Generation of Mouse FANCL Antibody and Analysis of FANCL Protein Expression Profile in Mouse Tissues (original) (raw)

Expression and purification of human FANCI and FANCD2 using Escherichia coli cells

Protein expression and purification, 2014

The DNA interstrand crosslink (ICL) is an extremely deleterious DNA lesion that covalently crosslinks complementary strands and prevents the strand-separation reaction. In higher eukaryotes, the Fanconi anemia proteins, FANCI and FANCD2, form a heterodimer and play essential roles in ICL repair. Human FANCI and FANCD2 are large proteins with molecular masses of 149kDa and 164kDa, respectively, and were reportedly purified using a baculovirus expression system with insect cells. We have established a novel expression and purification procedure for human FANCD2 and FANCI, using Escherichia coli cells. The human FANCD2 and FANCI proteins purified by this bacterial expression method formed a stable heterodimer, and exhibited DNA binding and histone chaperone activities, as previously reported for the proteins purified by the baculovirus system. Therefore, these purification methods for human FANCI and FANCD2 provide novel procedures to facilitate structural and biochemical studies of hu...

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...

Function of the Fanconi anemia pathway in Fanconi anemia complementation group F and D1 cells

Experimental Hematology, 2001

Objective. Fanconi anemia (FA) is a human autosomal-recessive cancer susceptibility disorder characterized by multiple congenital abnormalities, progressive bone marrow failure, and cellular sensitivity to mitomycin C (MMC). FA has at least eight complementation groups (A, B, C, D1, D2, E, F, G), and six of the FA genes have been cloned. Several FA proteins, including FANCA, FANCC, FANCF, and FANCG, interact in a nuclear complex, and this complex is required for the activation (monoubiquitination) of the downstream FANCD2 protein. Activation of FANCD2 results in the assembly of FANCD2/BRCA1 foci. The aim of this study was to analyze the FA pathway in several FA patient-derived cell lines.

The Fanconi Anemia Gene Product FANCF Is a Flexible Adaptor Protein

Journal of Biological Chemistry, 2004

The Fanconi anemia (FA) protein FANCF is an essential component of a nuclear core complex that protects the genome against chromosomal instability, but the specific function of FANCF is still poorly understood. Based upon the homology between human and Xenopus laevis FANCF, we carried out an extensive mutagenesis study to examine which domains are functionally important and to gain more insight into the function of FANCF. In contrast to previous suggestions, we show that FANCF does not have a ROM-like function. We found that the C terminus of FANCF interacts directly with FANCG and allows the assembly of other FA proteins into a stable complex. The N terminus appears to stabilize the interaction with FANCA and FANCG and is essential for the binding of the FANCC/FANCE subcomplex. We identified several important amino acids in this N-terminal region but, surprisingly, many amino acid changes failed to affect the function of the FANCF protein. Our data demonstrate that FANCF acts as a flexible adaptor protein that plays a key role in the proper assembly of the FA core complex.