Ubiquitination-Linked Phosphorylation of the FANCI S/TQ Cluster Contributes to Activation of the Fanconi Anemia I/D2 Complex

Abstract

Summary: Repair of interstrand crosslinks by the Fanconi anemia (FA) pathway requires both monoubiquitination and de-ubiquitination of the FANCI/FANCD2 (FANCI/D2) complex. In the standing model, the phosphorylation of six sites in the FANCI S/TQ cluster domain occurs upstream of, and promotes, FANCI/D2 monoubiquitination. We generated phospho-specific antibodies against three different S/TQ cluster sites (serines 556, 559, and 565) on human FANCI and found that, in contrast to the standing model, distinct FANCI sites were phosphorylated either predominantly upstream (ubiquitination independent; serine 556) or downstream (ubiquitination-linked; serines 559 and 565) of FANCI/D2 monoubiquitination. Ubiquitination-linked FANCI phosphorylation inhibited FANCD2 de-ubiquitination and bypassed the need to de-ubiquitinate FANCD2 to achieve effective interstrand crosslink repair. USP1 depletion suppressed ubiquitination-linked FANCI phosphorylation despite increasing FANCI/D2 monoubiquitination, providing an explanation of why FANCD2 de-ubiquitination is important for function of the FA pathway. Our work results in a refined model of how FANCI phosphorylation activates the FANCI/D2 complex. : How the Fanconi anemia pathway is activated to repair interstrand crosslinks is incompletely understood. Cheung et al. use FANCI-phosphorylation-specific antibodies to identify ubiquitination-linked FANCI phosphorylation. Ubiquitination-linked FANCI phosphorylation fully activates the FANCI/D2 complex so that it can function without needing to undergo de-ubiquitination. Keywords: Fanconi anemia, DNA repair, FANCD2, FANCI, USP1, phosphorylation, monoubiquitination, interstrand crosslink, DNA damage response, ATR