The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes.

范可尼贫血 (FA) 途径可修复人类 DNA 链间交联 (ICL)。该途径的激活依赖于将 FANCD2/FANCI 复合物加载到染色体上，并通过随后的单泛素化完全激活。然而，将复合物加载到染色体上的机制仍不清楚。在这里，我们鉴定了 FANCD2 上的 10 个 SQ/TQ 磷酸化位点，这些位点在 ICL 的响应下被 ATR 磷酸化。使用一系列生化检测并辅以活细胞成像（包括超分辨率单分子追踪），我们表明这些磷酸化事件对于将复合物加载到染色体上及其随后的单泛素化至关重要。我们揭示了磷酸化事件如何在细胞中受到严格调控，并且模仿其恒定磷酸化会导致 FANCD2 处于不受控制的活性状态，从而以不受限制的方式加载到染色体上。综上所述，我们描述了 ATR 触发 FANCD2/FANCI 加载到染色体上的机制。