Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase

Abstract

Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2–FANCI complex versus the monomeric proteins are. We show that the FANCD2–FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2–FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to—and independently of—FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase.

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

“…Conversely, a phosphorylationmimetic mutant failed to associate with FANCD2 and exhibited increased monoubiquitination. 106 Taken together, these findings indicate that phosphorylation of FANCI at the conserved S/TQ cluster is a key mechanism in the activation of the FA-BRCA pathway. The introduction of a cluster of negatively charged phosphate groups may result in a localized charge repulsion that promotes ID2 disassociation, enabling access of the previously occluded ubiquitin ligase machinery.…”

“…The latter model would more closely align with recent findings from the Sobeck laboratory indicating that activation of the FA-BRCA pathway coincides with dissociation of FANCD2 and FANCI. 74 ID2 dissociation is triggered by ATM/ATR-mediated phosphorylation of a cluster of at least 6 FANCI SQ/TQ motifs, and is followed by the monoubiquitination of FANCD2, see below. 33,74 The NEDD4 E3 ubiquitin ligase can mono-and polyubiquitinate substrates, and the balance between these 2 posttranslational modifications is determined, at least in part, by the nature of the enzyme-substrate interaction.…”

“…74 ID2 dissociation is triggered by ATM/ATR-mediated phosphorylation of a cluster of at least 6 FANCI SQ/TQ motifs, and is followed by the monoubiquitination of FANCD2, see below. 33,74 The NEDD4 E3 ubiquitin ligase can mono-and polyubiquitinate substrates, and the balance between these 2 posttranslational modifications is determined, at least in part, by the nature of the enzyme-substrate interaction. The tryptophan-tryptophan (WW) motif of NEDD4 can interact with relatively high affinity to a substrate PPxY (where x is any amino acid) motif, leading to substrate polyubiquitination.…”

“…Phosphorylation could also promote the interaction of FANCD2 with DNA in chromatin, which has been shown to lead to increased efficiency of monoubiquitination. 56,106 FANCI Phosphorylation FANCI was originally identified as an ATM/ATR substrate in a large-scale SILAC (stable isotope labeling with amino acids in cell culture) proteomics screen. 15 As described above, 6 S/TQ motifs are positioned proximal to K523, the sites of monoubiquitination site.…”

“…Recently, it has been suggested that activation of the pathway coincides with disassociation of the ID2 heterodimer, and that ID2 disassociation is triggered by ATM/ATR-mediated phosphorylation of the S/TQ cluster. 106 Using cell-free Xenopus laevis egg extracts, Sareen and colleagues demonstrated that a FANCI S/TQ cluster phosphorylationdead mutant failed to dissociate from FANCD2 and failed to undergo monoubiquitination. Conversely, a phosphorylationmimetic mutant failed to associate with FANCD2 and exhibited increased monoubiquitination.…”