The DNA Damage Checkpoint Response Requires Histone H2B Ubiquitination by Rad6-Bre1 and H3 Methylation by Dot1

Abstract

The cellular response to DNA lesions entails the recruitment of several checkpoint and repair factors to damaged DNA, and chromatin modifications may play a role in this process. Here we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses. We demonstrate that ubiquitination of histone H2B on lysine 123 by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest. We found a similar requirement for Dot1-dependent methylation of histone H3. Loss of H3-Lys79 methylation does not affect Mec1 activation, whereas it renders cells checkpoint-defective by preventing phosphorylation of Rad9. Such results suggest that histone modifications may have a role in checkpoint function by modulating the interactions of Rad9 with chromatin and active Mec1 kinase. The cellular response to DNA lesions entails the recruitment of several checkpoint and repair factors to damaged DNA, and chromatin modifications may play a role in this process. Here we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses. We demonstrate that ubiquitination of histone H2B on lysine 123 by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest. We found a similar requirement for Dot1-dependent methylation of histone H3. Loss of H3-Lys79 methylation does not affect Mec1 activation, whereas it renders cells checkpoint-defective by preventing phosphorylation of Rad9. Such results suggest that histone modifications may have a role in checkpoint function by modulating the interactions of Rad9 with chromatin and active Mec1 kinase. Eukaryotic cells respond to DNA damage by activating a variety of DNA repair pathways and by triggering the DNA damage checkpoint, a surveillance mechanism required to control cell cycle progression in response to genotoxic stress (1Elledge S.J. Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1790) Google Scholar). A considerable amount of information is now available relative to the key protein factors, which act in eukaryotic repair pathways. In the last few years, a lot has been learnt on the signal transduction cascade leading to cell cycle arrest following DNA damage (2Melo J. Toczyski D. Curr. Opin. Cell Biol. 2002; 14: 237-245Crossref PubMed Scopus (402) Google Scholar, 3Rouse J. Jackson S.P. Science. 2002; 297: 547-551Crossref PubMed Scopus (578) Google Scholar). However, the physical and functional interplay connecting activation of the checkpoint response with specific repair mechanisms has started to emerge only recently (3Rouse J. Jackson S.P. Science. 2002; 297: 547-551Crossref PubMed Scopus (578) Google Scholar, 4Giannattasio M. Lazzaro F. Longhese M.P. Plevani P. Muzi Falconi M. EMBO J. 2004; 23: 429-438Crossref PubMed Scopus (104) Google Scholar, 5van den Bosch M. Bree R.T. Lowndes N.F. EMBO Rep. 2003; 4: 844-849Crossref PubMed Scopus (214) Google Scholar). Since damage recognition and repair can be strongly influenced by the chromosome organization (6Mello J.A. Sillje H.H. Roche D.M. Kirschner D.B. Nigg E.A. Almouzni G. EMBO Rep. 2002; 3: 329-334Crossref PubMed Scopus (231) Google Scholar, 7Green C.M. Almouzni G. EMBO Rep. 2002; 3: 28-33Crossref PubMed Scopus (181) Google Scholar), it is now essential to extend this knowledge considering the context of a chromatin environment. Critical checkpoint factors are phosphorylated in response to DNA damage, and the order of function in the pathway has been mainly inferred by monitoring their phosphorylation state (8Longhese M.P. Foiani M. Muzi-Falconi M. Lucchini G. Plevani P. EMBO J. 1998; 17: 5525-5528Crossref PubMed Scopus (143) Google Scholar). In the yeast Saccharomyces cerevisiae, the Mec1-Ddc2 and the Rad17-Mec3-Ddc1 complexes are independently loaded in proximity of a DNA lesion (9Melo J.A. Cohen J. Toczyski D.P. Genes Dev. 2001; 15: 2809-2821Crossref PubMed Google Scholar, 10Kondo T. Wakayama T. Naiki T. Matsumoto K. Sugimoto K. Science. 2001; 294: 867-870Crossref PubMed Scopus (227) Google Scholar). Phosphorylation of Ddc2 is the first biochemical event detectable after checkpoint activation, and this modification does not require any checkpoint factor other than the Mec1 kinase itself (11Paciotti V. Clerici M. Lucchini G. Longhese M.P. Genes Dev. 2000; 14: 2046-2059PubMed Google Scholar, 12Rouse J. Jackson S.P. EMBO J. 2000; 19: 5801-5812Crossref PubMed Scopus (118) Google Scholar, 13Wakayama T. Kondo T. Ando S. Matsumoto K. Sugimoto K. Mol. Cell. Biol. 2001; 21: 755-764Crossref PubMed Scopus (109) Google Scholar). The Rad17-Mec3-Ddc1 complex, which shows a structural similarity to PCNA, 1The abbreviations used are: PCNA, proliferating cell nuclear antigen; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; PRR, postreplication repair; ISA, in situ kinase activity; MMS, methyl methane sulfonate; HU, hydroxyurea; ZEO, zeocin; FACS, fluorescence-activated cell sorting; YEPD, yeast extract peptone dextrose; DSB, double strand break. is also loaded near a DNA lesion and is phosphorylated on Ddc1 by the kinase activity of Mec1. Another critical target of Mec1 is the protein Rad9, whose phosphorylation may modulate its local concentration on DNA and is required for the recruitment and activation of Rad53 kinase (14Gilbert C.S. Green C.M. Lowndes N.F. Mol. Cell. 2001; 8: 129-136Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 15Vialard J.E. Gilbert C.S. Green C.M. Lowndes N.F. EMBO J. 1998; 17: 5679-5688Crossref PubMed Scopus (231) Google Scholar, 16Emili A. Mol. Cell. 1998; 2: 183-189Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 17Sun Z.X. Hsiao J. Fay D.S. Stern D.F. Science. 1998; 281: 272-274Crossref PubMed Scopus (338) Google Scholar). This is a key step in the signal transduction cascade, and it is generally used as a marker to monitor checkpoint activation (18Pellicioli A. Lucca C. Liberi G. Marini F. Lopes M. Plevani P. Romano A. Di Fiore P.P. Foiani M. EMBO J. 1999; 18: 6561-6572Crossref PubMed Scopus (315) Google Scholar). The cellular response to DNA damage is probably influenced by the structure of chromatin, much like all other DNA transactions (19Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2750) Google Scholar, 20Koundrioukoff S. Polo S. Almouzni G. DNA Repair (Amst.). 2004; 3: 969-978Crossref PubMed Scopus (53) Google Scholar), and it is tempting to speculate that local changes in chromatin structure near a DNA lesion may facilitate its identification contributing to the recruitment of repair and checkpoint proteins. Histones are the targets of a number of covalent modifications, such as acetylation, methylation, phosphorylation, and ubiquitination, which influence a variety of DNA metabolic processes. It is well established that, in mammalian cells, Ser139 in the C-terminal tail of the H2AX histone variant is phosphorylated in response to DSBs, treatments with genotoxic agents, and stalled replication forks (21Redon C. Pilch D. Rogakou E. Sedelnikova O. Newrock K. Bonner W. Curr. Opin. Genet. Dev. 2002; 12: 162-169Crossref PubMed Scopus (637) Google Scholar, 22Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4297) Google Scholar). Similarly, phosphorylation of Ser129 in the C-terminal tail of H2A occurs both in budding and fission yeast following DNA damage (23Downs J.A. Lowndes N.F. Jackson S.P. Nature. 2000; 408: 1001-1004Crossref PubMed Scopus (547) Google Scholar). In Saccharomyces cerevisiae, the only histone species currently known to be ubiquitinated is histone H2B (24Robzyk K. Recht J. Osley M.A. Science. 2000; 287: 501-504Crossref PubMed Scopus (540) Google Scholar). Protein ubiquitination requires the activity of three different enzymes. A ubiquitin-activating enzyme (UBA or E1) covalently binds an ubiquitin molecule and transfers it to a ubiquitin-conjugating enzyme (UBC or E2). This physically associates with a ubiquitin ligase (E3), which allows conjugation of the ubiquitin moiety to specific substrates. It is well established that a single E2 can play relevant roles in a variety of pathways by interacting with different E3 enzymes. 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In this we demonstrate that a and key role in the activation of the DNA damage checkpoint in response to a variety of genotoxic stresses. is not with in checkpoint such a role is by ubiquitination of histone DNA damage to Rad53 kinase requires methylation of H3-Lys79 by a role of the histone in the cellular response to DNA Loss of such histone modifications a checkpoint by the function of in Mec1 kinase is and and the A. A. A. P. 1998; 14: PubMed Scopus Google Scholar). of the used in this for are of The double by a of in of V. and M. A. and and D. E. is a a and used are in can K. Recht J. Osley M.A. Science. 2000; 287: 501-504Crossref PubMed Scopus (540) Google K. Recht J. Osley M.A. Science. 2000; 287: 501-504Crossref PubMed Scopus (540) Google 4Giannattasio M. Lazzaro F. Longhese M.P. Plevani P. Muzi Falconi M. EMBO J. 2004; 23: 429-438Crossref PubMed Scopus (104) Google 4Giannattasio M. Lazzaro F. Longhese M.P. Plevani P. Muzi Falconi M. 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Cell and in to a concentration of and with or of or cells in of YEPD, and on a with a after cells in for protein extract with cells and in methyl methane or and for protein extract and used and cells in the of the for and as in by cell with cells in by cell with in checkpoint the cells with and nuclear by and treatments as M. Lazzaro F. Longhese M.P. Plevani P. Muzi Falconi M. EMBO J. 2004; 23: 429-438Crossref PubMed Scopus (104) Google Scholar). DNA with a after with Green is implicated in ubiquitination of and cells are to a variety of S. P. Genet. PubMed Scopus Google Scholar, S. A. C. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). E2 activity is necessary for postreplication repair P. S. S. A. 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The in cells than that found in a the cell cycle by in the not checkpoint detectable in other such as and We the role of in checkpoint activation in and cells Rad53 phosphorylation after is strongly in cells, whereas it is only in of Rad53 kinase activity with the that activation of the checkpoint in cells is by with the whereas in Rad53 activity is only to The different for in the and of the cell cycle that a on is active in Rad53 phosphorylation found to be in and both in and that function in checkpoint is its function in We to a variety of other than it is shown in cells are in Rad53 phosphorylation also in response to MMS, HU, and treatments that DNA replication and DSBs, in this cells show a that of a is an E2 enzyme with in several cellular and its E3 in PRR, does not to be in checkpoint We the response to DNA damage in S. cerevisiae in all genes in the yeast not The only E3 gene required for Rad53 in response to different is The in cells the found in in cells and in Rad53 phosphorylation is or strongly We the of on the control of cell cycle progression following DNA cells, and a control with and a and their and cell cycle progression by shows that cells, to a not arrest the in response to In to a DNA after the whereas is in cells also to the of after in to is found in the checkpoint-defective M.P. Plevani P. Lucchini G. Mol. Cell. Biol. 1996; PubMed Scopus Google not also the DNA damage In cells are a arrest in the of of MMS, to to cells The checkpoint of cells is by the of Rad53 In cells are with and the Rad53 is phosphorylated the the Rad53 is only after in and checkpoint-defective cells are in the and are in the nuclear after The of the checkpoint in cells may their to MMS, and treatments not cells are in the and DNA damage and cells in with with or and the arrest. checkpoint to the after the for Rad53 phosphorylation state by after the the in A. the used in A in with and after the of cells is a protein with structural with other ubiquitin and it recently found as the E3 in the ubiquitination of by A. J. J. J. M.A. K. A. M. A. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. C. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This H2B modification has been to in S. cerevisiae C. T. K. S. 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The checkpoint is specific for a in does not affect Rad53 phosphorylation not that the in checkpoint function is not to of and the histone influence activation of the checkpoint, we step of the cascade histone modifications are required for the of the Since modification of Rad53 is a event in the we the phosphorylation state of other critical protein Mec1-Ddc2 and Rad17-Mec3-Ddc1 are independently loaded near DNA lesions (9Melo J.A. Cohen J. Toczyski D.P. Genes Dev. 2001; 15: 2809-2821Crossref PubMed Google Scholar, M. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Mec1 kinase and Rad9. This last event is required for of Rad9 and activation of We have shown that of lesions by repair factors is required for Ddc2 and Ddc1 phosphorylation, and it is a for of the Mec1-Ddc2 and the complexes to M. Lazzaro F. Longhese M.P. Plevani P. Muzi Falconi M. EMBO J. 2004; 23: 429-438Crossref PubMed Scopus (104) Google Scholar). 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Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), which leading to Rad9 recruitment and that ubiquitination and H3-Lys79 methylation influence the DNA damage response suggest that specific checkpoint factors may be to in chromatin structure or However, the of the histone changes in chromatin structure or and interactions near DNA damage a for We D. E. M. A. and V. for and and C. for We of the for and