Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (I_UbD2). Here, we present a 4.1 Å cryo-EM structure of I_UbD2 complex bound to double-stranded DNA. We show that this complex, like ID2_Ub and I_UbD2_Ub, is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the I_UbD2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2_Ub-DNA complex. The I_UbD2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2_Ub-DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an I_UbD2_Ub-DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, I_UbD2 complex.

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相互依赖的 FANCI-FANCD2 泛素化的结构和生化基础

FANCI-FANCD2 (ID2) 复合物的双单泛素化是通过范可尼贫血途径修复 DNA 链间交联的核心和关键步骤。虽然 FANCD2 泛素化先于 FANCI 泛素化，但 FANCD2 的去泛素化速度也快于 FANCI，这会导致 FANCI 泛素化 ID2 复合体 (I _Ub D2)。在这里，我们展示了与双链 DNA 结合的I _Ub D2 复合物的 4.1 Å 冷冻电镜结构。我们表明，该复合物与 ID2 _Ub和 I _Ub D2 _Ub一样，也处于闭合的 ID2 构象中并夹在 DNA 上。FANCD2 (K561) 的目标赖氨酸在 I _{Ub中完全暴露}D2-DNA 结构，因此为泛素化做好准备。同样，FANCI 的目标赖氨酸 (K523) 也准备好在 ID2 _Ub -DNA 复合物中进行泛素化。I _Ub D2-DNA 复合物表现出去泛素化抗性，由 DNA 和 FANCD2 的存在赋予。另一方面，ID2 _{Ub -DNA 可以被 USP1-UAF1 有效去泛素化，除非 FANCI 发生进一步泛素化。}因此，FANCI 泛素化以两种方式有效地维持 FANCD2 泛素化：它防止 I _Ub D2 _Ub -DNA 复合物中过度的 FANCD2 去泛素化，并且它使 FANCD2 在瞬态、闭合 DNA I _Ub D2 复合物中重新泛素化。