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 repa