Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination.
Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination.
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DOI：
10.15252/embj.2022111898
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发表时间：
2023-02-01
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FANCI-FANCD 2（ID 2）复合物的二单泛素化是通过范可尼贫血途径修复DNA链间交联的中心和关键步骤。虽然FANCD 2泛素化先于FANCI泛素化，但FANCD 2也以比FANCI更快的速率去泛素化，这可能导致FANCI-泛素化ID 2复合物（IUbD 2）。在这里，我们展示了与双链DNA结合的IUbD 2复合物的4.1 μ cryo-EM结构。我们发现，这种复合物，像ID 2Ub和IUbD 2Ub，也是在封闭的ID 2构象和钳DNA。FANCD 2（K561）的靶赖氨酸完全暴露在IUbD 2-DNA结构中，因此引发泛素化。类似地，FANCI的靶赖氨酸（K523）也被引发用于ID 2Ub-DNA复合物中的泛素化。IUbD 2-DNA复合物表现出去泛素化抗性，这是由DNA和FANCD 2的存在所赋予的。另一方面，ID 2Ub-DNA可以被USP 1-UAF 1有效地去泛素化，除非FANCI上发生进一步的泛素化。因此，FANCI泛素化以两种方式有效地维持FANCD 2泛素化：它防止IUbD 2Ub-DNA复合物内过度的FANCD 2去泛素化，并且它使FANCD 2能够在瞬时的、封闭的DNA IUbD 2复合物内重新泛素化。由更快的FANCD 2去泛素化动力学产生的仅在FANCI上泛素化的瞬时复合物保持夹在DNA上并有利于FANCD 2再泛素化。
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 (IUbD2). Here, we present a 4.1 Å cryo‐EM structure of IUbD2 complex bound to double‐stranded DNA. We show that this complex, like ID2Ub and IUbD2Ub, is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the IUbD2‐DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2Ub‐DNA complex. The IUbD2‐DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2Ub‐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 IUbD2Ub‐DNA complex, and it enables re‐ubiquitination of FANCD2 within a transient, closed‐on‐DNA, IUbD2 complex. A transient complex ubiquitinated only on FANCI, resulting from faster FANCD2 deubiquitination kinetics, remains clamped on DNA and favors FANCD2 re‐ubiquitination.