Structural and biochemical basis of FANCI-FANCD2 interdependent ubiquitination

Abstract

The Fanconi Anaemia pathway operates for the repair of interstrand crosslinks and the maintenance 11 of genomic stability upon replication stalling. Di‐monoubiquitination of the FANCI‐FANCD2 (ID2) 12 complex is a central and crucial step in this pathway. Evidence suggests that FANCD2 ubiquitination 13 precedes FANCI ubiquitination, and that both the FANCD2‐ubiquitinated (ID2Ub) and the di14 monoubiquitinated (IUbD2Ub) complex clamp on DNA. However, FANCD2 is deubiquitinated at a 15 faster rate than FANCI, which can result in a FANCI‐ubiquitinated ID2 complex (IUbD2). Here, we 16 present a 4.1 Å cryo‐EM structure of IUbD2 complex bound to double‐stranded DNA. We show that 17 this complex, like ID2Ub and IUbD2Ub, is also in the closed ID2 conformation and clamps on DNA. 18 While the target lysine of FANCD2 (K561) is partially buried in the non‐ubiquitinated ID2‐DNA 19 complex, it becomes fully exposed in the IUbD2‐DNA structure, and thus can be ubiquitinated at a 20 faster rate. The IUbD2‐DNA complex cannot easily revert to the non‐ubiquitinated ID2 state, due to 21 USP1‐UAF1‐resistance, conferred by the presence of DNA and FANCD2. ID2Ub‐DNA, on the other 22 hand, can be efficiently deubiquitinated by USP1‐UAF1, unless further ubiquitination on FANCI 23 occurs.