FANCI is a second monoubiquitinated member of the Fanconi anemia pathway (original) (raw)

The Fanconi anemia pathway and ubiquitin

BMC Biochemistry, 2007

Fanconi anemia (FA) is a rare genetic disorder characterized by aplastic anemia, cancer/leukemia susceptibility and cellular hypersensitivity to DNA crosslinking agents, such as cisplatin. To date, 12 FA gene products have been identified, which cooperate in a common DNA damage-activated signaling pathway regulating DNA repair (the FA pathway). Eight FA proteins form a nuclear complex harboring E3 ubiquitin ligase activity (the FA core complex) that, in response to DNA damage, mediates the monoubiquitylation of the FA protein FANCD2. Monoubiquitylated FANCD2 colocalizes in nuclear foci with proteins involved in DNA repair, including BRCA1, FANCD1/BRCA2, FANCN/PALB2 and RAD51. All these factors are required for cellular resistance to DNA crosslinking agents. The inactivation of the FA pathway has also been observed in a wide variety of human cancers and is implicated in the sensitivity of cancer cells to DNA crosslinking agents. Drugs that inhibit the FA pathway may be useful chemosensitizers in the treatment of cancer.

Evidence for subcomplexes in the Fanconi anemia pathway

Blood, 2006

Fanconi anemia (FA) is a genomic instability disorder, clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. Cells derived from patients with FA display a marked sensitivity to DNA cross-linking agents, such as mitomycin C (MMC). This observation has led to the hypothesis that the proteins defective in FA are involved in the sensing or repair of interstrand cross-link lesions of the DNA. A nuclear complex consisting of a majority of the FA proteins plays a crucial role in this process and is required for the monoubiquitination of a downstream target, FANCD2. Two new FA genes, FANCB and FANCL, have recently been identified, and their discovery has allowed a more detailed study into the molecular architecture of the FA pathway. We demonstrate a direct interaction between FANCB and FANCL and that a complex of these proteins binds FANCA. The interaction between FANCA and FANCL is dependent on FANCB, FANCG, and FANCM, but...

Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein

2012

The Fanconi anemia (FA)-BRCA pathway is critical for the repair of DNA interstrand crosslinks (ICLs) and the maintenance of chromosome stability. A key step in FA-BRCA pathway activation is the covalent attachment of monoubiquitin to FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI localize in chromatin-associated nuclear foci where they interact with several well-characterized DNA repair proteins. Importantly, very little is known about the structure, function, and regulation of FANCD2. Herein, we describe the identification and characterization of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain (UBD) in FANCD2, and demonstrate that the CUE domain mediates noncovalent binding to ubiquitin in vitro. We show that while mutation of the CUE domain destabilizes FANCD2, the protein remains competent for DNA damage-inducible monoubiquitination and phosphorylation. Importantly, we demonstrate that the CUE domain is required for interaction with FANCI, retention of monoubiquitinated FANCD2 and FANCI in chromatin, and for efficient ICL repair. Our results suggest a model by which heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently attached to FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation. For personal use only. on October 4, 2017. by guest www.bloodjournal.org From FANCN/PALB2, FANCO/RAD51C and FANCP/SLX4. The FA proteins together with BRCA1 function cooperatively in the FA-BRCA pathway to repair damaged DNA and prevent cellular transformation. 2 Disruption of the FA-BRCA pathway leads to cellular hypersensitivity to the cytotoxic and clastogenic effects of DNA interstrand crosslinking agents. 3 The FA-BRCA pathway is activated following exposure to DNA damaging agents and during S-phase of the cell cycle. 4,5 Activation of the pathway occurs when the core FA complex comprised of FANCA,-B,-C,-E,-F,-G,-L, and-M, and other proteins, assembles in the nucleus and monoubiquitinates the paralogous proteins FANCD2 and FANCI. 4,6,7 FANCL, a RING domain-containing protein is the catalytic E3 ubiquitin ligase subunit of the FA core complex, while UBE2T is the E2 conjugating enzyme. 8-10 Monoubiquitination of FANCD2 and FANCI targets these proteins to discrete chromatin-associated nuclear foci, where they interact with several key DNA repair proteins, including BRCA1, FANCD1/BRCA2, and RAD51. 4,5,11 Following DNA repair, FANCD2 and FANCI are deubiquitinated by the USP1/UAF1 complex facilitating the release of these proteins from chromatin. 12,13 Recent studies indicate that FANCD2 monoubiquitination is necessary for the recruitment of the FAN1 and SLX4/FANCP endonucleases to sites of DNA damage. 14-19 Despite the critical functions of FANCD2 and FANCI in ICL repair, very little is known about their structure, function, and regulation. For personal use only. on October 4, 2017. by guest www.bloodjournal.org From fail to correct the MMC hypersensitivity of FA-D2 (FANCD2-/-) patient cells. Taken together, our results suggest that the heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently linked to K523 of FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation. Methods Cell culture and antibodies PD20 (FA-D2 (FANCD2-/-)), HeLa and COS-7 cells were grown in DMEM media supplemented with 12% v/v FBS, L-glutamine and penicillin/streptomycin. Stable FA-D2 cells were generated by infection with pMMP Moloney murine leukemia or pLenti6.2/V5-DEST (Invitrogen) virus harboring wild type or mutant FANCD2 cDNAs. 4,25 Stable cell lines were grown in DMEM media supplemented with either 1 μg/ml puromycin or 2 μg/ml blasticidin. The following antibodies were used: rabbit polyclonal antisera against FANCD2 (NB100-182; Novus Biologicals), FANCI (Dr. Patrick Sung, Yale University and A300-212A; Bethyl Laboratories), H2A (07-146; Millipore), and mouse monoclonal antisera against α-tubulin (MS-581-PO; Lab Vision) and V5 (R96025; Invitrogen). Immunofluorescence microscopy For immunofluorescence microscopy (IF) freely soluble cellular proteins were pre-extracted with 0.3% v/v Triton X-100 and cells fixed in 4% w/v paraformaldehyde and 2% w/v sucrose at 4°C followed by permeabilization in 0.3% v/v Triton X-100 in PBS. Fixed cells were blocked for 30 minutes in antibody dilution buffer (5% v/v goat serum, 0.1% v/v NP-40, in PBS) and incubated For personal use only. on October 4, 2017. by guest www.bloodjournal.org From 6 with primary antibody for 1 h. Cells were washed three times in PBS and incubated for 30 minutes at room temperature with an Alexa fluor 488-conjugated secondary antibody. Nuclear foci were analyzed using a Zeiss AxioImager.A1 upright epifluorescent microscope with AxioVision LE 4.6 image acquisition software. Immunoprecipitation Cells were lysed in NETN100 (20 mM Tris-HCl pH 7.4, 0.1% v/v NP-40, 100 mM NaCl, 1 mM EDTA, 1 mM Na 3 O 4 V, 1 mM NaF, supplemented with protease inhibitors), incubated on ice and sonicated briefly. 800 µg whole-cell lysates (WCL) were incubated with 3 µg of antibodies against FANCD2 (FI-17; Santa Cruz), V5 (R96025; Invitrogen) or mouse IgG (12-371B; Millipore). Plasmids and site-directed mutagenesis The FANCD2-P204A,-LP215AA, and-LL234AA cDNAs were generated by site-directed mutagenesis of the wild type FANCD2 cDNA using the Quikchange Site-directed Mutagenesis Kit (Stratagene). The forward and reverse oligonucleotide sequences used are as follows: P204A

On the role of FAN1 in Fanconi anemia

Blood

Fanconi anemia (FA) is a rare bone marrow failure disorder with defective DNA interstrand crosslink repair. Still, there are FA patients without mutations in any of the 15 genes individually underlying the disease. A candidate protein for those patients, FA nuclease 1 (FAN1), whose gene is located at chromosome 15q13.3, is recruited to stalled replication forks by binding to monoubiquitinated FANCD2 and is required for interstrand crosslink repair, suggesting that mutation of FAN1 may cause FA. Here we studied clinical, cellular, and genetic features in 4 patients carrying a homozygous 15q13.3 micro-deletion, including FAN1 and 6 additional genes. Biallelic deletion of the entire FAN1 gene was confirmed by failure of 3'- and 5'-PCR amplification. Western blot analysis failed to show FAN1 protein in the patients' cell lines. Chromosome fragility was normal in all 4 FAN1-deficient patients, although their cells showed mild sensitivity to mitomycin C in terms of cell surviv...

Monoubiquitylation in the Fanconi anemia DNA damage response pathway

DNA Repair, 2009

a b s t r a c t The hereditary genetic disorder Fanconi anemia (FA) belongs to the heterogeneous group of diseases associated with defective DNA damage repair. Recently, several reviews have discussed the FA pathway and its molecular players in the context of genome maintenance and tumor suppression mechanisms [H. Joenje, K.J. Patel, The emerging genetic and molecular basis of Fanconi anaemia, Nat. Rev. Genet. 2 (2001) 446-457; W. Wang, Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins, Nat. Rev. Genet. 8 (2007) 735-748; L.J. Niedernhofer, A.S. Lalai, J.H. Hoeijmakers, Fanconi anemia (cross)linked to DNA repair, Cell 123 (2005) 1191-1198; K.J. Patel, Fanconi anemia and breast cancer susceptibility, Nat. Genet. 39 ]. This review assesses the influence of posttranslational modification by ubiquitin. We review and extract the key features of the enzymatic cascade required for the monoubiquitylation of the FANCD2/FANCI complex and attempt to include recent findings into a coherent mechanism. As this part of the FA pathway is still far from fully understood, we raise several points that must be addressed in future studies.

Clinical severity in Fanconi anemia correlates with residual function of FANCB missense variants

2019

ABSTRACTFanconi anemia (FA) is the most common genetic cause of bone marrow failure, and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry (IFAR). Those with FANCB deletion or truncation demonstrate earlier than average onset of bone marrow failure, and more severe congenital abnormalities compared to a large series of FA individuals in the published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Ab...

Fanconi Anemia Proteins and Their Interacting Partners: A Molecular Puzzle

Anemia

In recent years, Fanconi anemia (FA) has been the subject of intense investigations, primarily in the DNA repair research field. Many discoveries have led to the notion of a canonical pathway, termed the FA pathway, where all FA proteins function sequentially in different protein complexes to repair DNA cross-link damages. Although a detailed architecture of this DNA cross-link repair pathway is emerging, the question of how a defective DNA cross-link repair process translates into the disease phenotype is unresolved. Other areas of research including oxidative metabolism, cell cycle progression, apoptosis, and transcriptional regulation have been studied in the context of FA, and some of these areas were investigated before the fervent enthusiasm in the DNA repair field. These other molecular mechanisms may also play an important role in the pathogenesis of this disease. In addition, several FA-interacting proteins have been identified with roles in these "other" nonrepai...