title: Docking onto chromatin via the Saccharomyces cerevisiae Rad9 Tudor domain.
abstract: An integrated cellular response to DNA damage is essential for the maintenance of genome integrity. Recently, post-translational modifications to histone proteins have been implicated in DNA damage responses involving the Rad9 family of checkpoint proteins. In budding yeast, methylation of histone H3 on lysine 79 (H3-K79me) has been shown to be required for efficient checkpoint signalling and Rad9 localization on chromatin. Here, we have used a rad9 Tudor mutant allele and cells mutated for Dot1, the H3-K79 methylase, to analyse the epistatic relationship between RAD9 and DOT1 genes regarding the DNA damage resistance and checkpoint activation pathways. Our results show that RAD9 is epistatic to DOT1 and suggest that it acts downstream of the Dot1 methylase in the damage resistance and checkpoint response. We have also found that the Tudor domain of Rad9 is necessary for in vitro binding to H3-K79me as well as Rad9 focal accumulation in response to DNA damage in vivo. In summary, our study demonstrates that the interaction between Rad9, via its Tudor domain, and methylated H3-K79 is required at two different steps of the DNA damage response, an early step corresponding to checkpoint activation, and a late step corresponding to DNA repair. The study further shows that the function of this interaction is cell cycle-regulated; the role in checkpoint activation is restricted to the G(1) phase and its role in DNA repair is restricted to G(2).
authors.affiliation: Genome Stability Laboratory, Department of Biochemistry and National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland.
authors.forename: Muriel
authors.initials: M
authors.lastname: Grenon
authors.forename: Thomas
authors.initials: T
authors.lastname: Costelloe
authors.forename: Sonia
authors.initials: S
authors.lastname: Jimeno
authors.forename: Aisling
authors.initials: A
authors.lastname: O'Shaughnessy
authors.forename: Jennifer
authors.initials: J
authors.lastname: Fitzgerald
authors.forename: Omar
authors.initials: O
authors.lastname: Zgheib
authors.forename: Linda
authors.initials: L
authors.lastname: Degerth
authors.forename: Noel F
authors.initials: NF
authors.lastname: Lowndes
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D018816:Genes, cdc; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000378:metabolism; D008745:Methylation; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002843:Chromatin; D006657:Histones; C440014:LCD1 protein, S cerevisiae; D009687:Nuclear Proteins; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; C416235:Dot1 protein, S cerevisiae; D011495:Histone-Lysine N-Methyltransferase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae