Department of Pathology, The University of Chicago, Chicago, IL 60637, USA.
DNA Repair (Amst). 2009 Dec 3;8(12):1452-61. doi: 10.1016/j.dnarep.2009.09.010. Epub 2009 Oct 31.
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.
在酿酒酵母中，通过失活端粒重复结合因子 Cdc13 使端粒不稳定，会诱导细胞周期检查点，使细胞在有丝分裂中期到后期过渡时停滞——这很像对未修复的双链 DNA 断裂 (DSB) 的反应。在整个细胞周期中，多结构域衔接蛋白 Rad9 是激活检查点效应激酶 Rad53 对 DSB 反应所必需的，并且对于响应端粒去帽的检查点信号也是必需的。Rad53 在 G1 和 S 期的激活依赖于 Rad9 与邻近 DSB 的修饰染色质的关联，这是由结合组蛋白 H3 第 79 位二甲基化的 Tudor 结构域和结合组蛋白 H2A 第 129 位磷酸化的 BRCT 结构域介导的。尽管如此，Rad9 Tudor 或 BRCT 突变体可以在长春花碱处理的细胞中引发对 DNA 损伤的检查点反应。影响 H3 K79 二甲基化或其被 Rad9 识别的突变会增强端粒去帽时 5'链的切除，并可能暗示 Rad9 染色质结合在端粒去帽的检查点反应中起作用。事实上，我们报告说，Rad9 在端粒去帽后，在距端粒 10 kb 以内的亚端粒染色质上结合。Rad9 结合发生在失活 Cdc13 后 30 分钟内，早于 Rad53 磷酸化。反过来，Rad9 Tudor 和 BRCT 结构域突变阻断了染色质结合，并导致检查点信号减弱，表现为 Rad53 磷酸化减少和细胞周期阻滞受损。我们的工作确定了 Rad9 在有丝分裂期间与染色质的关联在端粒去帽的 DNA 损伤检查点反应中的作用，表明染色质结合可能是整个细胞周期检查点的起始事件。
