Dissection of Rad9 BRCT domain function in the mitotic checkpoint response to telomere uncapping.
In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition--much like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for the activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5' strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle.
Nnakwe
Chinonye C
CC
Department of Pathology, The University of Chicago, Chicago, IL 60637, USA.
Altaf
Mohammed
M
Côté
Jacques
J
Kron
Stephen J
SJ
D020816:Amino Acid Motifs; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics; D004249:DNA Damage; D006801:Humans; D008938:Mitosis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010768:Phosphoserine; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D016615:Telomere / Q000235:genetics*
D018797:Cell Cycle Proteins; D002843:Chromatin; C069930:rad9 protein; D010768:Phosphoserine