Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9.
We screened radiation-sensitive yeast mutants for DNA damage checkpoint defects and identified Dot1, the conserved histone H3 Lys 79 methyltransferase. DOT1 deletion mutants (dot1Delta) are G1 and intra-S phase checkpoint defective after ionizing radiation but remain competent for G2/M arrest. Mutations that affect Dot1 function such as Rad6-Bre1/Paf1 pathway gene deletions or mutation of H2B Lys 123 or H3 Lys 79 share dot1Delta checkpoint defects. Whereas dot1Delta alone confers minimal DNA damage sensitivity, combining dot1Delta with histone methyltransferase mutations set1Delta and set2Delta markedly enhances lethality. Interestingly, set1Delta and set2Delta mutants remain G1 checkpoint competent, but set1Delta displays a mild S phase checkpoint defect. In human cells, H3 Lys 79 methylation by hDOT1L likely mediates recruitment of the signaling protein 53BP1 via its paired tudor domains to double-strand breaks (DSBs). Consistent with this paradigm, loss of Dot1 prevents activation of the yeast 53BP1 ortholog Rad9 or Chk2 homolog Rad53 and decreases binding of Rad9 to DSBs after DNA damage. Mutation of Rad9 to alter tudor domain binding to methylated Lys 79 phenocopies the dot1Delta checkpoint defect and blocks Rad53 phosphorylation. These results indicate a key role for chromatin and methylation of histone H3 Lys 79 in yeast DNA damage signaling.
Wysocki
Robert
R
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
Javaheri
Ali
A
Allard
Stéphane
S
Sha
Fei
F
Côté
Jacques
J
Kron
Stephen J
SJ
D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D064447:Checkpoint Kinase 2; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000737:chemistry / Q000528:radiation effects; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D000939:Epitopes / Q000737:chemistry; D016193:G1 Phase; D017353:Gene Deletion; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins; D010766:Phosphorylation; D011496:Protein Methyltransferases; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011839:Radiation, Ionizing; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
D018797:Cell Cycle Proteins; D000939:Epitopes; D006657:Histones; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; D004247:DNA; D000076983:Histone Methyltransferases; D011496:Protein Methyltransferases; C416235:Dot1 protein, S cerevisiae; D011495:Histone-Lysine N-Methyltransferase; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D008239:Lysine