lee et al, 2002; Llorente and Symington, 2004). The nature of the nuclease(s) involved in DSB processing has been elusive; moreover, we lack information on the regulatory mechanisms governing DNA end resection (see Harrison and Haber, 2006 for a recent review). It has been recently shown that, both in human cells and in Schizosaccharomyces pombe, CtIP, a partner of the MRN complex, is required for efficient resection of DSB ends (Limbo et al, 2007; Sartori et al, 2007). In budding yeast, CtIP seems to correspond to Sae2, which also has a positive function in DSB processing (Clerici et al, 2005; Sartori et al, 2007). Recent studies show that CDK1 kinase is important for the generation of ssDNA tails; in fact, inhibition of CDK1 strongly interferes with resection (Aylon et al, 2004; Ira et al, 2004). ssDNA is bound by the RPA heterotrimer, generating a structure important for recruiting checkpoint factors (Kornbluth et al, 1992; Zou and Elledge, 2003; Zou et al, 2003; Majka et al, 2006). PI3-like kinases have essential functions in checkpoint signal transduction in all eukaryotes. In budding yeast, the Mec1/Ddc2 checkpoint protein kinase is stimulated both by binding to RPA-coated ssDNA and by the checkpoint sliding clamp (Rad17, Mec3 and Ddc1) (Zou and Elledge, 2003; Zou et al, 2003; Majka et al, 2006). Mec1 phosphorylates several targets, among these Ddc2, Ddc1, Rad9 and the protein kinases Chk1 and Rad53. Rad9 is a checkpoint adaptor molecule, linking the upstream Mec1 kinase with downstream Rad53 and Chk1 kinases, and it is essential for checkpoint function (Sanchez et al, 1996; Gardner et al, 1999; Blankley and Lydall, 2004). It is thought that Mec1-dependent phosphorylation of Rad9 recruits and catalyses Rad53 activation (Gilbert et al, 2001; Sweeney et al, 2005). As well as being essential for cell cycle arrest, Rad9 contributes to DNA damage metabolism because Rad9 inhibits the accumulation of ssDNA at uncapped telomeres (Lydall and Weinert, 1995). This effect of Rad9 is not simply checkpoint signal transduction dependent, because other checkpoint proteins, such as Rad24, are also required to signal cell cycle arrest at uncapped telomeres and yet have the opposite resection phenotype (Lydall and Weinert, 1995). However, so far, no biochemical mechanism by which Rad9 inhibits resection at uncapped telomeres has been discovered. In addition, it was not known whether Rad9 inhibits ssDNA accumulation at other types of lesion, such as DSBs. Budding yeast Rad9 interacts with the methylated K79 residue of histone H3, through the Rad9 Tudor domain, and this interaction regulates Rad9 function after DNA is damaged (Giannattasio et al, 2005; Wysocki et al, 2005; Grenon et al, 2007). Similar results were reported for S. pombe Crb2, where the binding target seems to be methylated H4-K20 (Sanders et al, 2004; Du et al, 2006), whereas for human 53BP1 binding to both residues has been reported (Huyen et al, 2004; Botuyan et al, 2006). Loss of methylation of the K79 residue of histone H3 impairs Rad9 phosphorylation and activation of Rad53, after DNA damage in G1 cells. Furthermore, a rad9Y798Q point mutation within the Tudor domain prevents Rad9 binding to chromatin and Rad9 hyper-phosphorylation after DNA damage (Giannattasio et al, 2005; Wysocki et al, 2005; Grenon et al, 2007; Hammet et al, 2007, and Supplementary Figure 1). The simplest explanation for these data is that methylated histone H3-K79 is involved in recruiting Rad9 to damaged chromosomes and that this contributes to Rad9 hyper-phosphorylation and checkpoint activation. As H3-K79 appears to be constitutively methylated by the Dot1 methyltransferase in undamaged cells (90% of H3 is methylated at K79; van Leeuwen et al, 2002), it has been proposed that the critical event for Rad9 recruitment may be a DNA-damage-induced change in the status of chromatin, allowing exposure of this methylated residue (Huyen et al, 2004). Another intriguing option would be that Rad9 is always weakly bound to methylated H3-K79; upon damage Rad9 oligomerization may cause its accumulation at the sites of lesion. Moreover, post-translational modifications may induce changes in Rad9-binding mode and allow it to interact with other partners (Du et al, 2006; Hammet et al, 2007). Here, we produce evidence that the interaction between histone H3-K79 and Rad9 inhibits accumulation of ssDNA at DSBs and at uncapped telomeres. This mechanism, requiring methylation of histone H3 and the Tudor domain of Rad9, regulates resection and appears to represent a strategy that coordinates cell cycle arrest with nuclease progression, thus limiting the amount of ssDNA generated during the cellular response to DNA damage. Results Methylation of H3-K79 controls Mec1 kinase activation after DNA damage Dot1 is required for the G1/S DNA damage checkpoint (Giannattasio et al, 2005; Wysocki et al, 2005). To investigate the effect of the loss of DOT1 on Mec1 kinase activity directly, we analysed the phosphorylation of its proximal target Ddc2, after DNA damage. Ddc2 is a stable partner of Mec1 and is directly phosphorylated by Mec1 in vivo and in vitro (Paciotti et al, 2000; Rouse and Jackson, 2000; Wakayama et al, 2001). Cells were arrested in G1 to avoid complications due to cell cycle-dependent phosphorylation of Ddc2 during the S/G2 phases (Paciotti et al, 2000). Analogous experiments in G2-arrested cells gave comparable results (Supplementary Figure 2). Surprisingly, in time-course analyses, we observed an increase in the phosphorylated form of Ddc2 in dot1Δ cells. G1-arrested wild-type (WT) and dot1Δ cells, expressing HA-tagged Ddc2, were treated with zeocine, which induces DSBs, and Ddc2 phosphorylation was evaluated at different times after the treatment. We consistently found that dot1Δ cells showed a hyper-modification of Ddc2, after induction of DSBs (Figure 1A and B). To verify that the increase in Ddc2 phosphorylation was due to Mec1 and not to another kinase, we compared Ddc2 phosphorylation in WT, dot1Δ, mec1-1 and dot1Δ mec1-1 mutants. Figure 1C shows that all the phospho-Ddc2 signal, detectable in WT and dot1Δ cells, disappears when Mec1 is defective, demonstrating that the increase in Ddc2 phosphorylation observed in dot1Δ cells is indeed due to the Mec1 kinase. These results suggest that more Mec1–Ddc2 kinase complexes can be activated after DSB induction, in the absence of Dot1, and hence H3-K79 methylation. Figure 1.Loss of DOT1 or RAD9 leads to Mec1 kinase hyper-activation after induction of DSBs. (A) WT (YLL683.8/3b) and dot1Δ (YFL403/10b) cells carrying an HA-tagged version of DDC2 at its chromosomal locus were arrested in G1 and treated with 50 μg/ml zeocine to induce DSBs. At the indicated times (–: untreated cells) samples were taken, protein extracts were prepared and the phosphorylation-dependent mobility shift of Ddc2 in SDS–PAGE was monitored by western blotting with 12CA5 antibodies (top panels). FACS profiles of the cultures (bottom panels) show that both cultures did not escape the G1 block throughout the experiment. (B) Quantification of the percentage of phosphorylated form relative to the total Ddc2 protein (from (A)). (C) Ddc2 phosphorylation was monitored in WT (YFL693), dot1Δ (YFL694), mec1-1 (YFL219/9b), mec1-1 dot1Δ (YFL571.1) cells after treatment with zeocine, as in (A). (D) Western blots of Ddc2 in WT (YLL683.8/3b), rad9Y798Q (YFL502) and rad9Δ (YFL407/5a) cells, at different times after induction of DSBs. (E) Quantification of the bands from (D). Download figure Download PowerPoint Failure to recruit Rad9 to histone H3 leads to an increase in Mec1 activation Previous reports suggested that Dot1-dependent methylation of H3-K79 is critical for docking Rad9 to damaged chromatin (Wysocki et al, 2005; Toh et al, 2006). To test whether impairment of Rad9 recruitment to histone H3 would, similarly to a dot1Δ mutation, lead to an increase in Mec1 activity, we introduced a RAD9 allele carrying a Y798Q point mutation in its Tudor domain; this mutation prevents Rad9 recruitment to damaged DNA, damage-dependent phosphorylation of Rad9 and the activation of Rad53 (Wysocki et al, 2005; Grenon et al, 2007, and Supplementary Figure 1). The kinetics of Mec1 activation after DSB induction was analysed in rad9Y798Q and rad9Δ cells. Figure 1D shows that, similarly to what was found in a dot1Δ strain, cells completely lacking Rad9 or expressing rad9Y798Q exhibit faster Ddc2 phosphorylation after DSB induction; quantification of the phospho-Ddc2 form confirmed the observation (Figure 1E). Taken together, these results strongly suggest that loss of Rad9 binding to methylated H3-K79 leads to a faster and more robust activation of Mec1 kinase in response to DSBs. A robust DNA damage checkpoint is not triggered by DSBs themselves, but rather by processed DNA ends, containing long stretches of ssDNA, which recruit Mec1–Ddc2 kinase complexes (White and Haber, 1990; Lydall et al, 1996; Lee et al, 1998; Usui et al, 2001; Harrison and Haber, 2006). Therefore, the Mec1 hyper-activation detected in dot1Δ and rad9 mutants could be explained if DSBs were more rapidly processed to ssDNA when Rad9 does not bind methylated H3. Dot1 and Rad9 limit resection of DNA DSB ends To test the hypothesis that more rapid activation of Mec1 kinase results from a faster production of ssDNA intermediates in dot1Δ, rad9Y798Q and rad9Δ cells, we investigated the kinetics of ssDNA formation after a single unrepairable DSB in these mutants, using an inducible HO endonuclease. Cells were arrested in G2, to prevent cell cycle-dependent effects on resection, and samples were collected at various time points after induction of the nuclease. ssDNA regions in genomic DNA were revealed by the loss of restriction sites distal to the HO-cut site, leading to the accumulation of uncut DNA fragments that were detected with a strand-specific probe, after alkaline electrophoresis (White and Haber, 1990; Shroff et al, 2004; Clerici et al, 2006. See Figure 2B for a map of the MAT locus and the location of probe used in these experiments). The kinetics of appearance of longer DNA fragments suggests that dot1Δ, rad9Y798Q and rad9Δ cells all showed more rapid resection than WT cells (Figure 2A). This finding was confirmed using a different assay where resection leads to the disappearance of a specific DNA restriction fragment in Southern blots (see Figures 5 and 6). These data suggest that the impairment of Rad9 binding to methylated H3-K79, as seen in rad9Δ, rad9Y798Q, dot1Δ and rad9Y798Q dot1Δ (not shown), leads to faster resection at an HO-induced DSB. Figure 2.Resection of a DSB is faster in dot1Δ, rad9Y798Q and rad9Δ cells. (A) WT (JKM179), dot1Δ (YFL399), rad9Y798Q (YFL504) and rad9Δ (YFL419) cells, carrying a unique HO-cut site at the MAT locus and expressing the HO endonuclease under the inducible GAL1 promoter, were grown in presence of lactate and arrested with nocodazole. HO was induced with galactose 2%. Genomic DNA, extracted from samples collected at the indicated times, was digested with SspI and separated on alkaline denaturating gels. Resection was monitored by Southern blotting using a ribo-probe specific for the 3′strand. (B) Scheme of the MAT locus. The figure shows the positions of the HO-cut site, and of the probe (asterisk) used for the experiments shown in (A). The black vertical bars indicate the SspI sites. The products of the digestion of differently resected molecules are shown above the scheme. Download figure Download PowerPoint Figure 3.Dot1 protects subtelomeric DNA from degradation in cdc13-1 mutants. (A) Schematic of ChrVR telomere. (B) Accumulation of ssDNA on the TG strand at PDA1. (C) Accumulation of ssDNA on the AC strand at PDA1. (D) Key to (B, C). In (B, C), ssDNA was measured by QAOS. A single representative experiment is shown with error bars indicating the error of the mean of three independent measurements of the same DNA samples, in most cases the error bars are small and are hidden by the symbols. Download figure Download PowerPoint Figure 4.Dot1 contributes to checkpoint-dependent arrest of cdc13-1 mutants. (A) Five-fold dilutions of strains with indicated phenotypes were spotted onto YEPD plates and grown for 3 days at the temperatures shown. Strains were CDC13+ (DLY640), cdc13-1 (DLY1195), cdc13-1 rad9Δ (DLY1256), rad9-Y798Q (YFL502), cdc13-1 rad9-Y798Q (DLY3083) and cdc13-1 dot1Δ (DLY2880). (B) Single cells were spread onto agar plates and colony formation of CDC13+ (DLY640), cdc13-1 (DLY1195), cdc13-1 dot1Δ (DLY2881) and cdc13-1 rad9Δ (DLY1256) cells was monitored after 20 h at temperatures shown (see Supplementary Figure 4). Number of colonies containing more than 20 cells is plotted against temperature. (C) Cultures of cdc13-1 (DLY1195), cdc13-1 rad9Δ (DLY1256) and cdc13-1 dot1Δ (DLY2881) were grown at the indicated temperatures for 5 h and Rad53 levels in protein extracts were immunoblotted with anti-Rad53 antibodies or anti-tubulin antibodies, as a loading control. Download figure Download PowerPoint Dot1 and Rad9 control DNA processing at uncapped telomeres The results reported so far suggest that a complex containing methylated H3 and Rad9 on damaged DNA inhibits ssDNA accumulation at DSBs. Consistent with this interpretation, earlier studies showed that Rad9 inhibits the accumulation of ssDNA at uncapped telomeres (Lydall and Weinert, 1995; Zubko et al, 2004). To assess the role of methylated H3-K79, and its relationship with Rad9, at uncapped telomeres, we analysed both DNA processing and checkpoint activation in a cdc13-1 mutant background. At temperatures higher than 26°C, cdc13-1 cells accumulate ssDNA and block cell division at the G2/M checkpoint (Garvik et al, 1995). We first examined whether, as found above at DSBs, Dot1 inhibited resection at uncapped telomeres. We used QAOS (quantitative amplification of ssDNA) (Booth et al, 2001; Zubko et al, 2006) to measure the accumulation of ssDNA in synchronous cultures of dot1Δ cdc13-1 strains. A bar1Δ mutation was present in strains to ensure efficient G1 cell cycle arrest with alpha factor and a cdc15-2 mutation was present to ensure that checkpoint-deficient cells did not initiate more than one round of DNA replication during the course of an experiment because at 36°C cdc15-2 mutants arrest in late anaphase (Zubko et al, 2006). We measured ssDNA at the PDA1 locus, which lies about 30 kb away from the end of ChV-R (Figure 3A), because ssDNA does not accumulate at this locus in RAD+ strains but does accumulate if Rad9 function is compromised (Figure 3B). As shown in Figure 3B, dot1Δ cdc13-1 cells, similarly to rad9Δ cdc13-1 strains, had significantly increased levels of ssDNA at PDA1, relative to cdc13-1 strains suggesting that Dot1, similar to Rad9, protects subtelomeric DNA from nucleolytic degradation in cdc13-1 mutants. Figure 5.Loss of Rad9 bypasses CDK1 requirement for SSA and resection. (A) Map of the YMV80 Chr III region, containing the HO-cut site. Vertical bars show the relevant KpnI sites. The thick lines above the map indicate the positions where the probe hybridizes. After HO cleavage, DNA is resected. When the left and right leu2 sequences have been converted to ssDNA, repair by SSA can take place and can be monitored by the appearance of a SSA product in a Southern blot. (B) Exponentially growing YEP+raffinose cell cultures of WT (YMV80) and isogenic rad9Δ (Y31), GAL1-SIC1 (Y20), rad9Δ GAL1-SIC1 (Y293), dot1Δ (YFL736) and dot1Δ GAL-SIC1 (YFL738) strains, carrying an HO-cut site and a gal-inducible HO gene, were arrested with nocodazole; galactose was added at time zero. KpnI-digested DNA, prepared from cells collected at the indicated times, was analysed by Southern blotting with a LEU2 probe. Two fragments, 8 and 6 kb long (his4∷leu2, leu2∷HOcs) are evident in the absence of HO cut, whereas the HO-induced DSB causes the disappearance of the 6-kb species and the formation of a 2.5-kb fragment (HO-cut fragment). Repair by SSA converts such fragment to a repair product of 3.5-kb (SSA product). (C) Speed of resection was evaluated in rad52Δ (YMV037) and rad52Δ rad9Δ (YMV038) cells under similar conditions by monitoring the disappearance of the his4∷leu2 signal. Download figure Download PowerPoint Dot1 affects checkpoint activation in response to telomere uncapping As Dot1, the H3-K79 methylase, is important for activating Rad9 in response to DSBs (Giannattasio et al, 2005; Wysocki et al, 2005; Toh et al, 2006), we tested the checkpoint role of Dot1 after telomere uncapping. Impairing checkpoint pathways, for example rad9Δ, partially suppresses the temperature sensitivity of cdc13-1 strains (Weinert et al, 1994; Zubko et al, 2004). Figure 4A shows that Dot1 inhibits growth of cells with uncapped telomeres because dot1Δ cdc13-1 strains grow better than cdc13-1 strains at 26.5°C. We note that rad9Δ cdc13-1 strains grow better than dot1Δ cdc13-1 strains. To test whether Dot1 growth inhibition of cdc13-1 cells is mediated by the interaction of Rad9-Tudor domain with H3-K79me, we analysed the effect of the rad9Y798Q mutation. Even though we routinely observed that rad9Y798Q cdc13-1 strains grew better than dot1Δ cdc13-1 strains, rad9Y798Q cdc13-1 mutants behave most similarly to dot1Δ cdc13-1 cells and grow better than cdc13-1 cells but less well than cdc13-1 rad9Δ cells (Figure 4A). This can be explained if the rad9Y798Q point mutation affects the structure of Rad9 or its interaction with other proteins. Moreover, loss of DOT1 causes redistribution of the SIR factors and this could also influence the vitality of cdc13-1 cells. The epistatic relationships between these mutations are shown in Supplementary Figure 3. Taken together, these data suggest that the role of Dot1 in responding to telomere uncapping is mediated by the Tudor domain of Rad9. However, our finding that rad9Y798Q cdc13-1 and dot1Δ cdc13-1 mutants do not grow as well as cdc13-1 rad9Δ strains, at semi-permissive temperature, suggests the existence of a Rad9-dependent mechanism acting independently of the H3-K79me/Rad9 Tudor domain at uncapped telomeres. Figure 6.Loss of RAD9 or DOT1 increase resection at DSBs bypassing the requirement for Cdk1. The graphs represent quantifications of data shown in Figure 5. (A) Quantification of the his4∷leu2 band, measuring resection at the distal site. The intensity of each band was normalized with respect to loading. The signal present before HO cutting was set to 100%. (B) Quantification of the SSA product band. ▪ rad9Δ; □ rad9Δ GAL-SIC1; • dot1Δ; ○ dot1Δ GAL-SIC1; ♦ WT; ⋄ WT GAL-SIC1. Download figure Download PowerPoint Our observations suggest a role for H3-K79 methylation in checkpoint activation following telomere uncapping. To address this directly, single cells were monitored for their ability to form colonies at different temperatures (Figure 4B, and Supplementary Figure 4). Figure 4B plots the fraction of colonies that contain more than 20 cells for checkpoint and nuclease-deficient cdc13-1 strains over a range of temperatures. The higher the temperature the smaller the colony size for both checkpoint-proficient and -deficient cells. At temperatures higher than 26°C, checkpoint proficient cdc13-1 cells divide slowly and form smaller colonies than checkpoint- or nuclease-deficient strains (Figure 4B). At high levels of telomere uncapping, for example, 30°C and 36°C, the colonies of dot1Δ cdc13-1 strains are of similar size as those of cdc13-1. At lower levels of uncapping, for example, 27°C and 28°C, dot1Δ cdc13-1 cells form larger colonies than cdc13-1 strains (Figure 4B). However, dot1Δ cdc13-1 colonies are smaller than cdc13-1 rad9Δ cells at 27°C (Supplementary Figure 4). This, and the spots tests shown in Figure 4A, suggest that Dot1 has a partial function in checkpoint activation, a function that is only detectable at low levels of DNA damage. Consistent with this interpretation, we saw complete and efficient cell cycle arrest of dot1Δ cdc13-1 mutants in the first cycle after G1 release in the ssDNA measurement experiment shown in Figure 3 (data not shown). The role of Dot1 in checkpoint activation at intermediate temperatures was confirmed by measuring phosphorylation of Rad53. At 23°C, telomeres in cdc13-1 strains are capped and Rad53 is largely hypo-phosphorylated (Figure 4C, lanes 1–3). In response to low levels of telomere uncapping (28°C), Rad53 is hyper-phosphorylated in a Dot1- and Rad9-dependent manner (Figure 4C, lanes 4–6). However, at higher levels of telomere uncapping (36°C) a significant fraction of Rad53 is hyper-phosphorylated in dot1Δ cdc13-1 strains (Figure 4C, lane 9). These observations suggest that at low levels of telomere uncapping, Dot1 has a more important function in Rad9-dependent checkpoint activation that it does at higher levels of uncapping, when presumably alternative mechanisms ensure that Rad9 is activated. Loss of Rad9 or Dot1 partially bypasses the requirement for CDK1 in DSB resection The molecular mechanisms controlling DNA end resection at DSBs and uncapped telomeres are poorly understood. Recent reports showed that CDK1 activity is necessary to obtain effective resection, both at DSBs and uncapped telomeres (Aylon et al, 2004; Ira et al, 2004; Vodenicharov and Wellinger, 2006). Indeed, inhibition of CDK1, by overexpression of the inhibitor Sic1, leads to a notable reduction of 5′–3′ processing of DSB ends (Aylon et al, 2004; Ira et al, 2004, and Figure 5B). To understand the relationships between the Rad9-dependent and the CDK1-dependent mechanisms that regulate resection, we analysed the processing of a DSB in rad9Δ and dot1Δ cells after inhibition of CDK1 activity. We used a yeast strain where a sit" @default.
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• W2034949406 title "Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres" @default.
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