Fanconi anemia (FA) binding protein FAAP20 stabilizesFA complementation group A (FANCA) and participatesin interstrand cross-link repairJustin Wai Chung Leunga, Yucai Wanga, Ka Wing Fonga, Michael Shing Yan Huenb, Lei Lia, and Junjie Chena,1

aDepartment of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030; and bGenome Stability ResearchLaboratory, Department of Anatomy, University of Hong Kong, Hong Kong Special Administrative Region, China

Edited* by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved February 6, 2012 (received for review November 13, 2011)

The Fanconi anemia (FA) pathway participates in interstrand cross-link (ICL) repair and the maintenance of genomic stability. The FAcore complex consists of eight FA proteins and two Fanconi anemia-associated proteins (FAAP24 and FAAP100). The FA core complexhas ubiquitin ligase activity responsible for monoubiquitination ofthe FANCI-FANCD2 (ID) complex, which in turn initiates a cascade ofbiochemical events that allow processing and removal of cross-linked DNA and thereby promotes cell survival following DNAdamage. Here, we report the identification of a unique componentof the FA core complex, namely, FAAP20, which contains a RAD18-like ubiquitin-binding zinc-finger domain. Our data suggest thatFAAP20 promotes the functional integrity of the FA core complexvia its direct interaction with the FA gene product, FANCA. Indeed,somatic knockout cells devoid of FAAP20 displayed the hallmarksof FA cells, including hypersensitivity to DNA cross-linking agents,chromosome aberrations, and reduced FANCD2 monoubiquitina-tion. Taking these data together, our study indicates that FAAP20is an important player involved in the FA pathway.

mitomycin C | DNA repair | foci

Fanconi anemia (FA) is a rare recessive genetic disorder char-acterized by bone marrow failure, congenital developmental

defects, and cancer predisposition (1–4). Cellular features of FAinclude chromosomal instability and hypersensitivity to cross-linking agents (5). Fifteen FA complementation genes have beenidentified so far. These genes form several complexes to orches-trate interstrand cross-linking (ICL) repair. The FA core complexis composed of eight of the FA gene products (FANCA, FANCB,FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM), inaddition to FAAP24 and FAAP100 (6, 7), and acts as an E3 ligaseto ubiquitinate FANCI/FANCD2 (I/D2) complex (8–11). Themonoubiquitinated FANCI/FANCD2 complex interacts withFanconi anemia-associated nuclease 1 (FAN1), which has exo-nuclease and endonuclease activity that may unhook the ICL,facilitate translesion synthesis, and promote downstream homol-ogous recombination (HR) repair (12–15).Besides the FA core complex and FANCI/FANCD2, there are

several other FA proteins that likely act downstream of FANCI/FANCD2 and participate in HR repair. These proteins includeBRCA2 (FANCD1) and PALB2 (FANCN), both of which areessential for HR repair (16–18). Another downstream FA pro-tein, BACH1 (FANCJ), is also a bona fide double-strand breakrepair factor (19). BRCA2/FANCD1, PALB2/FANCN, andBACH1/FANCJ are all recruited to ICL sites (20), indicating thatthey are directly involved in ICL repair. More recently, mutationsin two other genes, RAD51C/FANCO and SLX4/FANCP, wereidentified in patients with FA phenotypes (21–23), suggesting thatthere may be additional FA genes responsible for this disease.Not all FA proteins function in a linear pathway involved in ICL

repair. Many of the downstream FA proteins are involved in HRrepair and associated with breast cancer susceptibility (16–19, 24,25). These proteins all have functions besides ICL repair. Morerecently, anotherDNA repair protein, RAD18, which is best knownfor its role in UV lesion bypass, has also been shown to participatein the activation of the FA pathway via its ability to promote

proliferating cell nuclear antigen (PCNA) monoubiquitination(26). It remains to be resolved how PCNA monoubiquitination islinked with the activation of the FA pathway.Among all of the FA genes, mutations in FANCA (∼60%),

FANCC (∼14%), and FANCG (∼10%) account for over 80% ofthe mutations identified in patients (27). However, FANCA,FANCC, and FANCG are orphan proteins that do not shareextensive sequence homology with other proteins. Thus, it is stillunknown how these proteins function in the FA pathway. Wereason that the identification of new FA-associated proteins mayhelp us understand how these orphan proteins participate inDNA repair. In this study, we report the identification of C1orf86isoform2 as a previously undescribed FANCA-interacting protein(Fanconi anemia-associated protein 20 kDa, hereafter referred asFAAP20). Genetic inactivation of FAAP20 revealed many fea-tures of FA cells, highlighting that FAAP20 is a key component ofthe FA core complex and participates in ICL repair.

ResultsFAAP20 Is a Unique Component of the FA Core Complex. We per-formed tandem affinity purification (TAP) using FANCA as bait toidentify FANCA-associated proteins. After excluding general con-taminants, such as heat-shock proteins and ribosomal proteins, weidentifiedFAAP20 as a potential FANCA-binding partner (Fig. 1A).FAAP20 (LOC1999990 isoform 2) encodes a 20-kDa protein withunknown function (genename:C1orf86 isoform2; accessionnumber:NP_872339.2). To confirm its association with FANCA, we per-formed a reverse purification using FAAP20 as bait and showed thatFANCAand FANCG copurified with FAAP20 (Fig. 1A), indicatingthat FAAP20 is a potential component of the FA core complex.

FAAP20 Binds Directly to FANCA. To verify that FAAP20 interactswith FANCA, we coexpressed triple-tagged (SFB-tag: S-protein tag,FLAG epitope, tag and streptavidin-binding peptide tag) FANCAwith Myc-tagged FAAP20 or FANCG. As expected, we observeda robust binding of SFB-tagged FANCA with Myc-tagged FANCG.In addition, we found a strong interaction between FANCA andFAAP20 (Fig. 1B), suggesting that they exist in the same complex.Moreover, we showed that endogenous FANCA coimmunopreci-pitated with endogenous FAAP20 and this interaction occurs in-dependently of mitomycin C (MMC) treatment (Fig. 1C).We generated a series of internal deletion mutants of FANCA

(FANCA-D1 to FANCA-D5) (Fig. 1D) and observed that two ofthem, FANCA-D3 and FANCA-D4, failed to interact withFAAP20 (Fig. 1E), indicating that FAPP20 binds to the middleregion of FANCA. In addition, we performed a pull-down assayusing bacterial expressed GST-fused FAAP20 and maltose-

Author contributions: J.W.C.L., M.S.Y.H., L.L., and J.C. designed research; J.W.C.L., Y.W.,M.S.Y.H., and K.W.F. performed research; J.W.C.L., Y.W., K.W.F., M.S.Y.H., L.L., and J.C.analyzed data; and J.W.C.L., Y.W., K.W.F., M.S.Y.H., L.L., and J.C. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: jchen8@mdanderson.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1118720109/-/DCSupplemental.

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binding protein (MBP)-fused FANCA (aa674-1208) orMBP-fusedFANCG. GST-FAAP20 pulled down MBP-fused FANCA (resi-dues 674–1208), but not MBP-fused FANCG (Fig. 1F), suggestingthat FAAP20 binds directly to FANCA, but not to FANCG.We then used a series of FAAP20 internal-deletion mutants to

map a FANCA-binding region on FAAP20 (Fig. 2A). Althoughwild-type FAAP20 coprecipitated with FANCA, two N-terminaldeletion mutants of FAAP20 (FAAP20-D1 and FAAP20-D2)failed to do so (Fig. 2B). To further narrow down the residues onFAAP20 that are responsible for FAAP20-FANCA interaction,we aligned the human FAAP20 sequence with those of FAAP20from other species and noted three highly conserved motifs atthe N terminus of FAAP20 (residues 40–45, residues 76–81, andresidues 83–87). Thus, we constructed three alanine substitutionmutants, 6A1 (40WAELLR/AAAAAA45), 6A2 (76EVFTVG/AAAAAA81), and 6A3 (83 KTFSWT/AAAAAA87), to disrupteach of these conserved motifs (Fig. 2C), respectively, and ex-amined their ability to interact with FANCA. The 6A1 mutantcompletely abolished the interaction between FAAP20 and

FANCA (Fig. 2D), suggesting that this N-terminal motif withinFAAP20 is necessary for FAAP20-FANCA interaction.

FAAP20 Contains an Evolutionarily Conserved RAD18-like Ubiquitin-Binding Zinc-Finger Domain. We noticed that FAAP20 contains anevolutionarily conserved ubiquitin-binding zinc-finger (UBZ) 4-typedomain that belongs to the RAD18 family of zinc-finger (ZNF)domains (Fig. 3A). This domain is also present in other DNAdamage-repair proteins, such as FAN1, RAD18, and SLX4/FANCP, which are involved in the FA pathway (Fig. 3B). Promptedby the ability of these zinc-finger domains in recognizing ubiquitin,we examined whether FAAP20 would also bind to ubiquitinthrough its UBZ domain. Indeed, GST-FAAP20 was able to pulldown endogenous ubiquitin chains, whereasGST alone or theGST-FAAP20 C147/150A mutant failed to do so (Fig. 3C). FAAP20interacted with both K48- and K63-linked ubiquitin chains withoutany notable preference (Fig. 3D). Given that monoubiquitinatedFANCD2 is important for the function of the FA pathway (9), wetested but did not observe any appreciable binding of FAAP20 with

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Fig. 1. FAAP20 is a FANCA-binding protein. (A) 293T cellsstably expressed SFB-FANCA or SFB-FAAP20, respectively,were subjected to TAP and mass spectrometry analysis. Redindicates the bait protein and blue indicates the known orputative-associated proteins. Number of peptides recoveredfrom mass spectrometry analysis is also presented. (B) 293Tcells were transfected with constructs encoding SFB-FANCAalong with constructs encoding Myc-FANCG or Myc-FAAP20.Coprecipitation and immunoblotting were carried out asindicated. (C) Lysates prepared from control or MMC-treated293T cells were incubated with protein A agarose beadsconjugated with rabbit IgG or anti-FAAP20 antibodies.Western blotting was performed using indicated antibodies.(D) Schematic illustration of wild-type and deletion mutantsof FANCA used in this study. (E) 293T cells were transfectedwith constructs encoding SFB-FAAP20 along with constructsencoding Myc-tagged wild-type or deletion mutants ofFAAP20. Precipitation and immunoblotting were conductedas indicated. (F) Pull-down assays were performed usingbacterially expressed and purified GST-FAAP20 and MBP-fused FANCA (residues 674–1032) or FANCG. Immunoblot-ting were conducted using anti-MBP antibody.

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Fig. 2. A conserved N-terminal region of FAAP20 is required for its interaction with FANCA. (A) Illustration of deletion mutants of FAAP20 used in this study.(B and D) 293T cells were transfected with plasmids encoding SFB-FANCA along with those encoding Myc-tagged wild-type or mutants of FAAP20. Pre-cipitation was conducted using streptavidin beads and immunoblotting was performed using anti-Flag or anti-Myc antibodies as indicated. (C) Alignment ofthe N terminus of FAAP20 from different species. The conserved amino acids are shaded in black. Three mutants of FAAP20 with six alanine substitutions weregenerated (6A1, 6A2, and 6A3).

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monoubiquitinated FANCD2 in vitro, whereas the FAN1-UBZ(residues 1–100) domain was able to do so (Fig. 3E). We also ex-amined whether or not FAAP20 would bind to monoubiquitin. Inthis regard, we performed an in vitro pull-down assay using bacte-rial-expressed carboxyl terminal-tagged ubiquitin (ub-GST). Weobserved that MBP-FAAP20 and MBP-FAN1-UBZ interactedwith ub-GST, but MBP or MBP-FAAP20-C147/150A did not (Fig.3F). Thus, although FAAP20 contains a ubiquitin-binding domain,the physiological partners of this UBZ domain remain unknown.

FAAP20 Is Required for ICL Repair. To further substantiate the roleof FAAP20 in the FA pathway, we used the eChIP assay with adefined ICL (20) to test whether FAAP20 localize to ICL sites(Fig. 4A). Indeed, we observed a fourfold enrichment of FAAP20at cross-linked substrates (Fig. 4A), suggesting that FAAP20 isrecruited to ICLs in vivo. As a positive control, FANCD2 was alsoenriched at ICL substrates.To study the function of FAAP20 in the FA pathway and ICL

repair, we transiently knocked down FAAP20 inU2OS using siRNAand examined FANCD2 monoubiquitnation, as well as FANCD2foci formation followingMMCtreatment (Fig. S1A).U2OScellswithFAAP20 down-regulation showed a reduction of FANCD2 mono-ubiquitination and FANCD2 foci formation (Fig. S1). However,monoubiquitnation of FANCD2 was not absent in FAAP20 knock-down cells. This finding could be because of incomplete knockdownof FAAP20 by siRNA. Alternatively, this finding may suggest thatFAAP20 is not as important as other FA core components.To test whether FAAP20 is essential for the activation of the FA

pathway, we decided to generate a FAAP20-deficient cell line inHCT116 colon carcinomas and a control FANCL-deficientHCT116-derivative cell line. After targeting both alleles with virus vectorscontaining homology arms of exon 2 and 3 of FAAP20 and exon 2and 3 of FANCL, we screened for clones with the correctly targetedalleles by PCR analysis (Fig. S2) and confirmed the absence ofFAAP20 or FANCL in these cell lines byWestern blotting (Fig. 4C).Because the FA pathway is important for ICL repair, de-

pletion of any of the FA proteins leads to hypersensitivity toDNA cross-linking agents. As expected, we observed that bothFAAP20- and FANCL-deficient cells exhibited enhanced sensi-tivity to MMC. It is worth noting that FAAP20-deficient cellsshowed lower sensitivity than FANCL-deficient cells (Fig. 4B).

The FA core complex acts as an ubiquitin ligase that mono-ubiquitinates the FANCI/FANCD2 complex during S phase andalso upon exposure to cross-linking agents. Although only FANCLhas intrinsic E3 ligase activity, depletion of any component of the FAcore complex compromises FANCI/FAND2 monoubiquitination(1, 8, 28). Thus, monoubiquitination of FANCD2 has been used asa surrogate marker for the integrity or the activation of the FApathway. Because FAAP20 is a FANCA-associated protein, wereasoned that FAAP20-deficient cells might display defect inFANCD2 ubiquitination. Western blotting analysis revealed thepresence of both unmodified and monoubiquitinated forms ofFANCD2 in parental HCT116 cells, and an increase of mono-ubiquitinated FANCI and FANCD2 after MMC treatment (Fig.4C). In contrast, ubiquitination of FANCI and FANCD2 waslargely abrogated in FAAP20- and FANCL-deficient cells (Fig.4C), suggesting that like FANCL, FAAP20 is also a component ofthe FA core complex and contributes to the activation of the FApathway following MMC treatment. Interestingly, we observed areduction in FANCA protein level in FAAP20-deficient cells(Fig. 4C), indicating that as a FANCA-binding protein, FAAP20may stabilize FANCA in the cell. In addition to reducedFANCD2 monoubiquitnation, FAAP20-deficient cells also dis-played defective FANCD2 foci formation (Fig. 4 D and E).Moreover, increased G2/M accumulation and genomic instability,including radial chromosome formation and chromosome breaks,are also the hallmarks of FA cells (29). As a matter of fact, weobserved all of these phenotypes in FAAP20-deficient cells (Fig. 4G and H). These data further validated that FAAP20 is a com-ponent of the FA pathway.

Binding to FANCA Is Required for FAAP20 Function in the FA Pathway.We reconstituted FAAP20-deficient cells with wild-type FAAP20and observed that the FANCA level, MMC hypersensivity, andMMC-induced FANCD2 monoubiquitination and foci formationwere all restored (Fig. 5). These data strongly suggest that thedefects observed in FAAP20-deficient cells are a result of the lossof FAAP20 in these cells.To understand whether the FANCA-binding activity and the

UBZ domain of FAAP20 are important for its function in ICLrepair, we also reconstituted the FAAP20-deficient cells withFAAP20-6A1 and FAAP20-C147/150A mutants, respectively. Asexpected, FAAP20-6A1 could not restore FANCA level and thus

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this mutant of FAAP20 could not rescue MMC sensitivity orMMC-induced FANCD2 monoubiquitination and foci formationin FAAP20-deficient cells (Fig. 5). On the other hand, the C147/150A mutant of FAAP20 rescued all of the above defects ob-served in FAAP20-deficient cells (Fig. 5). Taken together, thesedata indicate that FANCA-binding, but not its UBZ domain, iscritical for FAAP20 function in ICL repair.

DiscussionIn this study, we identified FAAP20 as a unique component ofthe FA core complex. FAAP20 binds directly to FANCA andstabilizes FANCA in the cell. The FANCA binding is essentialfor its function in the FA pathway, which is at least one mech-anism of how it functions in the FA core complex.The FA core complex comprises several subcomplexes, including

FANCL-FANCB-FAAP100 (4), FANCC-FANCE-FANCF (30),FANCA-FANCG (31, 32), and FANCM-FAAP24 (33). Although

it is known that the loss of any component in the FA core complexwould lead to similar defects in FANCD2 ubiquitination andMMCsensitivity, it is not yet clear how these subcomplexes function to-gether. An early study indicates that FANCAand FANCG stabilizeeach other and promote the nuclear localization of the FA corecomplex (34). This finding is similar to the situation in the presentstudy. FANCA and FANCG consistently copurified with FAAP20(Fig. 1A), suggesting that these three components likely forma stable subcomplex. The destabilization of FANCA in FAAP20-deficient cells could, in part, contribute to the defects observed inthese cells. It is noteworthy to point out that unlike other FA cells,residual FANCD2monoubiquitination was detectable in FAAP20-deficient cells. This phenotype is similar to that observed inFANCMΔ2/Δ2 mouse embyrionic fibroblasts (35). The significanceof this observation is currently unclear.FAAP20 contains a RAD18-like ZNF domain that binds to

ubiquitin. At least threeRAD18UBZdomain-containing proteins,

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Fig. 4. FAAP20 deficiency sensitizes cells to ICL damage and leads to genomic instability. (A) Schematic representation of the plasmid substrates used in theeChIP assay. The presence of psoralen-ICL and PCR primer locations are indicated. Relative enrichment of FAAP20 at ICLs was calculated by normalizing com-parative concentration from real-time PCRof each sample against that of its input. Error bars represent SD from three independent experiments. CTL, control; XL,cross-linked. (B) Clonogenic survival assay of HCT116 cells, FANCL-deficient cells, and FAAP20-deficient cells following MMC treatment. (C) Whole-cell extractswere prepared from HCT116 cells, FAAP20-deficient cells, or FANCL-deficient cells mock-treated or treated with MMC for 24 h. Western blotting was conductedusing indicated antibodies. (D) HCT116 cells, FAAP20-deficient cells, and FANCL-deficient cells were mock-treated or treated with 1 μM MMC for 24 h. Immu-nostaining was performed using anti-FANCD2 antibody and cells were counterstained with DAPI, as indicated. (Magnification: 100×.) (E) Quantification resultswere the average of two independent experiments and were presented as mean ± SEM. More than 300 cells were counted in each experiment. (F) HCT116 cellsor FAAP20-deficient cells were exposed to a lowdose ofMMCand then treatedwith colcemid. A representativemicrograph shows radial chromosome formationand chromosome breaks marked by arrows that were observed in FAAP20-deficient cells. (Magnification: 100×.) (G) Quantification of chromosome aberrationwere the average of two independent experiments using wild-type, FAAP20−/− cells, and FANCL−/− cells. The data were presented as mean ± SEM. (H) HCT116cells or FAAP20-deficient cells were mock-treated or treated with 50 nMMMC. Cell-cycle distributions were analyzed by FACS and presented as percentages ofcells in G1, S, or G2/M phases.

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RAD18, FAN1, and SLX4/FANCP, are known to play critical rolesin the FA pathway (12–15, 21, 22, 26, 36, 37). In particular, theUBZ domain of FAN1 binds directly to monoubiquitinatedFANCD2 and thus recruits FAN1 to ICL sites to carry out itsfunction in ICL repair (12–15). However, in the case of FAAP20,we showed that the UBZ domain of FAAP20 is not critical forFANCD2monoubiquitination andMMC sensitivity. Further studyis required to elucidate the cellular function of this highly con-served UBZ domain of FAAP20.In conclusion, our data suggest that FAAP20 interacts with

FANCA and participates in the regulation of the FA pathway. Itis likely that via stabilizing FANCA, FAAP20 modulates theubiquitin ligase activity of the FA core complex, which in turnregulates the FANCI/FANCD2 monoubiquitination followingDNA damage. Up to now, no patient was found having mutationwith several genes encoding FA pathway-related proteins (in-cluding FAN1, FAAP100, and FAAP24). Because the cellularphenotypes of FAAP20-deficient cells are rather mild comparedwith FANCL-deficient cells, it is possible that patients withFAAP20 mutation would display mild FA phenotypes and thusbe difficult to diagnose. Knocking out the FAAP20 gene in micemay provide some clues to the function of this protein inmammals and may help the diagnosis of these patients.

Materials and MethodsPlasmids. FAAP20, FANCG, FANCA, and FAN1 cDNAs were purchased fromOpen Biosystems. The cDNAs were cloned into the pDONR201 vector usingGateway cloning technology (Invitrogen). All of the deletions and point mu-tations were generated by site-directedmutagenesis using standard protocols.The corresponding entry vectors were transferred into a Gateway-compatibledestination vector harboring an N-terminal triple-tag (S-protein, Flag, and astreptavidin-binding peptide), HA-Flag epitope tag, or a myc epitope tag for

expression in mammalian cells, and GST or MBP tag for expression in bacteria,respectively.

TAP of Protein Complexes. TAP was performed as previously described (38).Briefly, 293T cells were transfected with constructs encoding SFB-taggedFANCA or FAAP20 and selected with media containing puromycin (2 μg/mL).Cell lines stably expressing these tagged proteins were confirmed by West-ern blotting and immunofluorescence staining. For TAP, cells were lysed inNETN buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% Non-idet P-40, 1 mM MgCl2) for 20 min in 4 8C. The crude lysates were cleared bycentrifugation at 18,407 × g (Eppendorf 5424, Hamburg, Germany) at 4 8Cfor 30 min and rocked with streptavidin-conjugated beads (Amersham) for2 h at 4 8C. The immunocomplexes were washed with NETN three times andeluted with 2 mg/mL biotin. The eluent was then incubated with S-proteinAgarose beads (Novagen) for 2 h at 4 8C. The beads were then washed threetimes. The protein mixtures were eluted and analyzed by the Taplin MassSpectrometry Facility at Harvard Medical School (Boston, MA).

Antibodies. The primary antibodies used in this study were as follows: poly-clonal anti-C1orf86 isoform2 (FAAP20) antibody (Sigma-Aldrich; HPA038829);anti-myc antibody (Santa Cruz Biotechnology; sc-40); anti-FLAG antibody(Sigma-Aldrich; F1804); polyclonal anti-FANCA and anti-FANCI antibodies(Bethyl Laboratories; A301-980A and A301-254A); monoclonal anti-FANCD2antibody (Santa Cruz Biotechnology; sc-20022); polyclonal anti-FANCD2 an-tibody (Novus Biologicals; NB100-182); polyclonal anti-MBP antibody (Milli-pore; 05–912); monoclonal anti-Ub antibody (Millipore; 04–263); monoclonalanti-GST (Santa Cruz; SC-138); polyclonal anti-FANCL antibodies were a gen-erous gift from Weidong Wang (National institute on Aging, National Insti-tutes of Health, Baltimore, MD).

Cell Cultures and Transfection. Human embryonic kidney 293T cells and hu-man colorectal cancer HCT116 cells were cultured in RPMI 1640 and DMEM,respectively, supplemented with (vol/vol) FBS, 100 units/mL penicillin, and

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Fig. 5. FANCA-binding is required for FAAP20 function in the FA pathway. (A) Clonogenic survival assay of HCT116 cells, FAAP20-deficient cells, and FAAP20-deficient cells reconstitutedwithwild-type FAAP20, the 6A1, or the C147/150Amutant of FAAP20. (B)Whole-cell extractswere prepared fromHCT116 cells, FAAP20-deficient cells, and FAAP20-deficient cells complementedwith indicated constructswithorwithoutMMCtreatment.Westernblottingwas conductedusing indicatedantibodies. (C) FAAP20-deficient cells reconstituted with indicated SFB-tagged wild-type or mutant FAAP20 were mock-treated or treated with MMC for 24 h.Immunostaining was performed using anti-Flag and anti-FANCD2 antibodies. Cells were counterstained with DAPI as indicated. (Magnification: 100×.) (D) Quan-tification results were the average of two independent experiments and were presented as mean ± SEM. More than 100 cells were counted in each experiment.

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100 μg/mL streptomycin, and maintained in 5% CO2 at 37 8C. Plasmid andsiRNA transfection was performed using Lipofectamine 2000 and oligo-fectamineb (Invitrogen), respectively, according to the manufacturer’sinstructions. The coding strand for control siRNA was UCCAGUGAAUCCUU-GAGGUUU and that for FAAP20 siRNA was UCCGAAAGCACAGAA-GACGUUU. All siRNA were purchased from Dharmacon.

Immunoprecipitation, GST Pull-Down, and Western Blotting Analysis. Cells werelysed inNETNbuffercontainingprotease inhibitors. For immunoprecipitationofendogenous protein complexes, cell extracts were incubated with protein-Abeads and antibody against FAAP20 for 2 h at 4 8C. For precipitation of SFB-tagged proteins or pull-down experiments, cell extracts were incubated witheither streptavidin beads or GST-fusion proteins immobilized on glutathionebeads for 2 h at 4 8C. For in vitro binding assay, ub-GST were eluted with glu-tathione and then incubated with beads coated with bacterial expressed MBP,MBP-FAAP20, MBP-FAAP20 C147/150A, or MBP FAN1-1-100. The beads werewashed with NETN buffer and proteins were eluted by boiling in 1× Laemmlibuffer. Samples were resolved by SDS/PAGE, transferred to polyvinylidenedifluoride membrane, and immunoblotted with antibodies as indicated.

Immunofluorescence Staining. Cells cultured on coverslips werewashed in PBS,fixed in 3% paraformaldehyde for 15 min and then permeabilized in 0.5%triton solution for 5 min at room temperature. Samples were incubated withprimary antibodies for 30 min, washed, and incubated with secondary anti-bodies for 30min. Samples were then counterstainedwithDAPI andmountedon the glass slides with an antifade solution and visualized using a NikonEclipse 90i fluorescence microscope.

Somatic Knockout of FAAP20 and FANCL. For the generation of somaticknockout cells, adeno-associated virus-based strategy was used as previouslydescribed (39). The targeting adeno-associated viruses were packaged in 293Tcells by transfecting 3 μg of the targeting vector, pHelper, and pRC plasmids.Viruses were harvested at 72 h after transfection. Human colon cancer cellline HCT116 was infected for 48 h and selected with geneticin for 20 d. Thegeneticin-resistant clones were then screened using genomic PCR with

primers derived from the neomycin-resistant gene and the upstream regionof the left homologous arm or the downstream region of the right homol-ogous arm. After the first allele was targeted, the neomycin-resistant genewas excised using viruses expressing Cre-recombinase. Second targeting wasperformed using the same approach.

Clonogenic Assay. Cells were seeded at a density of 700 cells onto 6-cm dishesin triplicate. Twenty-four hours after seeding, the cells were treated with theindicated concentrations of MMC for 24 h. Cells were then washed free ofdrugs and incubated in fresh medium for another 10–14 d. The cells werethen fixed and stained with 0.5% crystal violet in 20% ethanol. Coloniescontaining more than 50 cells were counted.

MMC-Induced Radial Chromosome Analysis. Cells were plated in 10-cm dishesand treated with 0.063 μM MMC for 48 h. After treatment, cells were ex-posed to colcemid for 8 h, swollen using 0.075 M KCl, and fixed with 3:1methanol:acetic acid. Slides were stained with Giemsa and 50 metaphasespreads were scored for radials in two independent experiments.

eChIP Assay. Control substrates or substrates containing a defined cross-linkwere introduced into HEK293T cells stably expressing SFB-tagged FAAP20.ChIP was carried out as previously described (20).

Cell Cycle Analysis. Cells were exposed to 50 nM MMC and were allowed togrow for 24 h. Cells were trypsinized andfixed in 70%ethanol overnight. Cellswere thenwashed inPBS, nucleiwere stainedwithpropidium iodide (4μg/mL),treated with RNase (2 μg/mL) at room temperature for 30 min, and analyzedin a flowcytometer using FACS Flow Jo software.

ACKNOWLEDGMENTS. We thank our colleagues in the J.C. laboratory forinsightful discussions and technical assistance. This work was supported inpart by the Cancer Prevention Research Institute of Texas, Multi-InvestigatorAward, Grant RP110465-P2 (to J.C.). J.C. is a recipient of Era of Hope ScholarAward W81XWH-05-1-0470 from the Department of Defense and a memberof the MD Anderson Cancer Center (CA016672).

1. Joenje H, Patel KJ (2001) The emerging genetic and molecular basis of Fanconianaemia. Nat Rev Genet 2:446–457.

2. Kennedy RD, D’Andrea AD (2005) The Fanconi anemia/BRCA pathway: New faces inthe crowd. Genes Dev 19:2925–2940.

3. Auerbach AD (2009) Fanconi anemia and its diagnosis. Mutat Res 668:4–10.4. Ling C, et al. (2007) FAAP100 is essential for activation of the Fanconi anemia-asso-

ciated DNA damage response pathway. EMBO J 26:2104–2114.5. Niedernhofer LJ, Lalai AS, Hoeijmakers JH (2005) Fanconi anemia (cross)linked to DNA

repair. Cell 123:1191–1198.6. Gurtan AM, D’Andrea AD (2006) Dedicated to the core: Understanding the Fanconi

anemia complex. DNA Repair (Amst) 5:1119–1125.7. Kee Y, D’Andrea AD (2010) Expanded roles of the Fanconi anemia pathway in pre-

serving genomic stability. Genes Dev 24:1680–1694.8. Smogorzewska A, et al. (2007) Identification of the FANCI protein, a mono-

ubiquitinated FANCD2 paralog required for DNA repair. Cell 129:289–301.9. Garcia-Higuera I, et al. (2001) Interaction of the Fanconi anemia proteins and BRCA1

in a common pathway. Mol Cell 7:249–262.10. Sims AE, et al. (2007) FANCI is a second monoubiquitinated member of the Fanconi

anemia pathway. Nat Struct Mol Biol 14:564–567.11. Dorsman JC, et al. (2007) Identification of the Fanconi anemia complementation

group I gene, FANCI. Cell Oncol 29:211–218.12. Kratz K, et al. (2010) Deficiency of FANCD2-associated nuclease KIAA1018/FAN1

sensitizes cells to interstrand crosslinking agents. Cell 142:77–88.13. Liu T, Ghosal G, Yuan J, Chen J, Huang J (2010) FAN1 acts with FANCI-FANCD2 to

promote DNA interstrand cross-link repair. Science 329:693–696.14. MacKay C, et al. (2010) Identification of KIAA1018/FAN1, a DNA repair nuclease re-

cruited to DNA damage by monoubiquitinated FANCD2. Cell 142:65–76.15. Smogorzewska A, et al. (2010) A genetic screen identifies FAN1, a Fanconi anemia-

associated nuclease necessary for DNA interstrand crosslink repair. Mol Cell 39:36–47.16. Howlett NG, et al. (2002) Biallelic inactivation of BRCA2 in Fanconi anemia. Science

297:606–609.17. Xia B, et al. (2007) Fanconi anemia is associated with a defect in the BRCA2 partner

PALB2. Nat Genet 39:159–161.18. Reid S, et al. (2007) Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N

and predispose to childhood cancer. Nat Genet 39:162–164.19. Litman R, et al. (2005) BACH1 is critical for homologous recombination and appears to

be the Fanconi anemia gene product FANCJ. Cancer Cell 8:255–265.20. Shen X, et al. (2009) Recruitment of fanconi anemia and breast cancer proteins to

DNA damage sites is differentially governed by replication. Mol Cell 35:716–723.21. Stoepker C, et al. (2011) SLX4, a coordinator of structure-specific endonucleases, is

mutated in a new Fanconi anemia subtype. Nat Genet 43:138–141.

22. Crossan GP, et al.; Sanger Mouse Genetics Project (2011) Disruption of mouse Slx4,a regulator of structure-specific nucleases, phenocopies Fanconi anemia. Nat Genet43:147–152.

23. Vaz F, et al. (2010) Mutation of the RAD51C gene in a Fanconi anemia-like disorder.Nat Genet 42:406–409.

24. Levitus M, et al. (2005) The DNA helicase BRIP1 is defective in Fanconi anemia com-plementation group J. Nat Genet 37:934–935.

25. Levran O, et al. (2005) The BRCA1-interacting helicase BRIP1 is deficient in Fanconianemia. Nat Genet 37:931–933.

26. Geng L, Huntoon CJ, Karnitz LM (2010) RAD18-mediated ubiquitination of PCNAactivates the Fanconi anemia DNA repair network. J Cell Biol 191:249–257.

27. Taniguchi T, D’Andrea AD (2006) Molecular pathogenesis of Fanconi anemia: Recentprogress. Blood 107:4223–4233.

28. Grompe M, van de Vrugt H (2007) The Fanconi family adds a fraternal twin. Dev Cell12:661–662.

29. Hanlon Newell AE, et al. (2008) Loss of homologous recombination or non-homolo-gous end-joining leads to radial formation following DNA interstrand crosslinkdamage. Cytogenet Genome Res 121:174–180.

30. Léveillé F, et al. (2004) The Fanconi anemia gene product FANCF is a flexible adaptorprotein. J Biol Chem 279:39421–39430.

31. Reuter T, Herterich S, Bernhard O, Hoehn H, Gross HJ (2000) Strong FANCA/FANCGbut weak FANCA/FANCC interaction in the yeast 2-hybrid system. Blood 95:719–720.

32. Garcia-Higuera I, Kuang Y, Näf D, Wasik J, D’Andrea AD (1999) Fanconi anemiaproteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex.Mol Cell Biol 19:4866–4873.

33. Ciccia A, et al. (2007) Identification of FAAP24, a Fanconi anemia core complex pro-tein that interacts with FANCM. Mol Cell 25:331–343.

34. Garcia-Higuera I, Kuang Y, Denham J, D’Andrea AD (2000) The Fanconi anemiaproteins FANCA and FANCG stabilize each other and promote the nuclear accumu-lation of the Fanconi anemia complex. Blood 96:3224–3230.

35. Bakker ST, et al. (2009) Fancm-deficient mice reveal unique features of Fanconianemia complementation group M. Hum Mol Genet 18:3484–3495.

36. Yamamoto KN, et al. (2011) Involvement of SLX4 in interstrand cross-link repair isregulated by the Fanconi anemia pathway. Proc Natl Acad Sci USA 108:6492–6496.

37. Williams SA, Longerich S, Sung P, Vaziri C, Kupfer GM (2011) The E3 ubiquitin ligaseRAD18 regulates ubiquitylation and chromatin loading of FANCD2 and FANCI. Blood117:5078–5087.

38. Leung JW, et al. (2011) SET nuclear oncogene associates with microcephalin/MCPH1and regulates chromosome condensation. J Biol Chem 286:21393–21400.

39. Zhang X, et al. (2008) Epitope tagging of endogenous proteins for genome-wideChIP-chip studies. Nat Methods 5:163–165.

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