Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan.
Cancer Res. 2012 Mar 1;72(5):1149-56. doi: 10.1158/0008-5472.CAN-11-2904. Epub 2012 Jan 18.
ATR kinase activates the S-phase checkpoint when replication forks stall at sites of DNA damage. This event also causes phosphorylation of the Fanconi anemia (FA) protein FANCI, triggering its monoubiquitination of the key DNA repair factor FANCD2 by the FA core E3 ligase complex, thereby promoting this central pathway of DNA repair which permits replication to be restarted. However, the interplay between ATR and the FA pathway has been unclear. In this study, we present evidence that their action is directly linked, gaining insights into this relationship in a DT40 mutant cell line that is conditionally deficient in the critical ATR-binding partner protein ATRIP. Using this system, we showed that ATRIP was crucial for DNA damage-induced FANCD2 monoubiquitination and FANCI phosphorylation. ATR kinase phosphorylated recombinant FANCI protein in vitro, which was facilitated by the presence of FANCD2. Mechanistic investigations revealed that the RPA region but not the TopBP1 region of ATRIP was required for FANCD2 monoubiquitination, whereas Chk1 phosphorylation relied upon both domains. Together, our findings identify ATR as the kinase responsible for activating the FA pathway of DNA repair.
ATR 激酶在复制叉停滞在 DNA 损伤部位时激活 S 期检查点。这一事件还导致范可尼贫血 (FA) 蛋白 FANCI 的磷酸化，触发 FA 核心 E3 连接酶复合物对关键 DNA 修复因子 FANCD2 的单泛素化，从而促进了允许复制重新启动的这一主要 DNA 修复途径。然而，ATR 和 FA 途径之间的相互作用尚不清楚。在这项研究中，我们提供了证据表明它们的作用是直接相关的，从而深入了解了在条件性缺乏关键 ATR 结合伴侣蛋白 ATRIP 的 DT40 突变细胞系中这种关系。使用该系统，我们表明 ATRIP 对于 DNA 损伤诱导的 FANCD2 单泛素化和 FANCI 磷酸化至关重要。ATR 激酶在体外磷酸化重组 FANCI 蛋白，这一过程得益于 FANCD2 的存在。机制研究表明，ATRIP 的 RPA 区域而不是 TopBP1 区域对于 FANCD2 单泛素化是必需的，而 Chk1 磷酸化则依赖于这两个结构域。总之，我们的研究结果确定了 ATR 是激活 FA 途径的 DNA 修复激酶。
