The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

SUMMARY Repair of interstrand crosslinks by the Fanconi anemia (FA) pathway requires both the 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.

Fanconi anemia (FA), the most common form of inherited bone marrow failure, predisposes to leukemia and solid tumors. FA is caused by the genetic disruption of a cellular pathway that repairs DNA interstrand crosslinks. The impaired function of this pathway, and the genetic instability that results, is considered the main pathogenic mechanism behind this disease. The identification of breast cancer susceptibility genes (for example, BRCA1/FANCS and BRCA2/FANCD1) as being major players in the FA pathway has led to a surge in molecular studies, resulting in the concept of the FA-BRCA pathway. In this review, we discuss recent advances in the molecular pathogenesis of FA from three viewpoints: i) new FA genes, ii) modifier pathways that influence the cellular and clinical phenotypes of FA and iii) non-canonical functions of FA genes that may drive disease progression independently of deficient DNA repair. Potential therapeutic approaches for FA that are relevant to each will also be proposed.