FAAP100 is a new intrinsic component of the FA core complex. (A) A silver-stained SDS gel showing the FA core complex purified by an FANCA antibody from HeLa nuclear extract (Meetei et al, 2003a). (B) Immunoblotting shows the presence of FAAP100 in the FANCA and FANCB immunoprecipitates. (C) Immunoblotting shows that the antibody against FAAP100 co-immunoprecipitated several FA core complex components. (D) Immunoblotting shows that the Superose 6 gel filtration profile of FAAP100 is coincidental with that of FANCL, and is somewhat different from that of FANCA and FANCG.

FAAP100 interacts with the FANCB and FANCL. (A) Mammalian two-hybrid assay shows direct interaction between FAAP100 and FANCB. BD-FAAP100 was cotransfected with AD-FA proteins and a luciferase reporter construct to test for direct interactions in HEK 293 cells. Fold induction of luciferase expression is relative to FAAP100 alone. Each experimental data set was obtained in triplicate. (B) Mammalian three-hybrid assay shows that the interaction between FAAP100 and FANCL depends on the presence of FANCB. HEK 293 cells stably expressing FLAG-FANCB were cotransfected with the indicated protein pairs and assayed for luciferase activation. Fold induction relative to reporter gene activation when full-length FAAP100 is expressed alone. Each experimental data set was obtained in triplicate. (C, D) Silver-stained SDS gels showing HF-FANCL-associated polypeptides purified from either cytosol (C), or nuclear extract (D), of HeLa cells by tandem-affinity purification. The major polypeptides identified by mass spectrometry are indicated by arrows. The ‘MOCK' lanes have control samples that were obtained by purification using HeLa cells that do not express HF-FANCL. The salt concentration of the washing buffer (250 or 500 mM) used in affinity purification was indicated.

FAAP100 is essential for FANCD2 monoubiquitination and the stability of the FA core complex. (A) Immunoblotting shows that HeLa cells depleted of FAAP100 by two different siRNA oligos not only have reduced levels of FAAP100, but also of several other FA core complex components, including FANCB, FANCL, FANCA, and FANCG. HeLa cells transfected with a scrambled siRNA oligo are shown as control. The image of FAAP100 depletion (top panel) was reproduced from the lanes 1–3 of the top panel in (B), to allow easy comparison of the reduction of FAAP100 protein level with those of other FA proteins. (B, C) Immunoblotting shows that HeLa cells depleted of FAAP100 display reduced level of monoubiquitinated FANCD2 in cells that are either untreated (−), or treated with mitomycin C (MMC), cisplatin, or hydroxyurea (+). The monoubiquitinated and non-ubiquitinated FANCD2 proteins are indicated by FANCD2-L and FANCD2-S, respectively. (D) Immunoblotting shows that HeLa cells depleted of FAAP100 by On-Targetplus SMARTpool siRNAs display a level of monoubiquitinated FANCD2 comparable to cells depleted of FANCM or FANCL (compare the relative ratio between FANCD2-L and FANCD2-S in lane 6 with those in lanes 3 and 4). In addition, cells depleted by On-Target siRNAs have lower levels of FAAP100 and monoubiquitinated FANCD2 (compare lane 6 with lane 2). A nonspecific polypeptide was marked with an asterisk, which was resolved from FAAP100 using a 6% SDS gel. (E) Immunoblotting shows that HeLa cells depleted of FAAP100 by On-Targetplus SMARTpool siRNAs (siFAAP100) reduced monoubiquitination of FANCD2 in the presence of several DNA-damaging drugs. Note that the FAAP100 level in depleted cells is similar that of a nonspecific polypeptide (marked by an asterisk), indicating that its level is very low and close to the detection limit of the antibody. The cell lysis buffer in (A), (B) and (C) includes 8 M urea, which allows more efficient extraction of FANCD2-L and FANCB. The cell lysis buffer in (D) and (E), and those used in previous studies of FANCL, FANCB, and FANCM, do not include urea. Thus, the results in (D) and (E) should be more suitable for comparison with the previous studies.

FAAP100 is unstable in FA patient cells deficient of FANCB or FANCL, and its nuclear localization is defective in cells deficient of FANCA or FANCM. (A) Immunoblotting shows that the FAAP100 protein level is reduced in nuclear (NE) and cytoplasmic (cyt) extracts of two different FA patient cell lines deficient of FANCB (FA-B). A cell line from a normal individual, HSC93, was included as a wild-type control (WT). BAF57, a component of SWI/SNF chromatin remodeling complex, was used as a loading control. (B) Immunoblotting shows that the FAAP100 protein level is reduced in the FA patient cell line deficient of FANCL (FA-L). Its level is increased when the same cell line was complemented by reintroduction of HF-FANCL (‘WT') (Supplementary Figure 1). ATR was used as a control for nuclear extract and a crossreactive band was used as a loading control. (C) Immunoblotting shows that the level of FAAP100 is strongly reduced in the nuclear extract of a FA patient cell line (HSC72) deficient of FANCA (FA-A). Importantly, in the same cell line complemented with expression of exogenous FANCA (HSC72+FANCA), the level of FAAP100 in nuclear extract becomes normal. (D) Immunoblotting reveals that the level of FAAP100 is reduced in the nuclear extract of a FA patient cell line (EUFA867) deficient of FANCM. BAF57 and caspase 3 are loading controls for nuclear and cytosolic extracts, respectively.

Chicken DT40 cells in which the FAAP100 gene was inactivated display hallmark features of FA cells. (A) Immunoblotting shows that DT40 cells nullizygous for FAAP100 have no detectable monoubiquitinated FANCD2. The whole-cell lysates prepared from wild-type (WT) and FANCL-deficient (fancl) cells were included as positive and negative controls, respectively. Cells were either treated with MMC (500 ng/ml) for 6 h or left untreated. (B, C) The colony survival assays show that faap100-null cells are hypersensitive to DNA crosslinking drugs cisplatin and MMC. The percentage of the surviving colonies following treatment with the drugs at the indicated concentrations are shown. Two clones of faap100 cells were included in the analysis. The data shown are mean±standard deviation of at least three separate experiments. Wild-type and FANCC-knockout (fancc) cells were used as controls. (D) The chromosomal breakage analysis revealed that faap100-null cells have increased chromosomal abberations. Wild-type and two clones of faap100 cells were treated with MMC (20 or 40 ng/ml) for 24 h or left untreated. Chromosome aberrations were counted blindly using coded slides as described (Yamamoto et al, 2003). The counting was performed only on large chromosomes (total 12). Error bars represent standard error of total aberrations per metaphase. At least 150 (for wild-type) or 50 cells (for faap100) were scored for each preparation.

A hypothetical model for association of four subcomplexes in the FA core complex. The four predicted subcomplexes are indicated by different colors. FAAP24 is a new component of the FA core complex that interacts with the C-terminus of FANCM and may target FANCM to structured DNA that mimics intermediates formed during the replication or repair of damaged DNA (Ciccia et al, 2007). FANCD2 (red) and the E2 ubiquitin conjugating enzyme UBE2T (pink) may interact with FANCE (Pace et al, 2002) and FANCL (Machida et al, 2006), respectively.