Genome Stability Unit, St. Vincent's Institute of Medical Research, Fitzroy, Australia.
Department of Medicine (St. Vincent's Health), The University of Melbourne, Melbourne, Australia.
Elife. 2020 Mar 13;9:e54128. doi: 10.7554/eLife.54128.
FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show using electron microscopy that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.
FANCD2 单泛素化是范可尼贫血（FA）DNA 修复途径稳定复制叉的关键事件。据推测，在停滞的复制叉处，单泛素化的 FANCD2 可募集含有泛素结合基序的 DNA 修复蛋白。在这里，我们在体外重建了 FA 途径，以研究 FANCI:FANCD2 单泛素化的功能后果。我们报告说，单泛素化不会促进任何特定的外源蛋白：蛋白相互作用，而是稳定 dsDNA 上的 FANCI:FANCD2 异二聚体。这种夹断仅需要 FANCD2 亚基的单泛素化。我们进一步使用电子显微镜显示，纯化的单泛素化 FANCI:FANCD2 在长 dsDNA 上形成丝状排列。我们的结果揭示了单泛素化的 FANCI:FANCD2（在许多癌症类型和所有 FA 病例中均有缺陷）如何在 DNA 结合后被激活。
