ATR-ATRIP Kinase Complex Triggers Activation of the Fanconi Anemia DNA Repair Pathway

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. Cancer Res; 72(5); 1149-56. (C) 2012 AACR.

当复制叉在DNA损伤位点停滞时，ATR激酶激活S期检查点。这一事件还导致范可尼贫血（FA）蛋白FANCI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，从而促进这一DNA修复的核心通路，使复制得以重新启动。然而，ATR与FA通路之间的相互作用一直不清楚。在这项研究中，我们提供的证据表明它们的作用是直接相关的，我们通过对一种DT40突变细胞系的研究深入了解了这种关系，该细胞系在关键的ATR结合伙伴蛋白ATRIP上有条件性缺陷。利用这一系统，我们表明ATRIP对DNA损伤诱导的FANCD2单泛素化和FANCI磷酸化至关重要。ATR激酶在体外可使重组FANCI蛋白磷酸化，FANCD2的存在促进了这一过程。机制研究表明，ATRIP的RPA区域而非TopBP1区域是FANCD2单泛素化所必需的，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ATR是负责激活FA DNA修复通路的激酶。《癌症研究》；72(5)；1149 - 1156。（C）2012美国癌症研究协会。