Dissection of Rad9 BRCT domain function in the mitotic checkpoint response to telomere uncapping

Abstract

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.

Figure 1

Comparative Protein Modeling for the Rad9 BRCT domain. (A)T-Coffee alignment of BRCT sequences from multiple checkpoint adaptor proteins identified a functionally conserved region, highlighted by a yellow box. Adaptor protein BRCT sequences include: S. pombe Crb2 (537–778), S. cerevisiae Rad9 (994–1122), Homo sapiens 53BP1 (1719–1977), H. sapiens BRCA1 (1649–1859) and H. sapiens MDC1 (1719–1977). Conserved arginine and lysine residues are highlighted in red. Crystal structures of MDC1 (B) and Crb2 (C) BRCT domains, in complex with a phospho-H2A peptide [66, 71]. Blue arrows denote conserved arginine residues that may contact the H2A carboxyl-terminal tail. Red arrows denote conserved lysine residues that may contact phosphoserine residues within the H2A tail. Backbone molecules are highlighted in dark blue, Nitrogen molecules are featured in light blue, oxygen molecules are featured in red and sulfur molecules are featured in yellow.

Figure 1

Comparative Protein Modeling for the Rad9 BRCT domain. (A)T-Coffee alignment of BRCT sequences from multiple checkpoint adaptor proteins identified a functionally conserved region, highlighted by a yellow box. Adaptor protein BRCT sequences include: S. pombe Crb2 (537–778), S. cerevisiae Rad9 (994–1122), Homo sapiens 53BP1 (1719–1977), H. sapiens BRCA1 (1649–1859) and H. sapiens MDC1 (1719–1977). Conserved arginine and lysine residues are highlighted in red. Crystal structures of MDC1 (B) and Crb2 (C) BRCT domains, in complex with a phospho-H2A peptide [66, 71]. Blue arrows denote conserved arginine residues that may contact the H2A carboxyl-terminal tail. Red arrows denote conserved lysine residues that may contact phosphoserine residues within the H2A tail. Backbone molecules are highlighted in dark blue, Nitrogen molecules are featured in light blue, oxygen molecules are featured in red and sulfur molecules are featured in yellow.

Figure 2

Rad9 BRCT mutants initiate variable responses to uncapped telomeres. (A) rad9Δ strains were transformed with constructs harboring wild type RAD9-MYC or MYC-tagged rad9 mutant alleles, and analyzed for Rad53 activation in response to 300 Gy of ionizing irradiation. Samples were treated with nocodazole, irradiated and were harvested at 0, 15 and 30 min after irradiation. rad9Δ cdc13-1 strains were transformed with constructs harboring wild type RAD9-MYC or MYC-tagged rad9 mutant alleles, and analyzed for (B) Rad53 activation, H2A phosphorylation and (C) DNA content. (D and E) Microcolony analysis of the R1085E and K1088E mutants with the cdc13-1 allele. The data shown are the average of 3 replicates, mean ± SD. Bar = 10 μm

Figure 3

Rad9 binds to sub-telomeric chromatin after telomere uncapping. (A) A schematic representation of the sites probed within Chromosome VI for chromatin immunoprecipitation assays. (B) rad9Δ cdd13-1 strains expressing Myc-tagged RAD9 or rad9 mutant constructs were treated with α-factor at room temperature for one hour and then shifted to 37°C for 30 minutes. Next cells were released into pre-warmed YPD and incubated at 37°C. Samples were collected at 30 min, 1 h and 2 h time points and then used for ChIP analysis. Error bars represent the standard deviation between at least two replicate experiments. (C) With the same time course used in ChIP assays, Rad53 activation was detected after incubating cells at restrictive temperature. Arrows indicate high molecular weight species that correspond to phosphorylated Rad53. (D) DNA content analysis of cells from figure 3C after incubation at restrictive temperature.

Figure 4

Additional rad9 mutants demonstrate that chromatin association and SCD phosphorylation are necessary but not sufficient for checkpoint activation. A series of rad9 alleles were assayed to determine if Rad9 phosphorylation is upstream of chromatin association. rad9Δ cdc13-1 strains were transformed with additional MYC-tagged rad9 alleles and assayed for (A) Rad53 activation in response to telomere uncapping. Microcolonies of cdc13-1 strains, also harboring several rad9 alleles, were also visualized (D) and quantified (E) after telomere uncapping. The data shown are the average of 3 replicates, mean ± SD. (B) G1-arrested rad9 mutants were also assayed for Rad53 activation in response to 300 Gy of ionizing radiation. Arrows in Figs. 4A and B indicate high molecular weight species that correspond to phosphorylated Rad53. Bar = 10 μm