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

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Nucleic Acids Research (Impact Factor: 9.11). 06/2012; 40(17):8425-39. DOI: 10.1093/nar/gks638
Source: PubMed


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.

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    • "First, the FA pathway is activated in vivo by a diverse range of DNA-damaging and replication-stress-inducing agents that are likely to elicit different DNA conformations, structures, and adducts (Garcia-Higuera et al., 2001; Howlett et al., 2005). Second , studies in Xenopus egg extracts reveal that an array of exogenous DNA substrates can induce FANCI/FANCD2 monoubiquitination (Rä schle et al., 2008; Sareen et al., 2012; Sobeck et al., 2007). Given that unstructured polyT had a reduced effect, structured and/or duplex DNA may be required to promote a conformational change that makes the K563 acceptor lysine in FANCD2 accessible. "
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    ABSTRACT: Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.
    Molecular Cell 06/2014; 54(5):858-869. DOI:10.1016/j.molcel.2014.05.001 · 14.02 Impact Factor
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    • "At this time, we cannot distinguish whether the lag represents a dependence of FANCI ubiquitination on prior FANCD2 ubiquitination or that the ID2 complex dissociates to release free FANCI at the later incubation time points. Nevertheless, we note that our in vitro results mirror those observed in Xenopus laevis cell-free systems in which FANCI monoubiquitination peaks at a much later time than FANCD2 modification (24,26). Moreover, we observed that FANCI monoubiquitination may be compromised by the FANCD2 K561R mutation, whereas the FANCI K523R mutation has little or no effect on FANCD2 modification. "
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    ABSTRACT: FANCD2 and FANCI function together in the Fanconi anemia network of deoxyribonucleic acid (DNA) crosslink repair. These proteins form the dimeric ID2 complex that binds DNA and becomes monoubiquitinated upon exposure of cells to DNA crosslinking agents. The monoubiquitinated ID2 complex is thought to facilitate DNA repair via recruitment of specific nucleases, translesion DNA polymerases and the homologous recombination machinery. Using the ubiquitin conjugating enzyme (E2) UBE2T and ubiquitin ligase (E3) FANCL, monoubiquitination of human FANCD2 and FANCI was examined. The ID2 complex is a poor substrate for monoubiquitination, consistent with the published crystal structure showing the solvent inaccessibility of the target lysines. Importantly, FANCD2 monoubiquitination within the ID2 complex is strongly stimulated by duplex or branched DNA, but unstructured single-stranded DNA or chromatinized DNA is ineffective. Interaction of FANCL with the ID2 complex is indispensable for its E3 ligase efficacy. Interestingly, mutations in FANCI that impair its DNA binding activity compromise DNA-stimulated FANCD2 monoubiquitination. Moreover, we demonstrate that in the absence of FANCD2, DNA also stimulates FANCI monoubiquitination, but in a FANCL-independent manner. These results implicate the role of a proper DNA ligand in FANCD2 and FANCI monoubiquitination, and reveal regulatory mechanisms that are dependent on protein-protein and protein-DNA interactions.
    Nucleic Acids Research 03/2014; 42(9). DOI:10.1093/nar/gku198 · 9.11 Impact Factor
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    • "One of Fanconi anemia (FA) proteins, FANCI forms a functional heterodimer by interacting with FANCD2 (Fanconi anemia group D2) and the complex is recruited to the branched DNA structures [36]. On the other hands, FANCI is dissociated from the complex and also functions individually during DNA repair [37]. BCL2-associated athanogene 2 (BAG2) plays a crucial role in cellular senescence in cancer cells by c-Myc-mediated regulation [38]. "
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    ABSTRACT: Twenty different aminoacyl-tRNA synthetases (ARSs) link each amino acid to their cognate tRNAs. Individual ARSs are also associated with various non-canonical activities involved in neuronal diseases, cancer and autoimmune diseases. Among them, eight ARSs (D, EP, I, K, L, M, Q and RARS), together with three ARS-interacting multifunctional proteins (AIMPs), are currently known to assemble the multi-synthetase complex (MSC). However, the cellular function and global topology of MSC remain unclear. In order to understand the complex interaction within MSC, we conducted affinity purification-mass spectrometry (AP-MS) using each of AIMP1, AIMP2 and KARS as a bait protein. Mass spectrometric data were funneled into SAINT software to distinguish true interactions from background contaminants. A total of 40, 134, 101 proteins in each bait scored over 0.9 of SAINT probability in HEK 293T cells. Complex-forming ARSs, such as DARS, EPRS, IARS, Kars, LARS, MARS, QARS and RARS, were constantly found to interact with each bait. Variants such as, AIMP2-DX2 and AIMP1 isoform 2 were found with specific peptides in KARS precipitates. Relative enrichment analysis of the mass spectrometric data demonstrated that TARSL2 (threonyl-tRNA synthetase like-2) was highly enriched with the ARS-core complex. The interaction was further confirmed by coimmunoprecipitation of TARSL2 with other ARS core-complex components. We suggest TARSL2 as a new component of ARS core-complex.
    PLoS ONE 12/2013; 8(12):e81734. DOI:10.1371/journal.pone.0081734 · 3.23 Impact Factor
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