Molecular Cell, Vol. 7, 249–262, February, 2001, Copyright 2001 by Cell Press

Interaction of the Fanconi Anemia Proteinsand BRCA1 in a Common Pathway

as mitomycin C (MMC) and, to a lesser extent, to ionizingradiation (IR) (Duckworth-Rysiecki and Taylor, 1985;Carreau et al., 1999). Based on somatic cell fusion stud-

Irene Garcia-Higuera,*# Toshiyasu Taniguchi,*#Shridar Ganesan,† M. Stephen Meyn,‡Cynthia Timmers,§ James Hejna,§

ies, FA is comprised of seven distinct complementationMarkus Grompe,§ and Alan D. D’Andrea*‖groups (Joenje et al., 1997, 2000). Six of the FA genes,*Department of Pediatric Oncologyincluding the genes for FANCA, FANCC, FANCD2, FANCE,Dana-Farber Cancer InstituteFANCF, and FANCG, have been cloned (Strathdee et al.,and Department of Pediatrics1992; de Winter et al., 1998, 2000a; 2000b; The FanconiChildren’s HospitalAnaemia/Breast Cancer Consortium, 1996; Lo Ten FoeHarvard Medical Schoolet al., 1996; Timmers et al., 2001 [this issue of MolecularBoston, Massachusetts 02115Cell]). The six cloned FA proteins have little homology†Department of Oncologyto each other or to other proteins in GenBank, and littleDana-Farber Cancer Instituteis known regarding their cellular function. Based on theBoston, Massachusetts 02115similar clinical and cellular phenotypes observed among‡The Hospital for Sick Childrenthe seven FA complementation groups, the FA proteinsToronto, Ontario M5G 1X8appear to cooperate in a common cellular pathway. Con-Canadaflicting data suggest that the FA proteins may function§Department of Molecular and Medical Geneticsin any one of several cellular processes, including celland Department of Pediatricscycle control, apoptosis, oxygen radical detoxification,Oregon Health Sciences Universityor DNA repair (D’Andrea and Grompe, 1997).Portland, Oregon 97201

In normal cells, several of the FA proteins includingFANCA, FANCG, FANCC, and FANCF assemble in amultisubunit nuclear complex (Kupfer et al., 1997b; Garcia-SummaryHiguera et al., 1999; Waisfisz et al., 1999; de Winter etal., 2000c). The FA complex is disrupted in cell linesFanconi anemia (FA) is a human autosomal recessivederived from other FA complementation groups, includ-cancer susceptibility disorder characterized by cellu-ing groups B and E (Yamashita et al., 1998), suggestinglar sensitivity to mitomycin C and ionizing radiation.that other FA genes may function upstream in the path-Although six FA genes (for subtypes A, C, D2, E, F,way, perhaps in the assembly or stabilization of theand G) have been cloned, their relationship to DNAcomplex (Garcia-Higuera et al., 2000; Kuang et al., 2000).repair remains unknown. In the current study, we show

Among the FA complementation groups, FA-D is dis-that a nuclear complex containing the FANCA, FANCC,tinct. Although FA-D patients are phenotypically indis-FANCF, and FANCG proteins is required for the activa-tinguishable from patients from other FA subtypes, thetion of the FANCD2 protein to a monoubiquitinatedFA protein complex assembles normally in FA-D cellsisoform. In normal (non-FA) cells, FANCD2 is monoubiq-(Yamashita et al., 1998). The FA-D complementationuitinated in response to DNA damage and is targetedgroup appears to be genetically heterogenous, con-to nuclear foci (dots). Activated FANCD2 protein colo-sisting of at least two genes, FANCD1 and FANCD2

calizes with the breast cancer susceptibility protein,(Timmers et al., 2001). While two FA-D cell lines (PD20

BRCA1, in ionizing radiation–induced foci and in syn-and VU008) have biallelic mutations in the FANCD2

aptonemal complexes of meiotic chromosomes. The gene, FANCD2 mutations were not detected in the groupFANCD2 protein, therefore, provides the missing link D reference cell line (HSC62), suggesting that HSC62 hasbetween the FA protein complex and the cellular a mutation in a distinct gene (FANCD1). These resultsBRCA1 repair machinery. Disruption of this pathway suggest that the protein products of the FANCD1 andresults in the cellular and clinical phenotype common FANCD2 genes function downstream or independentlyto all FA subtypes. of the FA protein complex.

Several lines of evidence suggest that FA cells haveIntroduction an underlying molecular defect in either cell cycle regu-

lation (Bigelow et al., 1979; Kaiser et al., 1982; KubbiesFanconi anemia (FA) is an autosomal recessive cancer et al., 1985; Digweed et al., 1995; Kupfer and D’Andrea,susceptibility syndrome characterized by multiple con- 1996; Kupfer et al., 1997a) or DNA repair (Gluckman etgenital anomalies and progressive bone marrow failure al., 1983; Diatloff-Zito et al., 1986; Kupfer and D’Andrea,(reviewed in Huibregtse et al., 1985) (D’Andrea and 1996; Thyagarajan and Campbell, 1997; Escarceller etGrompe, 1997). FA patients develop several types of al., 1998; Smith et al., 1998). Despite the extensive docu-cancers including acute myeloid leukemias and squa- mentation of these defects, the downstream biochemi-mous cell carcinomas of the head and neck (Alter, 1996). cal events in the FA pathway have remained elusive. InFA cells are sensitive to DNA cross-linking agents such the current study, we examined the role of the recently

cloned FANCD2 protein (Timmers et al., 2001) in thecellular response to DNA damage. Our studies indicate‖ To whom correspondence should be addressed (e-mail: alan_that the FANCD2 protein functions downstream of thedandrea@dfci.harvard.edu).

# These authors contributed equally to this work. FA protein complex. In the presence of the assembled

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Figure 1. The Fanconi Anemia Protein Complex Is Required for the Monoubiquitination of FANCD2

(A) Whole-cell extracts were prepared from the indicated lymphoblast lines, and cellular proteins were immunoblotted with an anti-FANCD2antiserum. Normal (wild-type [WT]) cells (lane 1) express two isoforms of the FANCD2 protein: a low molecular weight isoform (FANCD2-S)(155 kDa) and a high molecular weight isoform (FANCD2-L) (162 kDa). FA cell lines derived from type A, C, G, and F patients only expressthe FANCD2-S isoform (lanes 3, 7, 9, and 11). Correction of these FA cell lines with the corresponding FA cDNA results in functionalcomplementation (Table 1) and restoration of the high molecular weight isoform, FANCD2-L (lanes 4, 8, 10, and 12).(B) HeLa cells were transfected with a cDNA encoding HA-ubiquitin, as indicated. After transfection, cells were treated with the indicateddose of MMC. Cellular proteins were immunoprecipitated with a polyclonal antibody (E35) to FANCD2, as indicated. Immune complexes wererun on SDS–PAGE, transferred to nitrocellulose, and immunoblotted with anti-FANCD2 (FI17) or anti-HA (HA.11) monoclonal antibody. (C)shows the same experiment as in (B), only cells were treated with the indicated dose of ionizing radiation (IR).

FA protein complex, the FANCD2 protein is activated to to ionizing radiation. Interestingly, FA cells from multiplecomplementation groups (A, C, G, and F) expressed onlya high molecular weight, monoubiquitinated isoform.

The activated FANCD2 protein accumulates in nuclear the FANCD2-S isoform (Figure 1A, lanes 3, 7, 9, and 11).FA cells from complementation groups B and E alsofoci in response to DNA-damaging agents and colocal-

izes and coimmunoprecipitates with BRCA1. These re- expressed only the FANCD2-S isoform (data not shown).Functional correction of the MMC and IR sensitivity ofsults resolve previous conflicting models (D’Andrea and

Grompe, 1997) and demonstrate that the FA proteins FA cells with the corresponding cDNA restored the FAprotein complex (Table 1) (Garcia-Higuera et al., 1999) andcooperate in a novel cellular pathway activated in re-

sponse to DNA damage. restored the high molecular weight isoform (FANCD2-L)(Figure 1A, lanes 4, 8, 10, and 12). Taken together, theseresults demonstrate that the FA protein complex, con-Resultstaining FANCA, FANCC, FANCF, and FANCG, directlyor indirectly regulates the expression of the two isoformsThe FA Genes Interact in a Common Cellular Pathwayof FANCD2. The six cloned FA genes, therefore, interactNormal lymphoblasts express two isoforms of thein a common pathway.FANCD2 protein, a short form (FANCD2-S, 155 kDa) and

a long form (FANCD2-L, 162 kDa) (Figure 1A, lane 1). The155 kDa isoform (FANCD2-S) is the primary translation The FA Protein Complex Is Required

for the Monoubiquitination of FANCD2product of the cloned FANCD2 cDNA (Timmers et al.,2001). We evaluated a large series of FA lymphoblasts The high molecular weight isoform of FANCD2 could

result from one or more mechanisms, including alterna-and fibroblasts for expression of the FANCD2 isoforms(Table 1). As previously described, FA cells are sensitive tive splicing of the FANCD2 mRNA or posttranslational

modification(s) of the FANCD2 protein. Treatment withto the DNA cross-linking agent MMC and, in some cases,

The Fanconi Anemia Proteins and BRCA1251

Table 1. MMC and IR Sensitivity of FA Cell Lines

Cell Line/Plasmid FA Group FA Protein Complexa MMC Sensitivityb IR/Bleomycin Sensitivityc

Lymphoblasts PD7 WT 1 R RHSC72 A 2 SHSC721A A 1 RPD4 C 2 SPD41C C 1 REUFA316 G 2 SEUFA3161G G 1 REUFA121 F 2 S SEUFA1211F F 1 R RPD20 D2 1 S SPD20(R) D2 1 R R

Fibroblasts GM0637 WT 1 R RGM6914 A 2 S SGM6941A A 1 R RPD426 C 2 SPD4261C C 1 RFAG326SV G 2 SFAG326SV1G G 1 RPD20F D2 1 S S20-3-15 (1D) D2 1 R RNBS2/2 NBS 1 S SATM2/2 ATM 1 S S

Tumor lines BRCA12/2, HCC1937 BRCA1 1 S SHCC19371BRCA1 BRCA1 1 R R

a The presence of the FA protein complex (FANCA/FANCG/FANCC) was determined as previously described (Garcia-Higuera et al, 1999).b MMC sensitivity was determined by the XTT assay for lymphoblasts or by the crystal violet assay for fibroblasts.c IR/bleomycin sensitivity was determined by analysis of chromosome breakage (see Experimental Procedures).

phosphatase did not convert FANCD2-L to FANCD2-S, glycine-glycine derived from the carboxyl terminus ofubiquitin, thereby identifying the monoubiquitinationdemonstrating that phosphorylation alone does not ac-

count for the observed difference in their molecular site as K561. Interestingly, K561 is conserved amongFANCD2 sequences from human, Drosophila, and Arabi-mass (data not shown).

In order to identify other possible posttranslational dopsis (Timmers et al., 2001), suggesting that the ubiqui-tination of this site is critical to the FA pathway in multiplemodifications of FANCD2, we initially sought cellular

conditions that regulate the conversion of FANCD2-S organisms. Mutation of this lysine residue, FANCD2(K561R), resulted in loss of FANCD2 monoubiquitinationto FANCD2-L (Figures 1B and 1C). Since FA cells are

sensitive to MMC and IR, we reasoned that these agents (see below).might regulate the conversion of FANCD2-S to FANCD2-Lin normal cells. HeLa cells treated with MMC (Figure 1B, Formation of Nuclear Foci Containing FANCD2

Requires an Intact FA Pathwaylanes 1–6) or IR (Figure 1C, lanes 1–6) demonstrated adose-dependent increase in the expression of the We next examined the immunofluorescence pattern of

the FANCD2 protein in uncorrected, MMC-sensitive FAFANCD2-L isoform.To determine whether FANCD2-L is a ubiquitinated fibroblasts and functionally complemented fibroblasts

(Figures 2A and 2B). Again, the corrected FA cells ex-isoform of FANCD2-S, we transfected HeLa cells with acDNA encoding HA-ubiquitin (Treier et al., 1994). Cellular pressed both the FANCD2-S and FANCD2-L isoforms

(Figure 2A, lanes 2, 4, 6, and 8). The endogenousexposure to MMC (Figure 1B, lanes 7–10) or IR (Figure1C, lanes 7–10) resulted in a dose-dependent increase FANCD2 protein was observed exclusively in the nu-

cleus of human cells, and no cytoplasmic staining wasin the HA-ubiquitin conjugation of FANCD2. Only theFANCD2-L isoform and not the FANCD2-S isoform was evident (Figure 2B, a–h). The PD20 (FA-D) cells have

decreased nuclear staining(Figure 2B, d), consistentimmunoreactive with an anti-HA antibody. AlthoughFANCD2 was not ubiquitinated in FA cells, FANCD2 ubiq- with their decreased expression of FANCD2 protein by

immunoblot (Figure 2A, lane 7). In PD20 cells functionallyuitination was restored upon functional complementa-tion of these cells (data not shown). Since the FANCD2-S corrected with the FANCD2 gene by chromosome trans-

fer, the FANCD2 protein stained in two nuclear patterns.and FANCD2-L isoforms differ by 7 kDa, the FANCD2-Lappears to contain a single ubiquitin moiety (76 amino Most corrected cells had a diffuse nuclear pattern of

staining. A fraction of cells also stained for nuclear fociacids) covalently bound by an amide linkage to an inter-nal lysine residue of FANCD2. (see dots, Figure 2B, h). Both nuclear patterns were ob-

served with three independently derived anti-FANCD2To confirm the monoubiquitination, we next immuno-precipitated and gel purified FANCD2-L protein from antisera (one polyclonal, two monoclonal antisera) (data

not shown). FA fibroblasts from subtypes A, G, and CHeLa cells and analyzed its tryptic fragments by massspectrometry (data not shown). Ubiquitin tryptic frag- showed only the diffuse pattern of FANCD2 nuclear im-

munofluorescence (Figure 2B, a–c). Functional comple-ments were unambiguously identified. Also, one FANCD2tryptic fragment (KQLSSTVFK) contained an additional mentation of these cells with the FANCA, FANCG, or

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Figure 2. The Fanconi Anemia Complex Is Required for the Formation of FANCD2 Nuclear Foci

The indicated SV40-transformed fibroblasts were analyzed by either anti-FANCD2 immunoblot of whole-cell extracts (A) or immunofluorescencewith the affinity-purified anti-FANCD2 antiserum (B, a–h). The uncorrected (mutant, M) FA fibroblasts were FA-A (GM6914), FA-G (FAG326SV),FA-C (PD426), and FA-D (PD20F). The FA-A, FA-G, and FA-C fibroblasts were functionally complemented with the corresponding FA cDNA.The FA-D cells were complemented with neomycin-tagged human chromosome 3p (Whitney et al, 1995). Uncorrected FA fibroblasts weresensitive to MMC; corrected cells were resistant to MMC (see Table 1). PD20 cells express very low levels of FANCD2 (A, lane 7) and, therefore,showed only background staining (B, d).

FANCC cDNA, respectively, restored MMC resistance We next tested the effect of DNA damage on FA cells(Figure 3E). FA cells from multiple complementation(Table 1) and restored the nuclear foci in some cells

(Figure 2B, e–g). Cell cycle synchronization studies dem- groups (A, C, and G) failed to activate FANCD2-L andfailed to activate FANCD2 nuclear foci in response toonstrated that FANCD2 nuclear foci formed specifically

in S phase (data not shown). The presence of the MMC or IR exposure. These data suggest that the sensi-tivity of FA cells to MMC or IR results, at least in part,FANCD2-L isoform, therefore, correlates with the pres-

ence of FANCD2 nuclear foci, suggesting that the mono- from their failure to activate FANCD2-L nuclear foci.ubiquitinated FANCD2-L isoform is selectively targetedto these foci. Monoubiquitination of FANCD2 Is Required for Foci

Formation and Functional ComplementationWe next tested the functional importance of monoubiq-FANCD2 Is Localized to Nuclear Foci

in Response to DNA Damage uitination at lysine 561 on the formation of IR-inducibleFANCD2 foci and on the biological activity of FANCD2We next examined the accumulation of FANCD2-L and

FANCD2 nuclear foci in response to DNA damage (Fig- (Figure 4). PD20 (FA-D2) fibroblasts were retrovirallytransduced with the cDNA encoding either FANCD2(WT)ure 3). Previous studies have shown that FA cells are

sensitive to agents that cause DNA interstrand cross- or FANCD2(K561R). Expression of wild-type FANCD2resulted in monoubiquitination on lysine 561 (Figure 4A),links (MMC) or double-strand breaks (IR) but are rela-

tively resistant to ultraviolet light (UV) and monofunc- MMC- and IR-inducible FANCD2 focus formation (Figure4C, b and f), and functional complementation of MMCtional alkylating agents (Duckworth-Rysiecki and Taylor,

1985; Carreau et al., 1999). MMC activated the conver- sensitivity (Figure 4B). In contrast, the FANCD2(K561R)protein was not monoubiquitinated, did not form foci insion of FANCD2-S to FANCD2-L in asynchronous HeLa

cells (Figure 3A). Maximal conversion to FANCD2-L oc- response to MMC or IR (Figure 4C, c and g), and failed tocomplement the MMC sensitivity of FA-D2 cells. Takencurred 12–24 hr after MMC exposure, correlating with

the time of maximal accumulation of FANCD2 in nuclear together, these results demonstrate that monoubiquiti-nation of FANCD2 at lysine 561 is required for the tar-foci (Figure 3D, c and d). Thus, the increase in FANCD2-L

was paralleled by an increase in FANCD2 foci. Ionizing geting and accumulation of FANCD2 in nuclear foci andthe function of the FA pathway.radiation also activated a time-dependent and dose-

dependent increase in FANCD2-L in HeLa cells (Figure3B), with a corresponding increase in FANCD2 foci (Fig- Colocalization of Activated FANCD2

and BRCA1 Proteinure 3D, e and f). Surprisingly, ultraviolet light also acti-vated a time-dependent and dose-dependent conver- Like FANCD2, the breast cancer susceptibility protein

BRCA1 is upregulated in proliferating cells and is acti-sion of FANCD2-S to FANCD2-L (Figure 3C) as well asan increase in FANCD2 foci (Figure 3D, g and h). vated by posttranslational modifications during S phase

The Fanconi Anemia Proteins and BRCA1253

or in response to DNA damage (Scully et al., 1997a; (Figure 7a) and in diplonema (Figures 7d, 7e, and 7g).The M118 anti-BRCA1 antibody also stained the un-Lee et al., 2000). The BRCA1 protein colocalizes in IR-

inducible foci (IRIFs) with other proteins implicated in paired sex chromosomes in mouse pachytene and dip-lotene spermatocytes (Figures 7f and 7h). FANCD2 AbDNA repair, such as RAD51 or the NBS/Mre11/RAD50

complex (Zhong et al., 1999; Wang et al., 2000). Cells staining of the unsynapsed axes of the sex chromo-somes was interrupted, giving a beads-on-a-string ap-with biallelic mutations in BRCA1 have a defect in DNA

repair and are sensitive to DNA-damaging agents such pearance (Figure 7g). A consecutive examination of 20pachytene nuclei indicated that most (65%) of theseas IR and MMC (Table 1) (Moynahan et al., 1999; Scully

et al., 1999). Taken together, these data suggest a func- anti-FANCD2 foci colocalized with regions of intenseanti-BRCA1 staining, further supporting an interactiontional interaction between the FANCD2 and BRCA1 pro-

teins. between these proteins (Figures 7g–7i). These resultsconfirm the interaction of FANCD2 and BRCA1 and pro-In order to determine whether the activated FANCD2

protein colocalizes with the BRCA1 protein, we per- vide an example of an FA protein (activated FANCD2)that binds to chromatin.formed double immunolabeling of HeLa cells (Figure

5). In the absence of ionizing radiation, approximately30%–50% of cells contained BRCA1 nuclear foci (Figure Discussion5A), consistent with previous studies (Wu et al., 2000).In contrast, only rare cells traversing S phase contained Our results demonstrate that the newly cloned FANCD2FANCD2 dots (Figure 5A, b and e). These nuclear foci protein (Timmers et al., 2001) provides the missing linkwere also immunoreactive with antisera to both BRCA1 between the FA protein complex and BRCA1-containingand FANCD2 (Figure 5A, c and f). Following IR exposure, foci. According to this model, DNA damage (MMC, IR,there was an increase in the number of cells containing or UV) activates the FA complex–dependent monoubiq-nuclear foci and the number of foci per cell. These nu- uitination of the FANCD2-S protein to the FANCD2-Lclear foci were larger than foci observed in the absence isoform. Monoubiquitination of FANCD2 is a targetingof IR. Again, these foci contained both BRCA1 and signal, resulting in the relocalization of FANCD2 from aFANCD2 protein (Figure 5A, i and l). FANCD2 and BRCA1 diffuse nuclear compartment to foci containing BRCA1also strongly colocalized in foci during S phase (data and perhaps other DNA repair proteins. These foci alsonot shown). An interaction of FANCD2-L (the long iso- form during S phase of the cell cycle (data not shown).form only) and BRCA1 was further confirmed by coim- After DNA repair is completed, the FANCD2-L proteinmunoprecipitation of the proteins (Figure 5B) from ly- appears to be deubiquitinated (D’Andrea and Pellman,sates prepared from exponentially growing HeLa cells 1998) and recycled back to the FANCD2-S isoform,exposed to IR. allowing the cell cycle to resume.

We next examined the effect of BRCA1 expression Interestingly, the conserved lysine (K561) of FANCD2on the formation of FANCD2-L and nuclear foci (Figure is required for monoubiquitination of FANCD2, IR-induc-6). The BRCA12/2 cell line, HCC1937, expresses a mutant ible foci formation, and functional complementation ofform of the BRCA1 protein with a carboxy-terminal trun- FA-D2 cells. A mutant form, FANCD2(K561R), failed tocation (Tomlinson et al., 1998; Scully et al., 1999). Al- undergo monoubiquitination to the long isoform, failedthough these cells expressed a low level of FANCD2-L to develop IR-inducible foci, and failed to complement(Figure 6A), IR failed to promote an increase in FANCD2-L the MMC sensitivity of FA-D2 cells. Taken together,levels. Also, these cells had decreased IR-inducible these results demonstrate the importance of the con-FANCD2 foci (Figure 6B, c and d). Correction of the served K561 residue in the regulation of FANCD2 fociBRCA12/2 cells by stable transfection with the BRCA1 and in the function of the FA pathway.cDNA restored IR-inducible FANCD2 ubiquitination (Fig-ure 6A) and nuclear foci to normal levels (Figure 6B, Activation of the FA Pathway by DNA Damagek and l). These data suggest that wild-type BRCA1 is The monoubiquitination of the FANCD2 protein and therequired as an “organizer” for IR-inducible FANCD2 dot formation of FANCD2 foci is highly regulated and occursformation and further suggest a functional interaction during S phase of the cell cycle (data not shown) orbetween the proteins. following DNA damage (Figure 3). While the molecular

basis of this regulation is unknown, monoubiquitinationappears to require both an intact FA protein complexColocalization of FANCD2 and BRCA1

on Meiotic Chromosomes (Fanconi A, C, G, and F proteins) and the BRCA1 protein.Absence of the FA complex results in complete loss ofThe association of FANCD2 and BRCA1 in mitotic cells

suggested that these proteins might also colocalize dur- FANCD2 monoubiquitination; absence of BRCA1 resultsin loss of IR-inducible FANCD2 monoubiquitination.ing meiotic prophase. Previous studies have demon-

strated that the BRCA1 protein is concentrated on the The regulation of protein polyubiquitination has beenextensively studied (Ciechanover, 1994). Protein poly-unsynapsed/axial elements of human synaptonemal

complexes in zygotene and pachytene spermatocytes ubiquitination is mediated by the serial activation of anE1 (ubiquitin-activating enzyme), E2 (ubiquitin carrier(Scully et al., 1997b). To test for a possible colocalization

of FANCD2 and BRCA1 in meiotic cells, we examined protein), and E3 (ubiquitin ligase). In general, E3 ligasescomprise a large enzyme superfamily and determine thesurface spreads of late pachytene and early diplotene

mouse spermatocytes for the presence of FANCD2 and subunit specificity of ubiquitination. While some sub-families of E3 ligases contain conserved sequence mo-BRCA1 protein (Figure 7). The rabbit polyclonal anti-

FANCD2 antibody E35 specifically stained the unpaired tifs such as the HECT proteins (Huibregtse et al., 1985)and the RING finger proteins (Lorick et al., 1999; Joazeiroaxes of the X and Y chromosomes in late pachynema

Molecular Cell254

Figure 3. Formation of Activated FANCD2 Nuclear Foci Following Cellular Exposure to MMC, Ionizing Radiation, or Ultraviolet Light

Exponentially growing HeLa cells were either untreated or exposed to the indicated DNA damaging agents, (A), mitomycin C (MMC); (B),gamma irradiation (IR); or (C), ultraviolet light (UV) and processed for FANCD2 immunoblotting or FANCD2 immunostaining (D).(A) Cells were continuously exposed to 40 ng/ml MMC for 0–72 hr as indicated.(B and C) Cells were exposed to gamma irradiation (10 Gy, B) or UV light (60 J/m2, C) and collected after the indicated time (upper panels)or irradiated with the indicated doses and harvested 1 hr later (lower panels).(D) For immunofluorescence analysis, cells were similarly irradiated and fixed 8 hr later (a, b, and e–h) or treated with MMC for 24 hr beforefixing (c and d).(E) The indicated EBV-transformed lymphoblast lines from a normal individual (PD7) or from various Fanconi anemia patients were either

The Fanconi Anemia Proteins and BRCA1255

Figure 4. Monoubiquitination of FANCD2 Is Required for Formation of IR-Inducible Foci and Functional Complementation

PD20 (FA-D2) fibroblasts were transduced with retroviral constructs encoding either FANCD2(WT) or FANCD2(K561R), and stable transfectantswere selected in puromycin.(A) Immunoblot analysis of the indicated cells with the anti-FANCD2 monoclonal antibody.(B) The indicated PD20 fibroblast lines were analyzed for MMC sensitivity as previously described. PD20-3-15 cells were functionally comple-mented with human chromosome 3p (Whitney et al., 1995).(C) Anti-FANCD2 immunofluorescence of the indicated PD20F cells following MMC treatment (40 ng/ml, a–d) or IR exposure (20 Gy, e–h).

and Weissman, 2000), other E3 ligases have novel se- protein monoubiquitination is preceded by phosphory-lation or whether a specific subclass of E2 or E3 enzymesquences. Protein polyubiquitination of an internal lysine

residue frequently follows the phosphorylation of a mediates monoubiquitination remains unknown.Several models describing the regulated monoubiqui-nearby serine residue. Protein polyubiquitination is a

signal for proteasome-mediated degradation. tination of the FANCD2 protein are plausible. The moststraightforward model is that the FA complex itself hasLess is known about protein monoubiquitination, and

fewer examples are available (Mueller et al., 1985; Pham intrinsic monoubiquitin ligase activity that is activatedby cell cycle or DNA damage cues. For instance, it isand Sauer, 2000; Shih et al., 2000). Monoubiquitination,

such as that observed for FANCD2, appears to be a possible that one of the known subunits of the FA proteincomplex (A, C, F, or G proteins) has E2 or E3 activityreversible signal, regulating either protein targeting,

protein complex assembly, or intrinsic activity (Kaiser that is activated by DNA damage and specific for thedownstream FANCD2 protein. Alternatively, the FA pro-et al., 2000). Histone H2A and H2B, for example, are

monoubiquitinated and deubiquitinated in a cell cycle– tein complex may have additional subunits with E2 orE3 activity. For instance, RAD6 or another DNA damage–dependent manner (Mueller et al., 1985), and Ub-histone

H2A assembles in S phase nuclear foci (Vassilev et al., responsive E2 or E3 may associate with the FA proteincomplex.1995). The E2 enzyme RAD6 is required for the monoubiq-

uitination of H2B in vivo (Robzyk et al., 2000). Whether Interestingly, the BRCA1 protein is required for IR-

treated with 40 ng/ml of mitomycin C continuously (lanes 1–21) or exposed to 15 Gy of gamma irradiation (lanes 22–33) and processed forFANCD2 immunoblotting. The upregulation of FANCD-L after MMC or IR treatment was seen in PD7 (lanes 2–5) and in the corrected FA-Acells (lanes 28–33) but was not observed in any of the mutant Fanconi anemia cell lines. Similarly, IR-induced FANCD2 nuclear foci were notdetected in FA fibroblasts (FA-G 1 IR) but were restored after functional complementation (corrected FA-G 1 IR).

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Figure 5. Colocalization of Activated FANCD2 and BRCA1 in Discrete Nulear Foci Following DNA Damage

HeLa cells were untreated or exposed to ionizing radiation (10 Gy) as indicated and fixed 8 hr later.(A) Cells were double-stained with the D-9 monoclonal anti-BRCA1 antibody (green; a, d, g, and h) and the rabbit polyclonal anti-FANCD2antibody (red; b, e, h, and k), and stained cells were analyzed by immunofluorescence. Where green and red signals overlap (Merge; c, f, i,and l) a yellow pattern is seen, indicating colocalization of BRCA1 and FANCD2.(B) Coimmunoprecipitation of FANCD2 and BRCA1. HeLa cells were untreated (2IR) or exposed to 15 Gy of gamma rradiation (1IR) andcollected 12 hr later. Cell lysates were prepared, and cellular proteins were immunoprecipitated with either the monoclonal FANCD2 antibody(FI-17, lanes 9 and 10) or any one of three independently derived monoclonal antibodies to human BRCA1 (lanes 3–8): D-9 (Santa Cruz), Ab-1,and Ab-3 (Oncogene Research Products). The same amount of purified mouse IgG (Sigma) was used in control samples (lanes 1–2). Immunecomplexes were resolved by SDS–PAGE and were immunoblotted with anti-FANCD2 or anti-BRCA1 antisera. The FANCD2-L isoform preferen-tially coimmunoprecipitated with BRCA1.

inducible FANCD2 monoubiquitination and foci forma- 2000), providing a possible regulatory mechanism of itsE3 ligase activity. The BRCA1 E3 ligase may cooperatetion. BRCA1 has recently been shown to contain an E3

RING finger domain (Joazeiro and Weissman, 2000), and with the FA protein complex in the regulated monoubiq-uitination of the FANCD2 protein. The BRCA12/2BRCA1 can function as a RING finger E3 ligase in vitro

(Lorick et al., 1999). BRCA1 is phosphorylated by S HCC1937 cells do, however, express a low level of mono-ubiquitinated FANCD2-L. This suggests that the endog-phase or DNA damage-inducible kinases (Scully et al.,

1997a; Cortez et al., 1999; Ruffner et al., 1999; Li et al., enous carboxy-terminal truncated (mutant) BRCA1 pro-

The Fanconi Anemia Proteins and BRCA1257

Figure 6. BRCA1 Is Required for the Ioniz-ing Radiation–Induced Monoubiquitination ofFANCD2 and the Formation of FANCD2 Foci

(A) BRCA1–/– cells (HCC1937) express a trun-cated mutant form of the BRCA1 protein witha carboxy- terminal truncation. HCC1937 (ei-ther untransfected or stably transfected withthe BRCA1 cDNA) were left untreated or wereirradiated (20 Gy) and allowed to recover forthe indicated time. The FANCD2 protein wasthen analyzed by anti-FANCD2 immunoblotof whole-cell lysates.(B) The same cells were analyzed by immuno-fluorescence with the anti-FANCD2 antise-rum or anti-BRCA1 antiserum. The anti-BRCA1 was raised against the C terminusof BRCA1 and does not react with the en-dogenous truncated BRCA1 expressed inHCC1937 cells.

tein in these cells may have partial activity or that other coimmunoprecipitates with BRCA1, it is not knownwhether FANCD2 binds directly or indirectly to BRCA1E3 ligase(s) also regulate FANCD2 monoubiquitination.

Whether the FA complex, the BRCA1 protein, RAD6, or or to other “dot” proteins such as RAD50, Mre11, NBS,or RAD51. Recent studies demonstrate that BRCA1 focisome other ubiquitin-conjuating enzyme monoubiquiti-

nates the FANCD2 protein will require further in vitro are composed of a large (2 MDa) multiprotein complexof unknown function (Wang et al., 2000). This complexstudies.

Monoubiquitination of the FANCD2 protein may re- includes ATM, ATM substrates involved in DNA repairfunctions (BRCA1), and ATM substrates involved inquire a preceeding phosphorylation event rendering it

a suitable substrate for a monoubiquitin ligase. Interest- checkpoint functions (NBS), suggesting many possibledownstream roles for FANCD2. For instance, the acti-ingly, the FANCD2 protein is rapidly phosphorylated in

response to ionizing radiation, and phosphorylation of vated FANCD2 protein may play a direct role in DNAdamage recognition, cell cycle checkpoint regulation,FANCD2 appears to precede monoubiquitination (T. T.

and I. G.-H., unpublished observation). Several cellular or DNA repair. The carboxy-terminal 20 amino acidsof FANCD2 contains a highly acidic HMG-like domainkinases are known to be activated by DNA damage,

including the PIK kinase family members (ATM, ATR, (Bachvarov and Moss, 1991), suggesting a possiblemechanism for its chromatin association.CHK1, and CHK2) or JNK kinase. It will be interesting

to determine whether cells that are defective in any of Several lines of evidence suggest a functional interac-tion between FANCD2 and BRCA1. First, the BRCA1–/–these DNA damage–inducible kinases are also defective

in IR-inducible FANCD2 phosphorylation or monoubiq- cell line HCC1937 (Tomlinson et al., 1998; Scully et al.,1999) has a “Fanconi anemia–like” phenotype, withuitination.chromosome instability and increased triradial and tet-raradial chromosome formation in response to MMCA Physical and Functional Interaction

of FANCD2 and BRCA1 (Table 1 and data not shown). Second, as stated above,the functional complementation of BRCA1–/– cells withThe monoubiquitinated isoform of FANCD2 colocalizes

with nuclear foci containing BRCA1. Although FANCD2-L the BRCA1 cDNA restores IR resistance and restores

Molecular Cell258

Figure 7. FANCD Forms Foci on Synaptonemal Complexes that Can Colocalize with BRCA1 during Meiosis I in Mouse Spermatocytes

(A) Anti-SCP3 (white) and anti-FANCD (red) staining of synaptonemal complexes in a late pachytene mouse nucleus.(B) SCP3 staining of late pachytene chromosomes.(C) Staining of this spread with preimmune serum for the anti-FANCD E35 antibody.(D) Anti-SCP3 staining of synaptonemal complexes in a mouse diplotene nucleus.(E) Costaining of this spread with E35 anti-FANCD antibody. Note staining of both the unpaired sex chromosomes and the telomeres of theautosomes with anti-FANCD.(F) Costaining of this spread with anti-BRCA1 antibody. The sex chromosomes are preferentially stained.(G) Anti-FANCD staining of late pachytene sex chromosome synaptonemal complexes.(H) Anti-BRCA1 staining of the same complexes.(I) Anti-FANCD (red) and anti-BRCA1 (green) costaining (colocalization reflected by yellow areas).

BRCA1 foci, FANCD2 monoubiquitination, and FANCD2 to regulate subsequent recombinational events. The rel-atively synchronous manner in which FANCD2 assem-foci to normal levels. In contrast, FA cells do form BRCA1

foci normally in response to IR (data not shown), sug- bles on meiotic chromosomes and forms dot structuresin mitotic cells suggests a role of FANCD2 in both mitoticgesting that FANCD2 function is further downstream in

the pathway. and meiotic cell cycle control. Further studies will berequired to determine (1) the dynamic relationship be-FANCD2 and BRCA1 also colocalize in foci on the

unpaired axes of XY bivalents in late pachytene and in tween FANCD2 and BRCA1 foci during meiotic develop-ment and (2) whether the FANCD2 foci colocalize withdiplotene murine spermatocytes, further suggesting a

functional interaction between the proteins. Taken to- other proteins such as RAD51 or ATM in synaptonemalcomplexes.gether with the known fertility defects in FA patients

(Bargman et al., 1977) and FA-C knockout mice (Chen Interestingly, FANCD2 foci were also observed on au-tosomal telomeres in diplonema (Figure 7E), suggestinget al., 1996; Kelly et al., 1996), our observations suggest

that activated FANCD2 protein may be required for nor- that the FANCD2 protein may play a role in telomerelength maintenance. Fanconi anemia cells are known tomal progression of spermatocytes through meiosis I.

Most FANCD2 foci seen on the XY axes colocalized have accelerated telomere shortening (Leteurtre et al.,1999), and accelerated loss of telomeres may lead towith BRCA1 foci, suggesting that the two proteins may

function together in meiotic cells. Like BRCA1, FANCD2 the high predisposition of squamous cell carcinomas inFA patients. The NBS protein, another protein found inwas detected on the axial (unsynapsed) elements of

developing synaptonemal complexes (data not shown). IR-inducible foci, localizes to telomeres in somatic cells(Zhu et al., 2000). Whether the activated (monoubiquiti-Since recombination occurs in synapsed regions, FANCD2

may function prior to the initiation of recombination, nated) FANCD2 protein regulates telomere length in so-matic cells will require further studies.perhaps to help prepare chromosomes for synapsis or

The Fanconi Anemia Proteins and BRCA1259

source (Timmers et al., 2001). Two anti-FANCD2 monoclonal anti-Implications of the FA Pathwaybodies were generated as follows. Balb/c mice were immunizedFA patient–derived cells share some but not all of thewith a GST-FANCD2 (N-terminal) fusion protein, which was the samephenotypic characteristics of cell lines derived from pa-fusion protein used for the generation of the rabbit polyclonal antise-

tients with Bloom syndrome (BS), AT (ataxia telangiecta- rum (E35) against FANCD2 (Timmers et al., 2001). Animals weresia), and NBS (Nijmegen breakage syndrome). These boosted with immunogen for the 4 days before fusion, splenocytescells demonstrate spontaneous chromosome breakage, were harvested, and hybridization with myeloma cells was per-

formed. Hybridoma supernatants were collected and assayed usingalthough they differ in their specific pattern of drug sen-standard ELISA assay as the initial screen and immunoblot analysissitivity. For instance, AT and NBS cells have more spe-of FANCD2 as the secondary screen. Two anti-human FANCD2cific IR sensitivity, while FA cells have more specific DNAmonoclonal antibodies (FI17 and FI14) were selected for furthercross-linker sensitivity. Interestingly, the BASC proteinstudy.

complex contains the BRCA1, BLM, ATM, and NBS pro-teins (Wang et al., 2000) and perhaps the FANCD2 pro- Immunoblottingtein, suggesting a common mechanism in the pathogen- Cells were lysed with 13 sample buffer (50 mM Tris-HCl [pH 6.8],esis of these human chromosome instability syndromes. 86 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate [SDS]),

boiled for 5 min, and subjected to 7.5% polyacrylamide SDS gelHow selective deficiencies in any one of these proteinselectrophoresis. After electrophoresis, proteins were transferred toleads to the characteristic disease-specific spectrumnitrocellulose using a submerged transfer apparatus (BioRad) filledof drug sensitivities and clinical abnormalities remainswith 25 mM Tris base, 200 mM glycine, and 20% methanol. Afterunknown.blocking with 5% nonfat dried milk in TBS-T (50 mM Tris-HCl [pH

Unlike the proteins in the FA complex (A, C, F, and 8.0], 150 mM NaCl, 0.1% Tween 20), the membrane was incubatedG) that have no obvious lower eukaryote homologs, the with the primary antibody diluted in TBS-T (1:1000 dilution for thehuman FANCD2 protein (and its K561 monoubiquitina- affinity-purified anti-FANCD2 polyclonal antibody [E35] or anti-HA

[HA.11] [Babco], 1:200 dilution for the anti-FANCD2 mouse mono-tion site) is highly conserved in Arapidopsis, Drosophila,clonal antibody FI17), washed extensively, and incubated with theand C. elegans (Timmers et al., 2001). The FA complexappropriate horseradish peroxidase–linked secondary antibodyproteins (A, C, G, and F) are expressed at relatively low(Amersham). Chemiluminescence was used for detection.levels, consistent with their putative role in an enzymatic

function. In lower eukaryotes, an alternative monoubiq-Generation of DNA Damage

uitinating enzyme may modify FANCD2. In contrast, the Gamma irradiation was delivered using a Gammacell 40 apparatus.FANCD2 protein is abundant, consistent with its role UV exposure was achieved using a Stratalinker (Stratagene) afteras the critical downstream substrate in the pathway. gently aspirating the culture medium. For mitomycin C treatment,

cells were continuously exposed to the drug for the indicated time.Disruption of the FANCD2 homologs in Drosophila andC. Elegans may result in genomic instability and ionizing

Detection of Monoubiquitinated FANCD2radiation sensitivity.HeLa cells (or the FA-G fibroblasts, FAG326SV) were transfectedFinally, our results indicate that the monoubiquitina-using FuGENE6 (Roche) following the manufacturer’s protocol. HeLation of FANCD2 and the formation of FANCD2 nuclearcells were plated onto 15 cm tissue culture dishes and were trans-

foci are downstream events in the FA pathway, requiring fected with 15 mg of an HA-tagged ubiquitin expression vector (pMTthe function of several FA genes and perhaps other 123) (Treier et al., 1994) (a gift of Dr. Dirk Bohmann, EMBL, Heidel-tumor suppressor genes. Accordingly, screening FA pa- berg, Germany) per dish. Twelve hours following transfection, cells

were treated with the indicated concentration of MMC or the indi-tient–derived cell lines, tumors, or cancer cell lines bycated dose of IR. After 24 hr incubation with MMC or 2 hr after IRanti-FANCD2 immunoblotting and immunofluorescencetreatment, whole-cell extracts were prepared in lysis buffer (50 mMmay provide a simple diagnostic test for upstream de-Tris-HCl [pH 7.4], 150 mM NaCl, 1% (v/v) Triton X-100) supplementedfects in the FA pathway and a practical alternative to the with protease inhibitors (1 mg/ml leupeptin and pepstatin, 2 mg/

currently employed DEB/MMC chromosome breakage ml aprotinin, 1 mM phenylmethylsulfonylfluoride) and phosphatasetest for FA (Auerbach, 1993). inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride).

Using the polyclonal antibody to FANCD2 (E35), immunoprecipita-Experimental Procedures tion (IP) was performed essentially as described (Kupfer et al.,

1997a), except that each IP was normalized to contain 4 mg ofCell Lines and Culture Conditions protein. As a negative control, preimmune serum from the sameEpstein–Barr virus (EBV) transformed lymphoblasts were maintained rabbit was used in the IP reaction. Immunoblotting was done usingin RPMI media supplemented with 15% heat-inactivated fetal calf anti-HA (HA.11) or anti-FANCD2 (FI17) monoclonal antibody.serum (FCS) and grown in a humidified 5% CO2-containing atmo-sphere at 378C. A control lymphoblast line (PD7) and FA lymphoblast Mass Spectrometrylines (FA-A [HSC72], FA-C [PD4], FA-D [PD20], FA-F [EUFA121], and HeLa cells were synchronized at G1/S boundary with double thymi-FA-G [EUFA316]) have been previously described (Yamashita et

dine block, and whole-cell extracts were prepared in lysis buffer.al., 1994; Whitney et al., 1995; de Winter et al., 1998, 2000b). PD81

Synchronization at G1/S results in increased expression of theis a lymphoblast cell line from an FA-A patient. The SV40-trans-

FANCD2-L isoform (T. T., unpublished observation). IP was per-formed FA fibroblasts GM6914, PD426, FAG326SV, and PD20F asformed using purified monoclonal antibody against FANCD2 (FI17),well as HeLa cells were grown in DMEM supplemented with 15%and the IP product was separated by 7% SDS–PAGE. Gel piecesFCS. FA cells (both lymphoblasts and fibroblasts) were functionallycorresponding to FANCD2-L bands were cut from a Coomassie-complemented with pMMP retroviral vectors containing the corre-stained gel and subjected to tryptic digestion. The sequence analy-sponding FANC cDNAs, and functional complementation was con-sis was performed at the Harvard Microchemistry Facility by micro-firmed by the MMC assay (Naf et al., 1998; Garcia-Higuera et al.,capillary reverse-phase HPLC nano electrospray tandem mass1999; Kuang et al., 2000). The K561R mutant FANCD2 cDNA wasspectrometry on a Finnigan LCQ quadrupole ion trap mass spec-produced with the QuikChange site-directed mutagenesis kit (Stra-trometer.tegene).

Immunofluorescence MicroscopyGeneration of Anti-FANCD2 AntibodiesCells were fixed with 2% paraformaldehyde in PBS for 20 min fol-A rabbit polyclonal antiserum (E35) against FANCD2 was generated

using a GST-FANCD2 (N-terminal) fusion protein as an antigen lowed by permeabilization with 0.3% Triton X-100 in PBS (10 min).

Molecular Cell260

After blocking in 10% goat serum, 0.1% NP-40 in PBS (blocking Bigelow, S.B., Rary, J.M., and Bender, M.A. (1979). G2 chromosomalradiosenitivity in Fanconi’s anamia. Mutat. Res. 63, 189.buffer), specific antibodies were added at the appropriate dilution

in blocking buffer and incubated for 2–4 hr at room temperature. Carreau, M., Alon, N., Bosnoyan-Collins, L., Joenje, H., and Buch-FANCD2 was detected using the affinity-purified E35 polyclonal wald, M. (1999). Drug sensitivity spectra in Fanconi anemia lympho-antibody (1/100). For BRCA1 detection, we used a commercial blastoid cell lines of defined complementation groups. Mut. Res.monoclonal antibody (D-9, Santa Cruz) at 2 mg/ml. Cells were subse- 435, 103–109.quently washed three times in PBS 1 0.1% NP-40 (10–15 min each

Chen, M., Tomkins, D.J., Auerbach, W., McKerlie, C., Youssoufian,wash), and species-specific fluorescein or Texas red–conjugated

H., Liu, L., Gan, O., Carreau, M., Auerbach, A., Groves, T., et al.secondary antibodies (Jackson Immunoresearch) were diluted in

(1996). Inactivation of Fac in mice produces inducible chromosomalblocking buffer (anti-mouse 1/200, anti-rabbit 1/1000) and added.

instability and reduced fertility reminiscent of Fanconi anaemia. Nat.After 1 hr at room temperature, three more 10–15 min washes were

Genet. 12, 448–451.applied, and the slides were mounted in Vectashield (Vector Labora-

Ciechanover, A. (1994). The ubiquitin-proteasome proteolytic path-tories). Images were captured on a Nikon microscope and pro-way. Cell 79, 13–21.cessed using Adobe Photoshop software.Cortez, W.J., Wangt, Y., Qin, J., and Elledge, S.J. (1999). Require-ment of ATM-dependent phosphorylation of BRCA1 in the DNAMeiotic Chromosome Stainingdamage response to double-strand breaks. Science 286, 1162–Surface spreads of pachytene and diplotene spermatocytes from1166.male mice between the ages of 16–28 days old were prepared asD’Andrea, A.D., and Grompe, M. (1997). Molecular biology of Fan-described by Peters et al. (1997). A polyclonal goat antibody toconi anemia: implications for diagnosis and therapy. Blood 90, 1725–the mouse SCP3 protein was used to visualize axial elements and1736.synaptonemal complexes in the meiotic preparations. The M118

mouse monoclonal antibody against mouse BRCA1 was generated D’Andrea, A.D., and Pellman, D. (1998). Deubiquitinating enzymes:by standard techniques: by immunizing mice with murine BRCA1 a new class of biological regulators. Crit. Rev. Biochem. Mol. Biol.protein. The affinity-purified E35 rabbit polyclonal antibody was 33, 337–352.used in 1:200 dilution to detect FANCD. Antibody incubation and de Winter, J.P., Waisfisz, Q., Rooimans, M.A., van Berkel, C.G.M.,detection procedures were a modification of the protocol of Moens Bosnoyan-Collins, L., Alon, N., Carreau, M., Bender, O., Demuth, I.,et al. (1987) as described by Keegan et al. (1996). Combinations of Schindler, D., et al. (1998). The Fanconi anaemia group G gene isdonkey anti-mouse IgG-FITC-congugated, donkey anti-rabbit IgG- identical with human XRCC9. Nat. Genet. 20, 281–283.TRITC-congugated, and donkey anti- goat IgG-Cy5-congugated

de Winter, J.P., Leveille, F., van Berkel, C.G.M., Rooimans, M.A.,secondary antibodies were used for detection (Jackson ImmunoRe-van der Weel, L., Steltenpool, J., Demuth, I., Morgan, N.V., Alon, N.,search Laboratories). All preparations were counterstained with 49,69Bosnoyan-Collins, L., et al. (2000a). Isolation of a cDNA representingdiamino-2-phenylindole (DAPI, Sigma) and mounted in a DABCOthe Fanconi anemia Complementation group E gene. Am. J. Hum.(Sigma) antifade solution. The preparations were examined on aGenet. 67, 1306–1308.Nikon E1000 microscope (603 CFI Plan Apochromat and 1003 CFIde Winter, J.P., Rooimans, M.A., van der Weel, L., Van Berkel, C.M.,Plan Fluor oil-immersion objectives). Each fluorochrome (FITC,Alon, N., Bosnoyan-Collins, L., de Groot, J., Zhi, Y., Waisfisz, Q.,TRITC, Cy5, and DAPI) image was captured separately as an 800 3Pronk, J.C., et al. (2000b). The Fanconi anemia complementation1000 pixel 12-bit source image via IPLab software (Scanalytics)gene FANCF encodes a novel protein with homology to ROM. Nat.controlling a cooled-CCD camera (Princeton Instruments Micro-Genet. 24, 15–16.Max), and the separate 12-bit gray scale images were resampled,

24-bit pseudocolored, and merged using Adobe Photoshop. de Winter, J.P., van Der Weel, L., de Groot, J., Stone, S., Waisfisz,Q., Arwert, F., Scheper, R.J., Kruyt, F.A., Hoatlin, M.E., and Joenje,H. (2000c). The Fanconi anemia protein FANCF forms a nuclearAcknowledgmentscomplex with FANCA, FANCC and FANCG. Hum. Mol. Genet. 9,2665–2674.We gratefully acknowledge Dr. Dirk Bohmann for the ubiquitin ex-

pression plasmids, Junjie Chen for BRCA12/2 cells, and Joanne Diatloff-Zito, C., Papadopoulo, D., Averbeck, D., and Moustacchi,E. (1986). Abnormal response to DNA crosslinking agents of FanconiSweasy for the anti-SCP3 antibody. We thank Hans Joenje for the

cDNAs for FANCA, FANCG, and FANCF. We thank James DeCaprio, anemia fibroblasts can be corrected by transfection with normalhuman DNA. Proc. Natl. Acad. Sci. USA 83, 7034–7038.David Pellman, Xiaohua Wu, Dan Silver, David Livingston, and David

Nathan for helpful discussions. We thank Bill Lane for performing Digweed, M., Gunthert, U., Schneider, R., Seyschab, H., Friedl, R.,the mass spectrometry. We thank Lisa Moreau for analysis of chro- and Sperling, K. (1995). Irreversible repression of DNA Synthesis inmosome breakage and Wei Wang for preparation of the meiotic Fanconi anemia cells is alleviated by the product of a novel cyclin-spreads. This work was supported by National Institutes of Health related gene. Mol. Cell. Biol. 15, 305–314.grants RO1HL52725-04, RO1DK43889-09, and PO1HL54785-04

Duckworth-Rysiecki, G., and Taylor, A.M.R. (1985). Effects on ioniz-(A. D. D.). I. G.-H. is a Special Fellow of the Leukemia and Lymphoma

ing radiation on cells from Fanconi’s anemia patients. Cancer Res.Society. T. T. was supported by a grant from the Naito Foundation.

45, 416.

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