MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK; Department of Medicine, Level 5, Addenbrooke's Hospital, University of Cambridge, Cambridge CB2 0QQ, UK.
Mol Cell. 2014 Jun 5;54(5):858-69. doi: 10.1016/j.molcel.2014.05.001.
Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.
范可尼贫血症（FA）是一种癌症易感性综合征，其特征是细胞对 DNA 链间交联剂敏感。FA 的分子缺陷是 DNA 修复途径受损。激活该途径的关键事件是 FANCD2 的单泛素化。在体内，一个多亚基 FA 核心复合物催化这一步骤，但它的机制尚不清楚。在这里，我们报告了天然禽源 FA 核心复合物的纯化和 FANCD2 单泛素化的生化重建。这表明催化 FANCL E3 连接酶亚基必须嵌入复合物中才能发挥最大的活性和位点特异性。我们通过遗传和生化方法定义了一个仅由三个蛋白质（FANCB、FANCL 和 FAAP100）组成的最小亚基复合物，作为单泛素化模块。在其他 FA 核心复合物亚基缺失的细胞中保留了残留的 FANCD2 单泛素化活性。这项工作描述了这种多亚基单泛素 E3 连接酶的体外重建和表征，为保守的 FA DNA 修复途径提供了关键的见解。
