Structural and biochemical basis of interdependent FANCI‐FANCD2 ubiquitination

Abstract

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 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. The target lysine of FANCD2 (K561) becomes fully exposed in the I Ub D2‐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 Ub D2‐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 Ub D2 Ub ‐DNA complex, and it enables re‐ubiquitination of FANCD2 within a transient, closed‐on‐DNA, I Ub D2 complex. image Monoubiquitination of each subunit of the DNA‐clamping FANCI‐FANCD2 (ID2) complex is a central step in DNA interstrand crosslink repair via the Fanconi Anemia pathway. Here, structural and biochemical studies elucidate the interdependence of these two ubiquitination events. Ubiquitination on any of the two subunits of the FANCI‐FANCD2 complex transforms the complex into a DNA clamp and promotes ubiquitination of the other subunit. When ubiquitinated FANCI associates with DNA, both FANCI and FANCD2 are protected against deubiquitination by USP1‐UAF1. Ubiquitin conjugated to FANCI forms extensive interactions with FANCD2, which further protect the FANCI‐ubiquitinated FANCI‐FANCD2‐DNA complex from deubiquitination.