The Fanconi anemia ID2 complex: dueling saxes at the crossroads

Abstract

Fanconi anemia (FA) is a rare recessive genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility in early adulthood. FA is caused by biallelic germ-line mutation of any one of 16 genes. While several functions for the FA proteins have been ascribed, the prevailing hypothesis is that the FA proteins function cooperatively in the FA-BRCA pathway to repair damaged DNA. A pivotal step in the activation of the FA-BRCA pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Despite their importance for DNA repair, the domain structure, regulation, and function of FANCD2 and FANCI remain poorly understood. In this review, we provide an overview of our current understanding of FANCD2 and FANCI, with an emphasis on their posttranslational modification and common and unique functions.

Keywords: AML , acute myeloid leukemia; APC/C, anaphase-promoting complex/cyclosome; APH, aphidicolin; ARM, armadillo repeat domain; AT, ataxia-telangiectasia; ATM, ataxia-telangiectasia mutated; ATR, ATM and Rad3-related; BAC, bacterial-artificial-chromosome; BS, Bloom syndrome; CUE, coupling of ubiquitin conjugation to endoplasmic reticulum degradation; ChIP-seq, CHIP sequencing; CtBP, C-terminal binding protein; CtIP, CtBP-interacting protein; DNA interstrand crosslink repair; DNA repair; EPS15, epidermal growth factor receptor pathway substrate 15; FA, Fanconi anemia; FAN1, FANCD2-associated nuclease1; FANCD2; FANCI; FISH, fluorescence in situ hybridization; Fanconi anemia; HECT, homologous to E6-AP Carboxy Terminus; HJ, Holliday junction; HR, homologous recombination; MCM2-MCM7, minichromosome maintenance 2–7; MEFs, mouse embryonic fibroblasts; MMC, mitomycin C; MRN, MRE11/RAD50/NBS1; NLS, nuclear localization signal; PCNA, proliferating cell nuclear antigen; PIKK, phosphatidylinositol-3-OH-kinase-like family of protein kinases; PIP-box, PCNA-interacting protein motif; POL κ, DNA polymerase κ; RACE, rapid amplification of cDNA ends; RING, really interesting new gene; RTK, receptor tyrosine kinase; SCF, Skp1/Cullin/F-box protein complex; SCKL1, seckel syndrome; SILAC, stable isotope labeling with amino acids in cell culture; SLD1/SLD2, SUMO-like domains; SLIM, SUMO-like domain interacting motif; TIP60, 60 kDa Tat-interactive protein; TLS, Translesion DNA synthesis; UAF1, USP1-associated factor 1; UBD, ubiquitin-binding domain; UBZ, ubiquitin-binding zinc finger; UFB, ultra-fine DNA bridges; UIM, ubiquitin-interacting motif; ULD, ubiquitin-like domain; USP1, ubiquitin-specific protease 1; VRR-nuc, virus-type replication repair nuclease; iPOND, isolation of proteins on nascent DNA; ubiquitin.

Figure 1.

Domain architecture and structure of FANCD2 and FANCI. (A) Schematic of the FANCD2 protein indicating the amino-terminal NLS (nuclear localization signal) domain (green), CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) domain (maroon), PIP-box (PCNA-interacting protein motif) (orange), and the C-terminal EDGE motif (purple). Functionally-characterized phosphorylation sites (teal) and K561 monoubiquitination site (yellow) are indicated by small circles. (B) Schematic of the FANCI protein indicating the Leu (leucine zipper) domain (light blue), ARM (armadillo repeat) domain (pink), and C-terminal EDGE motif (purple) and NLS domain (green). The S/TQ motif (teal) and K523 site of monoubiquitination (yellow) are indicated by small circles. (C) Mouse Fanci-Fancd2 heterodimer crystal structure represented as both surface and ribbons with domains indicated. This structure was solved by the Pavletich group in 2011(PDB ID: 3S4W).

Figure 2.

Comparison of the SCF multi-subunit ubiquitin ligase protein complex and the FA core complex. (A) The Skp1/Cullin/F-box protein (SCF) complex includes the Cullin protein, which acts as a scaffold to bridge the catalytic E3 ubiquitin ligase RBX1 to the adaptor protein Skp1, and the F-box protein. The F-box protein recognizes and recruits the target protein for ubiquitination by the E2 ubiquitin-conjugating enzyme, UBC. (B) We propose that the FANCA protein is structurally analogous to Cullin, and may link the E3 ubiquitin ligase FANCL with the putative adaptor protein FANCC. FANCC has been shown to interact with both FANCA and FANCE, indicating that it may function analogously to Skp1. FANCE may be analogous to the F-box protein. FANCE is known to interact directly with FANCD2 and may facilitate its monoubiquitination of FANCL and UBE2T.

Figure 3.

Models for FANCD2 and FANCI monoubiquitination. The schematics depict several potential outcomes upon monoubiquitination of FANCD2 and FANCI, which would preclude further ubiquitination. (A) The ID2 heterodimer inactivation model. Following monoubiquitination, ID2 heterodimerization occurs and is stabilized through a noncovalent interaction between monoubiquitin covalently linked to FANCI K523 and the FANCD2 CUE domain. There is also possibly a reciprocal interaction between monoubiquitinated FANCD2 K561 and an UBD in the carboxy-terminus of FANCI, shielding FANCD2 from further ubiquitination. (B) The FANCD2 self-inactivation model. Monoubiquitination could promote an intramolecular association between ubiquitin covalently attached to K561 and the amino-terminal CUE domain, resulting in a closed conformation. (C) The E3 ubiquitin ligase dissociation model. Once FANCL is autoubiquitinated, the ubiquitin moiety may interact noncovalently with the CUE domain on FANCD2 enabling monoubiquitination of FANCD2 on K561. This interaction is predicted to be weak and short-lived, leading to rapid dissociation of FANCL and FANCD2, precluding further ubiquitination.