Structural and biochemical basis of FANCI-FANCD2 interdependent ubiquitination

The Fanconi Anaemia pathway operates for the repair of interstrand crosslinks and the maintenance of genomic stability upon replication stalling. Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step in this pathway. Evidence suggests that FANCD2 ubiquitination precedes FANCI ubiquitination, and that both the FANCD2-ubiquitinated (ID2 _Ub ) and the di- monoubiquitinated (I _Ub D2 _Ub ) complex clamp on DNA. However, FANCD2 is deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (I _Ub D2). Here, we present a 4.1 Å cryo-EM structure of I _Ub D2 complex bound to double-stranded DNA. We show that this complex, like ID2 _Ub and I _Ub D2 _Ub , is also in the closed ID2 conformation and clamps on DNA. While the target lysine of FANCD2 (K561) is partially buried in the non-ubiquitinated ID2-DNA complex, it becomes fully exposed in the I _Ub D2-DNA structure, and thus can be ubiquitinated at a faster rate. The I _Ub D2-DNA complex cannot easily revert to the non-ubiquitinated ID2 state, due to USP1-UAF1-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. FANCI ubiquitination also progresses at a faster rate in ID2 _Ub -DNA over ID2-DNA complex, and results in partial DNA-dependent protection from FANCD2 deubiquitination. Taken together, our results suggest that, while FANCD2 ubiquitination promotes FANCI ubiquitination, FANCI ubiquitination in turn maintains FANCD2 ubiquitination by two mechanisms: it prevents excessive FANCD2 deubiquitination within an I _Ub D2 _Ub -DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, I _Ub D2 complex.