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 (dot1 Delta) are G, and intra-S phase checkpoint defective after ionizing radiation but remain competent for G(2)/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 dot1 Delta checkpoint defects. Whereas dot1 Delta alone confers minimal DNA damage sensitivity, combining dot1 Delta with histone methyltransferase mutations set1 Delta and set2 Delta markedly enhances lethality. Interestingly, set1 Delta and set2 Delta mutants remain G, checkpoint competent, but set1 Delta 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 dot1 Delta 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.