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Mutation Research 668 (2009) 11–19

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /molmutCommuni ty address : www.e lsev ier .com/ locate /mutres

he genetic and molecular basis of Fanconi anemia

ohan P. de Winter ∗, Hans Joenjeepartment of Clinical Genetics, Section Oncogenetics, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

r t i c l e i n f o

rticle history:eceived 21 August 2008eceived in revised form 28 October 2008ccepted 6 November 2008

a b s t r a c t

The capacity to maintain genomic integrity is shared by all living organisms. Multiple pathways aredistinguished that safeguard genomic stability, most of which have originated in primitive life forms. Inhuman individuals, defects in these pathways are typically associated with cancer proneness. The Fanconianemia pathway, one of these pathways, has evolved relatively late during evolution and exists – in its

vailable online 14 November 2008

eywords:anconi anemiaNA cross-linking agentseplication

fully developed form – only in vertebrates. This pathway, in which thus far 13 distinct proteins havebeen shown to participate, appears essential for error-free DNA replication. Inactivating mutations in thecorresponding genes underlie the recessive disease Fanconi anemia (FA). In the last decade the geneticbasis of this disorder has been uncovered by a variety of approaches, including complementation cloning,genetic linkage analysis and protein association studies. Here we review these approaches, introduce the

cuss

enomic instabilityNA repair

encoded proteins, and dis

. Introduction

When the Swiss pediatrician Guido Fanconi first described aamily with physical abnormalities and aplastic anemia [1], heould not have realized that the bone marrow failure syndrome head discovered would reveal an important cellular defense mech-nism against genetic instability. Many years after Fanconi’s reportt became evident that defects in several genes can cause Fan-oni anemia (Table 1). The affected genes all encode proteins incellular pathway that is activated when DNA replication becomeslocked and that regulates mechanisms leading to recovery fromhis harmful situation. An effective way to block DNA replica-ion is by covalently connecting the two complementary DNAtrands with a DNA cross-linking agent. It is this type of agent tohich FA cells are exceptionally sensitive. Since DNA cross-linking

gents like cisplatin are often used to treat cancer patients, Fan-oni’s discovery had still broader implications than he could havemagined. In this review we will outline how the FA genes weredentified and what role these proteins may play in the FA path-ay.

. DNA cross-linker sensitivity, an important hallmark for

A cells

Almost 40 years after Fanconi’s report it was Traute Schroederho noticed spontaneous chromosomal breakage during routine

∗ Corresponding author. Tel.: +31 20 4448270; fax: +31 20 4448285.E-mail address: j.dewinter@vumc.nl (J.P. de Winter).

027-5107/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2008.11.004

their possible role in ensuring genomic integrity.© 2008 Elsevier B.V. All rights reserved.

cytogenetic analysis of FA patients [2], which classified FA as achromosomal instability syndrome. Ten years later, Masao Sasakidiscovered that the chromosomal abnormalities in FA cells weredrastically enhanced by the DNA cross-linking agent mitomycin C(MMC) [3]. This observation has provided the basis for the widelyused diagnostic chromosomal breakage test, as first introduced byArleen Auerbach ([4] and “Fanconi Anemia and its Diagnosis” Auer-bach, this issue). The cross-linker sensitivity of FA cells has alsobeen exploited to demonstrate the genetic heterogeneity amongFA patients by complementation analysis (Fig. 1) and has enabledthe identification of five FA genes by complementation cloning(Table 1).

Initial cell fusion experiments between different FA cell lines inManuel Buchwald’s lab demonstrated that four FA cell lines wereable to complement each other’s cross-linker sensitivity, indicatingthe involvement of at least four different FA genes: FANCA, FANCB,FANCC and FANCD [5]. The number of complementation groups wasfurther extended with groups E, F, G, H, I and J using the same tech-nique [6–8]. Although in general each FA complementation grouprepresented a separate disease gene, phenotypic reversion to wildtype cross-linker sensitivity by additional compensatory mutationsat the affected gene locus turned out to be an important pitfall inthe complementation assay. This has led to false classification of oneFA patient to group H and subsequent withdrawal of this comple-mentation group [9]. In retrospect, this patient had two pathogenicmutations in FANCA. One of these mutations was a missense muta-

tion (R951Q), which was functionally compensated by anothermissense mutation 15 amino acids further downstream (E966K)[10]. To avoid these misclassifications in the future, assignment ofpatients with FA to new complementation groups was subjectedto more stringent criteria. A new complementation group is now

12 J.P. de Winter, H. Joenje / Mutation Research 668 (2009) 11–19

Table 1Overview of the 13 FA genes identified so far.

Subtype Gene Location Protein (amino acids) Cloning method Features

A FANCA 16q24.3 1455 Complementation cloning,positional cloning

Partner of FANCG, contains nuclear localization signal

B FANCB Xp22.2 859 Protein association Partner of FANCL, contains nuclear localization signalC FANCC 9q22.3 558 Complementation cloning Partner of FANCED1 FANCD1/BRCA2 13q12.3 3418 Candidate-gene approach Supports RAD51 filament formation, contains BRC

repeats and OB-foldD2 FANCD2 3p26 1451 Positional cloning Monoubiquitinated and phosphorylated following DNA

damageE FANCE 6p21.3 536 Complementation cloning Partner of FANCC and FANCD2, Chk1 target, contains

nuclear localization signalF FANCF 11p15 374 Complementation cloning Adaptor protein stabilizing A/G and E/C interactionG FANCG 9p13 622 Complementation cloning Partner of FANCA, contains TPR motifsI FANCI 15q26.1 1328 Positional cloning;

candidate-gene approachPartner of FANCD2, monoubiquitinated andphosphorylated following DNA damage

J FANCJ/BRIP1 17q22-24 1249 Positional cloning 5′-to-3′ DEAH helicase, unwinds G-quadruplex DNAstructures

L FANCL 2p16.1 375 Protein association E3 ubiquitin ligase, contains RING-finger and WD40domains

M FANCM 14q21.3 2048 Protein association Translocase, contains DEAH helicase and ERCC4/XPFlike nuclease domain

N FANCN/PALB2 16p12.1 1186 Protein

Fig. 1. Complementation analysis revealed genetic heterogeneity among FApatients. MMC sensitive Epstein Barr virus immortalized B-lymphoblasts from dif-ferent FA patients are fused to form a hybrid cell line. The resulting hybrid is eitherMMC sensitive or MMC resistant. Complementation of the MMC sensitive pheno-type is only seen when the two cell lines have a defect in different genes (indicatedwith letters in lower case) and in that situation the patients are said to belong todifferent “complementation groups”.

Fig. 2. Outline of the complementation cloning strategy. MMC-sensitive lymphoblasts froma hygromycin selection marker. Cells are cultured in the presence of hygromycin to obtaina MMC dose that kills the uncorrected FA cells and selects for the cells in which the FA deextracted from the cells and the cDNA insert is sequenced. Finally, proof of identity for thmutations in the patient cell line.

association Partner of BRCA2, essential for stability andlocalization of BRCA2

based on at least two FA patients whose cell lines are excluded fromall known groups and that fail to complement each other in fusionhybrids, or, if only one such cell line is available, on a new gene thatcarries pathogenic mutations in this cell line [9].

To elucidate the molecular defect in FA cells, several labs startedto identify the gene defects in the various FA complementationgroups. In the beginning, cloning strategies utilized the cross-linkersensitive phenotype of FA cells and the ability to complement thisphenotype by introduction of a cDNA plasmid that expresses a nor-mal copy of the defective gene (Fig. 2). In 1992, Manuel Buchwald’slab identified the first FA gene, FANCC, using a home-made episo-mal cDNA expression library and the FA-C lymphoblast line HSC536[11]. This gene cloning method turned out to be quite powerful asit subsequently led to the discovery of FANCA, FANCE, FANCF, andFANCG in our lab [12–15]. Somewhat disappointingly, the proteinproducts of these FA genes were novel and hardly informative topinpoint the defective process in FA cells. Interestingly, the encodedproteins appeared specific for vertebrates, since no orthologs werefound in yeast, worms or flies.

Given the hypersensitivity to DNA cross-linking agents in FAcells, it was not a total surprise that FANCA and FANCE containednuclear localization signals and were present in a nuclear com-plex together with FANCC, FANCF and FANCG [16–19]. Besides thesenuclear localization signals, the only other functional domainsfound in these FA proteins were seven tetratricopeptide repeat

(TPR) motifs in FANCG, which function as scaffolds mediating inter-actions with the other FA proteins [20], but a connection betweenFA and DNA repair mechanisms was not revealed by the five FAproteins that were initially identified.

an FA patient are transfected with an episomal cDNA expression library containingcells that carry a cDNA construct. Selected cells are then cultured in medium withfect is complemented by the cDNA construct. Subsequently, the episomal vector ise insert representing the disease gene should come from the finding of pathogenic

J.P. de Winter, H. Joenje / Mutation Research 668 (2009) 11–19 13

Fig. 3. Positional cloning to identify FA genes. Inheritance of polymorphic markers (e.g. CA repeats) is determined along with the disease trait in families. These markers arespread over the entire genome, occur in variant forms and often differ between individuals. Each distinct variant allele is labeled with a number. In families with unrelatedparents, a marker can be associated (“linked”) with the disease locus when affected children share the same alleles, which should be different from the alleles of the unaffecteds cestorg sibs sf be dp

3D

[c1trcqlegdc3aairfgeb

cfksmtcwhitapriwF

ibs (A). In consanguineous families parents are related through sharing the same anene. The affected children will be homozygous for this allele, while the unaffectedrom the same complementation group, candidate regions for the disease gene canathogenic mutations in the affected children.

. Complementation group D, the first link between FA andNA repair

Positional cloning (Fig. 3), which also helped to identify FANCA21], was successfully utilized to discover the gene defective inomplementation group D, by Markus Grompe and co-workers. In995, microcell-mediated chromosome transfer had already shownhat the MMC-sensitive phenotype of the FA-D cell line PD20 wasestored to wild type levels by introducing chromosome 3 in theseells [22]. Several of the complemented PD20 clones lacked the-arm of chromosome 3, indicating that the defective gene was

ocated on the p-arm. Exclusion mapping using polymorphic mark-rs in affected and unaffected family members revealed that theene was located between 3p22 and 3p26. One of the PD20 clonesid contain the 3p22-3p26-candidate region, but failed to showomplementation. This clone lacked 1.2 Mb of sequence aroundp25 [23] and positional cloning was used to finally identify theffected gene in this region [24]. Surprisingly, in two of the patientsssigned to complementation group D by cell fusion, mutationsn the new gene were not found. One of these patients was theeference patient for complementation group D (HSC62). There-ore, complementation group D had to be split up into two groups;roup FA-D1 including patient HSC62 and group FA-D2. The discov-red gene was named FANCD2 as it was defective in the patientselonging to group FA-D2.

FANCD2 provided a first clue for a DNA repair defect in FAells, since upon DNA damage this protein co-localized in nuclearoci with the breast cancer susceptibility gene BRCA1 and theey protein for homologous recombination RAD51 [25,26]. Thistep occurred during S phase of the cell cycle and required theonoubiquitination of FANCD2 on lysine 561, which appeared

o be absolutely dependent on the nuclear FANCA, -C, -E, -F, -Gomplex. These observations suggested a role for the FA path-ay in an S-phase specific DNA repair pathway that involvedomologous recombination. The FANCD2 monoubiquitination step

tself is independent of BRCA1 or histone H2AX, but the forma-ion of damage-induced FANCD2 foci depends on these proteinsnd involves the BRCA1-dependent binding of FANCD2 to the

hosphorylated form of histone H2AX [27,28]. At the stalled DNAeplication fork, H2AX is phosphorylated by the DNA damage-nducible kinase ATR [29]. However, this is probably not the onlyay in which ATR can regulate FANCD2 focus formation, since

ANCD2 is also an ATR substrate and phosphorylation of FANCD2 by

. In this case the parents are heterozygous for the same mutated allele of the diseasehould carry different alleles (B). By comparing all the markers in different familiesetermined. Sequencing of the genes in these regions should finally reveal biallelic

ATR is essential for hydroxyurea- and MMC-induced FANCD2 foci[30,31].

The close link between FANCD2 and BRCA1 urged Alan D’Andreaand co-workers to investigate BRCA1 and BRCA2 in the FA-Dpatients lacking mutations in FANCD2. With this candidate geneapproach, they discovered that FA-D1 patients carried biallelicmutations in BRCA2 and expressed truncated BRCA2 proteins [32].BRCA2 supports the formation of RAD51 filaments, which are essen-tial for strand invasion during the homologous recombinationprocess. These filaments can be seen as foci at the sites of dam-age and in cells from FA-D1 patients the formation of these fociwas disturbed [33]. This finding now connected Fanconi anemiato homologous recombination repair. Additional FA patients withbiallelic BRCA2 mutations were identified and it turned out thatpatients in the D1 group are different from other FA patients inpresenting with a much more severe clinical phenotype, with early-onset and high rates of leukemia and specific solid tumors (Wilmstumors and medulloblastomas) at relatively young age [34]. This isnot surprising, since Brca2 knockout mice are embryonically lethal[35], whereas mice with disrupted Fanca [36,37], Fancc [38,39],Fancd2 [40], or Fancg [41,42] have a rather mild phenotype. For someresearchers these different phenotypes were a reason to considercomplementation group FA-D1 as a distinct clinical entity, althoughat a molecular level there are clear links between the FA pathwayand BRCA2. A direct interaction between FANCD2 and BRCA2 hasbeen described [43], which is important for chromatin loading ofBRCA2 and for the formation of nuclear BRCA2 foci [44]. To empha-size the connection between the FA proteins and BRCA1 and BRCA2,the FA pathway was renamed as the FA/BRCA pathway, in whichthe FA proteins may prepare the DNA lesion for repair by BRCA1and BRCA2. Because of the different phenotypes of FA and BRCA1or BRCA2 deficient mice, the FA proteins are probably not essen-tial for these BRCA proteins to function, but may assist them in thehandling of certain types of DNA damage. In the absence of the FAproteins, other DNA repair proteins, e.g. ATM may partially replacethe FA pathway [45], but this situation is possibly less efficient andmore error-prone.

4. Multiple FA proteins act in a nuclear core complex

While the connection between the FA pathway and DNA repairbecame more obvious after the identification of FANCD1 and

14 J.P. de Winter, H. Joenje / Mutation

Fig. 4. Identification of FA genes by protein association studies. An antibody againstFANCA is added to a HeLa cell nuclear extract to precipitate FANCA and the proteinsthat bind to FANCA. The precipitated proteins are separated on an SDS-PAGE gel andtSfg

FFmttFTwlws[Fma

wmwwfibtwFf

tcsolaFfobIpedars[

he individual proteins are excised from the gel and analyzed by mass spectrometry.ubsequently, the genes encoding the novel proteins in the precipitate are analyzedor pathogenic mutations in FA patients with no mutations in any of the known FAenes.

ANCD2, it was still a mystery what the nuclear FANCA, -C, -E, -, -G complex was actually doing and how it was involved in theonoubiquitination of FANCD2. To get a more complete picture of

he core complex Weidong Wang and co-workers purified a pro-ein complex from HeLa nuclear extracts using an antibody againstANCA and analyzed its components by mass spectrometry (Fig. 4).hey found several new proteins in association with FANCA, whichere labeled as Fanconi Anemia Associated Proteins (FAAPs) fol-

owed by their molecular weight in kDa. In addition, an associationith the helicase involved in Bloom syndrome was observed, again

uggesting that the FA core complex may have a role in DNA repair46]. Two FAAPs (FAAP43 and FAAP250) turned out to be genuineA proteins and thus led to the identification of two new comple-entation groups for the disease (FA-L and FA-M), while FAAP95

ppeared to be defective in FA-B patients.The first issue that was solved by this purification approach

as the connection between the FA core complex and FANCD2onoubiquitination [47]. From a previous study in Patel’s lab itas already clear that the FA core complex was able to interactith FANCD2 through FANCE [19], but since FAAP43 was a RING-nger protein containing E3 ubiquitin ligase activity in vitro, itecame evident that the FA core complex was directly involved inhe monoubiquitination of FANCD2. One FA patient was identifiedith a defect in FAAP43 and that is why FAAP43 was renamed as

ANCL (FANCK was skipped because it was difficult to discriminaterom FANCA in oral conversations).

Several FA patients, classified as FA-B, appeared to carry muta-ions in the gene encoding another novel subunit of the FA coreomplex, FAAP95, which was then renamed as FANCB [48]. Thepecial feature of this gene was not in its functional domains (itnly contained a bipartite nuclear localization signal), but in itsocation on the X-chromosome. As a consequence, FA-B patientsre exclusively male. Although in theory the existence of femaleA-B patients is to be expected, these patients have not beenound, because the affected FA-B males are unable to produceffspring. FANCB is subject to X-inactivation, which implies thatoth males and females have only one active copy of the gene.n theory, this would make FANCB a vulnerable component of theathway, as it would be inactivated by only one genetic hit. How-ver, there is as yet no evidence for a high proportion of FANCB

eficiency in sporadic tumors. This could be due to a growth dis-dvantage of cells that lack FANCB, which is probably also theeason why heterozygous female carriers of a FANCB defect do nothow a mosaic phenotype but demonstrated skewed X-inactivation48].

Research 668 (2009) 11–19

Interesting functional domains were present in FAAP250, whichcontains a DEAH-like helicase domain at its N-terminus and anERCC4/XPF like nuclease domain at its C-terminus [49] (see also“FANCM-FAAP24 and FANCJ: FA Proteins that Metabolize DNA” Aliet al., this issue). FAAP250 is the human ortholog of the archaeabacterial DNA repair protein Hef, but the linker region betweenthe helicase and nuclease domain in the human protein is muchlarger than in Hef. Possibly this region is used to interact with theother members of the FA core complex, which are absent in archaeabacteria. In contrast to Hef, FAAP250 only has a functional helicasedomain. FAAP250 acts as an ATP-dependent translocase rather thana helicase [49] and is able to promote branch migration of Holli-day junctions and replication forks [50]. FAAP250 probably acts asa replication fork remodeler that promotes fork reversal and cre-ates chicken-foot structures upon stalling of the replication fork[51]. The discovery of FAAP250 was a breakthrough, because it con-nected the FA core complex directly to DNA repair and stronglysuggested that this complex is able to prepare the stalled replicationfork for other DNA repair pathways. There was one FA patient withpathogenic mutations in the FAAP250 gene and therefore FAAP250became FANCM [49]. In this patient’s cell line FANCA and FANCGlevels were reduced and FANCA was not properly localized to thenucleus, suggesting that FANCM is essential for the assembly or sta-bilization of the FA core complex. However, ablation of FANCM inchicken DT40 cells [52] and siRNA knockdown experiments in HeLacells [53] clearly showed that not the stability of the FA core com-plex, but the chromatin association of the complex was affected inthe absence of FANCM. As a consequence, the monoubiquitinationof FANCD2 is strongly impaired in FANCM deficient cells [49,52–54].When FANCM is expressed, but inactivated by a point mutation inthe ATP-binding domain, FANCD2 is monoubiquitinated, but thismutant is not able to protect cells against DNA cross-linking agents[54]. These data suggest an important role for FANCM in the ATP-dependent processing of the lesion created by DNA cross-linkingagents. Interestingly, disruption of FANCM in FANCC-deficient DT40cells attenuates the effect of cross-linking agents [52], indicatingthat FANCM acts upstream of the FA core complex. In the absenceof FANCM the replication-blocking lesion may be repaired by analternative route, which bypasses the FA pathway.

The association of FANCM with chromatin seems to depend onits direct binding partner FAAP24, which also appears critical in theprotection against DNA cross-linking agents [53,55]. This predictedthe existence of FA patients with a defect in FAAP24, but until nowsuch patients have not been found. The same holds for FAAP100,another novel protein found in association with FANCA in the FAcore complex purification [56]. FAAP100 is a direct binding partnerof FANCB and needed for the stability of both FANCB and FANCL.DT40 knockouts for FAAP100 lack monoubiquitinated FANCD2 andshow cross-linker sensitivity and chromosomal instability, but noFA patient with a mutation in FAAP100 has been identified yet. Pos-sibly, mutations in FAAP24 and FAAP100 are exceedingly rare or,alternatively, may lead to a clinical phenotype that is different fromFanconi anemia.

5. FANCJ, a helicase downstream or independent of FANCD2

In 2005 the link between FA and DNA repair became more firmlyestablished, when in the same Nature Genetics issue that describedthe identification of FANCM also the BRCA1-interacting DEAH heli-case BRIP1 was reported as the gene affected in FA-J patients (seecommentary [57]). FANCJ was identified by genetic linkage analysis

and microcell-mediated chromosome transfer [58–60]. Genome-wide linkage analysis with polymorphic repeat markers on a singleconsanguineous FA-J patient revealed a large region of homozygousmarkers on chromosome 17, which prompted chromosome transferinto FANCJ-deficient fibroblasts [58]. Colony survival assays showed

tation

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the genome. Part of the picture has become clear in the past decade(Fig. 5).

The majority of the FA proteins function in a nuclear corecomplex and defects in one of each individual subunit affect thestability of the entire complex. The complex consists of several,

Fig. 5. Proteins participating in the FA/BRCA pathway. The FA core complex, whichrepresent the upstream part of the pathway, consists of FANCA, -B, -C, -E, -F, -G, -L, -M,and two FA-associated proteins FAAP24 and FAAP100. This complex is essential for

J.P. de Winter, H. Joenje / Mu

hat their MMC-hypersensitive phenotype had been corrected toild type, confirming that FANCJ was on chromosome 17. In an

ndependent study, a 50K single nucleotide polymorphism arraynalysis pointed at a similar region of homozygosity [59]. Withinhe homozygous candidate region the most convincing candidate-ene was BRIP1, since others had just shown that disruption of thisene in DT40 cells generated an FA-like phenotype [61]. Subsequentequence analysis identified pathogenic mutations in BRIP1 in allatients classified as FA-J, confirming that this gene was a true FAene and thus adding another helicase to the FA/BRCA pathway58,59].

FANCJ/BRIP1 was first identified as a novel BRCA1-interactingrotein in a pull-down assay with the C-terminal BRCT motifs ofRCA1 [62]. The protein contained a DEAH helicase domain and wasalled BRCA1-associated C-terminal helicase (BACH1). This nameas rather confusing, because a basic leucine zipper transcription

actor had been given the same name; the protein was thereforeenamed as BRCA1-interacting protein 1 (BRIP1). BRIP1 is a DNA-ependent helicase with 5′-to-3′ polarity [63], which in additiono BRCA1 also binds to the single-stranded DNA-binding proteinPA [64] and the mismatch repair proteins MLH1 and PMS2 [65].inding to these mismatch repair proteins seems essential for DNAross-link repair, whereas the interaction with BRCA1 appears to beispensable for this process [65]. FANCJ’s function probably goeseyond DNA cross-link repair, since it is essential for the stabil-

ty of G/C-rich regions in the genome of C. elegans [66,67] andnwinds G-quadruplex DNA structures formed in these G/C-tracts68]. This idea is consistent with an additive effect of FANCJ inacti-ation observed in FANCC-deficient DT40 cells [61]. Possibly, FANCJemoves road-blocking lesions during the repair process occurringt a stalled replication fork (see also “FANCM-FAAP24 and FANCJ: FAroteins that Metabolize DNA” Ali et al., this issue). In the absencef FANCJ monoubiquitination of FANCD2 still occurs, pointing to aole for FANCJ downstream or independent of FANCD2 [8].

. PALB2/FANCN, another FA protein identified by proteinssociation

The purification of the FA core complex clearly showed thatovel players in a given pathway can be identified by immuno-recipitation strategies. This approach was also critical in the

dentification of PALB2 as a new component in the downstreamart of the FA/BRCA pathway. David Livingston and co-workers

nvestigated the proteins that bind to BRCA2 in a large-scale co-mmunoprecipitation experiment [69]. They identified, in additiono RAD51, a novel BRCA2-interacting protein, which turned out toe essential for the association of BRCA2 with chromatin and forhe protein to function in recombination repair; the new proteinas called Partner and Localizer of BRCA2, or PALB2. Knockdownf PALB2 in HeLa cells resulted in a cross-linker-sensitive pheno-ype, pointing to a possible involvement in the FA/BRCA pathway.ndeed, several as yet unclassifiable FA patients appeared to carryathogenic mutations in PALB2 and lacked the full-length protein70,71]. Like FA-D1 patients with a defect in BRCA2, PALB2-deficientatients were severely affected with early childhood cancer in allases. Interestingly, in a cell line of one of these patients, geneticeversion was found, which was probably due to single strandnnealing on repeat sequences in the gene [70]. This removed aarge part of the PALB2 protein, but apparently this truncated pro-ein was functional in DNA cross-link repair. Impaired homologous

ecombination repair in BRCA2- and PALB2-deficient cells may beompensated by a more active single strand annealing process,hich might increase the probability for these genetic reversion

vents, as has been observed in the BRCA2-mutated pancreaticancer cell line, Capan-1 [72]. This phenomenon partially explains

Research 668 (2009) 11–19 15

acquired cisplatin resistance in BRCA2-deficient tumors, a featurethat is highly relevant for cancer therapy.

7. FANCI, the monoubiquitinated partner of FANCD2

The most recent addition to the FA/BRCA pathway is FANCI,which has been discovered in 2007 by different strategies [73–75].Genome-wide linkage analysis on four FA-I families resulted in fourcandidate regions for FANCI. From these regions candidate geneswere selected by bioinformatics and data mining. In candidateKIAA1794 pathogenic mutations were found in all patients classi-fied as FA-I by complementation analysis, including the referencepatent for this group [73]. At the same time, Steve Elledge and co-workers found KIAA1794/FANCI in a large-scale screen for ionizingradiation-induced ATM/ATR kinase substrates, using phosphospe-cific antibodies and mass spectrometry [74]. Others found FANCIbased upon its homology with the FANCD2 monoubiquitination sitein a screen for substrates of the FA core complex [75].

Like FANCD2, FANCI was monoubiquitinated in an FA corecomplex-dependent manner and co-localized with FANCD2 in DNAdamage-induced nuclear foci [74,75]. FANCD2 and FANCI werefound to interact in the so-called ‘ID complex’. The monoubiqui-tination of FANCI appeared to depend on the monoubiquitinationof FANCD2, whereas the monoubiquitination of FANCD2 occurredindependent of the monoubiquitination of FANCI. These ubiquiti-nation steps seem to require the phosphorylation of FANCI, whichmay be a molecular switch to turn on the FA pathway [76] (see also“The Fanconi Anemia Pathway: Insights from Somatic Cell GeneticsUsing DT40 Cell Line” Takata et al., this issue). Since in FA-I cells,FANCD2 is hardly detectable in the chromatin fraction [77], FANCIcould act as a localizer of FANCD2, which probably depends on itsphosphorylation by ATM or ATR.

8. A model for the FA/BRCA pathway

Although there may still be more new players in the FA/BRCApathway that remain to be identified [78], the field now faces thechallenge to define the functional relationships amongst the FA pro-teins and to precisely determine how the pathway acts to protect

the monoubiquitination of the FA proteins FANCD2 and FANCI. In this reaction, FANCLis the E3 ubiquitin ligase and UBE2T is the E2-conjugating enzyme. Monoubiquiti-nated FANCD2 and FANCI co-localize in damage-induced nuclear foci with BRCA2,which, together with its binding partner PALB2 and BRIP1 belong to the down-stream branch of the pathway. Ubiquitin is removed from FANCD2 and FANCI bythe deubiquitinating enzyme USP1.

16 J.P. de Winter, H. Joenje / Mutation Research 668 (2009) 11–19

Fig. 6. Model for the FA/BRCA pathway. (A) The DNA interstrand cross-link prevents further DNA replication and this recruits FANCM and the FANCD2/FANCI complexindependently. (B) The FA core complex associates with FANCM and together with UBE2T monoubiquitinates FANCD2/FANCI. (C) FANCM translocates along the DNA andp iquitinu ne-enu stranb e cros

meF[nnaF[nciFtswtF

p[ptFFuoc

cF[aitwtFs

cpPptTcbs-plc

rocesses the stalled replication fork. This creates ssDNA to which more monoubnhook the cross-linked DNA. By cutting 3′ of the lesion MUS81/EME1 generates a onhooking step, while FANCD2 recruits BRCA2 and Rad51 to the one-ended doublereak, which may be stimulated by USP1 mediated deubiquitination of FANCD2. Th

ore or less stable, subcomplexes that directly bind and stabilizeach other. FANCA and FANCG form such a subcomplex, in whichANCG binds to the N-terminal nuclear localization signal of FANCA17,79,80]. The binding of FANCG may be a way to regulate theuclear import of FANCA, by shielding the nuclear localization sig-al. There are many pathogenic missense mutations in FANCA thatffect its nuclear accumulation and interaction with FANCC andANCF, but these mutations do not influence the binding of FANCG81]. It is conceivable that these mutants are unable to expose theiruclear localization signal. This step may involve a conformationalhange induced by phosphorylation, since FANCA phosphorylations required for nuclear accumulation of FANCA and affected in theANCA mutants that fail to enter the nucleus [81,82]. In contrasto the entire FA core complex, the FANCA/FANCG subcomplex istable in the absence of the other FA proteins, but it is not clearhether this subcomplex has a function on its own, especially since

he nuclear accumulation of FANCA also depends on FANCB andANCL [18,83].

FANCB, FANCL, and FAAP100 are partners in another subcom-lex, in which FANCB directly interacts with FANCL and FAAP10056,83]. The nuclear localization of this subcomplex requires theresence of FANCA, which points to an interdependence of thesewo subcomplexes [47,48,56]. FANCA, FANCB, FANCG, FANCL, andAAP100 probably form a larger complex, which does not needANCC, FANCE, and FANCF for its stability [83]. Although still spec-lative, this complex may function as an E3 ubiquitin ligase forther substrates than FANCD2 and FANCI during repair processesoordinated by the FA pathway.

FANCD2, probably in complex with FANCI, is presented to the FAore complex for monoubiquitination by direct interaction betweenANCE and FANCD2, which involves the amino terminus of FANCD219,84]. FANCE has another direct binding partner in FANCC [84,85]nd these two proteins are interdependent for their nuclear local-zation [86,87]. A subcomplex of FANCC and FANCE interacts withhe other subcomplexes through a weak direct interaction of FANCEith both FANCA and FANCG [85]. This interaction is stabilized by

he FANCF protein, which uses its C-teminus for interaction withANCG and its N-terminus for interaction with the FANCC/FANCEubcomplex [88].

These data altogether suggest that the assembly of the FA coreomplex is a highly regulated process, in which different subcom-lexes may have different functions. However, recent work fromatel’s lab in DT40 cells has shown that the FA core complex isresent during the entire cell cycle and that the regulation seemso be restricted to the complex’ association with chromatin [89].he FA core complex localizes to chromatin in a DNA damage- andell cycle-dependent fashion [90], which seems to be orchestratedy FANCM [52,53]. The current view is that replication fork stalling

timulates FANCM to recruit a stable complex consisting of FANCA,B, -C, -E, -F, -G, and -L (Fig. 6). Through FANCE, this core com-lex interacts with its substrate FANCD2, only after FANCD2 has

oaded onto the chromatin, which occurs independent of the coreomplex itself [89] and most likely upon phosphorylation of FANCI

ated FANCD2 is loaded and generates space for MUS81/EME1 and XPF/ERCC1 toded double strand break. (D) Translesion synthesis (TLS) fills the gap formed by thed break. Homologous recombination repair (HR) is finally used to restore the DNAs-link adduct is removed by nucleotide excision repair or by other mechanisms.

[76]. The core complex, with FANCL as the catalytic subunit, actsas an E3-ubiquitin ligase to monoubiquitinate FANCD2 and FANCIin conjunction with the E2-conjugating enzyme UBE2T [91]. ThisE2 enzyme is loaded onto the chromatin independent of FANCD2and the FA core complex [89]. The monoubiquitination of FANCD2is considered as an activating step in the pathway, but it actuallyseems a targeting signal needed for stable association of FANCD2with the damaged DNA. As long as the precise function of the FApathway has not been resolved or an activity for FANCD2 has notbeen described it is not clear whether the monoubiquitination ofFANCD2 is really activating the pathway, since it may also targetFANCD2 and inhibit the protein util it is activated by USP1 throughdeubiquitination (see below).

We speculate that FANCM, through its translocase activity,may move away the core complex from the lesion that stalledthe replication fork to allow other repair factors to access theDNA (Fig. 6). Inactivation of this process by a point mutation inthe ATP-binding domain of FANCM results in reduced FANCD2monoubiquitination and cross-linker sensitivity [54]. By remod-eling the stalled replication fork [51], we propose that FANCMcreates single-stranded DNA, which could activate the DNA dam-age kinase ATR and its downstream target Chk1. ATR may thenphosphorylate FANCI and FANCD2, loading more FANCD2/FANCIcomplex to the site of damage, which can in turn be monoubiq-uitinated by the FA core complex. Chk1 phosphorylates FANCE atT346 and S374, targeting FANCE for degradation by the protea-some [92], which in our view could function to avoid overloadingof monoubiquitinated FANCD2. The phosphorylation and degrada-tion of FANCE may leave the FANCA, -B, -G, -L, FAAP100 subcomplexat the site of damage for additional functions, e.g. ubiquitinationof other substrates. Interference with the Chk1-mediated phos-phorylation of FANCE, by inactivating the phosphorylation sites onFANCE, results in increased levels of monoubiquitinated FANCD2, anaccumulation of nuclear FANCD2 foci, and an MMC-hypersensitivephenotype [92]. Monoubiquitinated FANCD2 may recruit BRCA2[44], which was loaded onto the chromatin by PALB2 and possiblythis event is important to prepare the lesion for repair by homol-ogous recombination. Finally, ubiquitin is removed from FANCD2by the deubiquitinating enzyme USP1 [93], a step that is essen-tial for cross-link repair [94]. This probably removes FANCD2 fromthe damaged site and may be a trigger for BRCA2 to start load-ing RAD51 and activate homologous recombination. In this wholeprocess, FANCJ may function to remove DNA structures that wouldimpede repair of the lesion.

Besides the FA proteins, several other proteins have been impli-cated in the repair of lesions generated by DNA cross-linking agentsand the FA/BRCA pathway may play a role in regulating these pro-teins. Unhooking of the DNA cross-link requires the endonuclease

MUS81-EME1 [95], which creates a one-ended double-strand break(Fig. 6), while XPF-ERCC1 [96] incises on the other side of the cross-link. This generates a substrate for translesion synthesis to bypassthe DNA cross-link. Studies in chicken DT40 cells have shown thatRev3, the catalytic subunit of the low-fidelity DNA repair poly-

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J.P. de Winter, H. Joenje / Mu

erase zeta, is involved in cross-link repair in a pathway that ispistatic with FANCC [97]. In the same system FANCC was epistaticith XRCC2, a RAD51 paralog involved in homologous recombina-

ion repair, which again underlines the cross-talk between the FAnd homologous recombination pathways. Like XRCC2, the otherAD51 paralogs XRCC3, RAD51B, RAD51C, and RAD51D are also

nvolved in cross-link repair [98].In summary, we can conclude that the repair of damage induced

y DNA cross-linking agents is a highly complex process requiringultiple repair pathways. It is because of Fanconi’s description, 80

ears ago, that we now have a better picture of how this process isoordinated by a set of proteins that, when defective, cause the clin-cal entity Fanconi anemia. Although we have just seen the tip of theceberg, Fanconi anemia already provides a nice illustration of howrare genetic disorder can help to uncover a basic cellular process.uring the next decade, the pathway will be further unraveled and

he new knowledge will hopefully help to improve the prognosisor FA patients and lead to better treatment options not only for FAatients, but also for patients suffering from sporadic cancer.

onflict of interest statement

Nothing to declare.

cknowledgements

The work of our group has been supported by the Dutch Can-er Society, the Netherlands Organization for Health Research andevelopment and the FA Research Fund Inc.

eferences

[1] G. Fanconi, Familiäre infantile perniziosartige anämie (pernizioses blutbild undkonstitution), Jahrb. Kinderh. 117 (1927) 257–280.

[2] T.M. Schroeder, F. Anschütz, A. Knopp, Spontane chromosomenaberrationen beifamiliärer panmyelopathie, Humangenetik 1 (1964) 194–196.

[3] M. Sasaki, Is Fanconi’s anaemia defective in a process essential for the repair ofDNA cross links? Nature 257 (1975) 501–503.

[4] A.D. Auerbach, S.R. Wolman, Susceptibility of Fanconi’s anaemia fibroblasts tochromosome damage by carcinogens, Nature 261 (1976) 494–496.

[5] C.A. Strathdee, A.M. Duncan, M. Buchwald, Evidence for at least four Fanconianaemia genes including FACC on chromosome 9, Nat. Genet. 1 (1992) 196–198.

[6] H. Joenje, J.R. Lo Ten Foe, A.B. Oostra, C.G. van Berkel, M.A. Rooimans, T.Schroeder-Kurth, R.D. Wegner, J.J. Gille, M. Buchwald, F. Arwert, Classificationof Fanconi anemia patients by complementation analysis: evidence for a fifthgenetic subtype, Blood 86 (1995) 2156–2160.

[7] H. Joenje, A.B. Oostra, M. Wijker, F.M. di Summa, C.G. van Berkel, M.A. Rooimans,W. Ebell, M. van Weel, J.C. Pronk, M. Buchwald, F. Arwert, Evidence for at leasteight Fanconi anemia genes, Am. J. Hum. Genet. 61 (1997) 940–944.

[8] M. Levitus, M.A. Rooimans, J. Steltenpool, N.F. Cool, A.B. Oostra, C.G. Mathew,M.E. Hoatlin, Q. Waisfisz, F. Arwert, J.P. de Winter, H. Joenje, Heterogeneityin Fanconi anemia: evidence for 2 new genetic subtypes, Blood 103 (2004)2498–2503.

[9] H. Joenje, M. Levitus, Q. Waisfisz, A. D’Andrea, I. Garcia-Higuera, T. Pearson,C.G.M. van Berkel, M.A. Rooimans, N. Morgan, C.G. Mathew, F. Arwert, Com-plementation analysis in Fanconi anemia: assignment of the reference FA-Hpatient to group A, Am. J. Hum. Genet. 67 (2000) 759–762.

10] M. Gross, H. Hanenberg, S. Lobitz, R. Friedl, S. Heterich, R. Dietrich, B. Gruhn, D.Schindler, H. Hoehn, Reverse mosaicism in Fanconi anemia: natural gene ther-apy via molecular self-correction, Cytogenet. Genome Res. 98 (2002) 126–135.

11] C.A. Strathdee, H. Gavish, W.R. Shannon, M. Buchwald, Cloning of cDNAs forFanconi’s anaemia by functional complementation, Nature 356 (1992) 763–767.

12] J.R. Lo Ten Foe, M.A. Rooimans, L. Bosnoyan-Collins, N. Alon, M. Wijker, L. Parker,J. Lightfoot, M. Carreau, D.F. Callen, A. Savoia, N.C. Cheng, C.G. van Berkel, M.H.Strunk, J.J. Gille, G. Pals, F.A. Kruyt, J.C. Pronk, F. Arwert, M. Buchwald, H. Joenje,Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA, Nat.Genet. 14 (1996) 320–323.

13] J.P. de Winter, Q. Waisfisz, M.A. Rooimans, C.G.M. van Berkel, L. Bosnoyan-Collins, N. Alon, M. Carreau, O. Bender, I. Demuth, D. Schindler, J.C. Pronk, F.Arwert, H. Hoehn, M. Digweed, M. Buchwald, H. Joenje, The Fanconi anaemia

group G gene FANCG is identical with XRCC9, Nat. Genet. 20 (1998) 281–283.

14] J.P. de Winter, M.A. Rooimans, L. van der Weel, C.G.M. van Berkel, N. Alon, L.Bosnoyan-Collins, J. De Groot, Y. Zhi, Q. Waisfisz, J.C. Pronk, F. Arwert, C.G.Mathew, R.J. Scheper, M.E. Hoatlin, M. Buchwald, H. Joenje, The Fanconi anaemiagene FANCF encodes a novel protein with homology to ROM, Nat. Genet. 24(2000) 15–16.

[

Research 668 (2009) 11–19 17

15] J.P. de Winter, F. Léveillé, C.G.M. van Berkel, M.A. Rooimans, L. van der Weel, J.Steltenpool, I. Demuth, N.V. Morgan, N. Alon, L. Bosnoyan-Collins, J. Lightfoot,K. Komatsu, F. Arwert, P.A. Leegwater, Q. Waisfisz, J.C. Pronk, C.G. Mathew, M.Digweed, M. Buchwald, H. Joenje, Isolation of a cDNA representing the Fan-coni anemia complementation group E gene, Am. J. Hum. Genet. 67 (2000)1306–1308.

16] G.M. Kupfer, D. Näf, M. Pulsipher, A.D. D’Andrea, The Fanconi anaemia proteins,FAA and FAC, interact to form a nuclear complex, Nat. Genet. 17 (1997) 487–490.

[17] I. Garcia-Higuera, Y. Kuang, D. Näf, J. Wasik, A.D. D’Andrea, Fanconi anemiaproteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclearcomplex, Mol. Cell Biol. 19 (1999) 4866–4873.

[18] J.P. de Winter, L. van der Weel, F.A.E. Kruyt, J. de Groot, Y. Zhi, Q. Waisfisz, F.Arwert, R.J. Scheper, M.E. Hoatlin, H. Joenje, The Fanconi anaemia protein FANCFforms a nuclear complex with FANCA, FANCC and FANCG, Hum. Mol. Genet. 9(2000) 2665–2674.

19] P. Pace, M. Johnson, W.M. Tan, G. Mosedale, C. Sng, M. Hoatlin, J. de Winter, H.Joenje, F. Gergely, K.J. Patel, FANCE: the link between Fanconi anemia complexassembly and activity, EMBO J. 21 (2002) 3414–3423.

20] E. Blom, H.J. van der Vrugt, Y. de Vries, J.P. de Winter, F. Arwert, H. Joenje, Mul-tiple TPR motifs characterize the Fanconi anemia FANCG protein, DNA Repair 3(2004) 77–84.

21] The Fanconi anaemia/breast cancer consortium, Positional cloning of the Fan-coni anaemia group A gene, Nat. Genet. 14 (1996) 324–328.

22] M. Whitney, M. Thayer, C. Reifsteck, S. Olson, L. Smith, P.M. Jacobs, R. Leach, S.Naylor, H. Joenje, M. Grompe, Microcell mediated chromosome transfer mapsthe Fanconi anaemia group D gene to chromosome 3p, Nat. Genet. 11 (1995)341–343.

23] J.A. Hejna, C.D. Timmers, C. Reifsteck, D.A. Bruun, L.W. Lucas, P.M. Jacobs, S. Toth-Fejel, N. Unsworth, S.L. Clemens, D.K. Garcia, S.L. Naylor, M.J. Thayer, S.B. Olson,M. Grompe, R.E. Moses, Localization of the Fanconi anemia complementationgroup D gene to a 200-kb region on chromosome 3p25.3, Am. J. Hum. Genet.66 (2000) 1540–1551.

24] C. Timmers, T. Taniguchi, J. Hejna, C. Reifsteck, L. Lucas, D. Bruun, M. Thayer, B.Cox, S. Olson, A.D. D’Andrea, R. Moses, M. Grompe, Positional cloning of a novelFanconi anemia gene, FANCD2, Mol. Cell 7 (2001) 241–248.

25] I. Garcia-Higuera, T. Taniguchi, S. Ganesan, M.S. Meyn, C. Timmers, J. Hejna, M.Grompe, A.D. D’Andrea, Interaction of the Fanconi anemia proteins and BRCA1in a common pathway, Mol. Cell 7 (2001) 249–262.

26] T. Taniguchi, I. Garcia-Higuera, P.R. Andreassen, R.C. Gregory, M. Grompe, A.D.Grompe, S-phase-specific interaction of the Fanconi anemia protein, FANCD2,with BRCA1 and Rad51, Blood 100 (2002) 2414–2420.

27] C.J. Vandenberg, F. Gergely, C.Y. Ong, P. Pace, D.L. Mallery, K. Hiom, K.J.Patel, BRCA1-independent ubiquitination of FANCD2, Mol. Cell 12 (2003)247–254.

28] M. Bogliolo, A. Lyakhovich, E. Callen, M. Castellà, E. Cappelli, M.J. Ramirez, A.Creus, R. Marcos, R. Kalb, K. Neveling, D. Schindler, J. Surrallés, Histone H2AXand Fanconi anemia FANCD2 function in the same pathway to maintain chro-mosome stability, EMBO J. 26 (2007) 1340–1351.

29] I.M. Ward, J. Chen, Histone H2AX is phosphorylated in an ATR-dependent man-ner in response to replicational stress, J. Biol. Chem. 276 (2001) 47759–47762.

30] P.R. Andreassen, A.D. D’Andrea, T. Taniguchi, ATR couples FANCD2 monoubiq-uitination to the DNA-damage response, Genes Dev. 18 (2004) 1958–1963.

31] G.P. Ho, S. Margossian, T. Taniguchi, A.D. D’Andrea, Phosphorylation of FANCD2on two novel sites is required for mitomycin C resistance, Mol. Cell Biol. 26(2006) 7005–7015.

32] N.G. Howlett, T. Taniguchi, S. Olson, B. Cox, Q. Waisfisz, C. De Die-Smulders, N.Persky, M. Grompe, H. Joenje, G. Pals, H. Ikeda, E.A. Fox, A.D. D’Andrea, Biallelicinactivation of BRCA2 in Fanconi anemia, Science 297 (2002) 606–609.

33] B.C. Godthelp, F. Arwert, H. Joenje, M.Z. Zdzienicka, Impaired DNA damage-induced nuclear Rad51 foci formation uniquely characterizes Fanconi anemiagroup D1, Oncogene 21 (2002) 5002–5005.

34] B.P. Alter, P.S. Rosenberg, L.C. Brody, Clinical and molecular features associatedwith biallelic mutations in FANCD1/BRCA2, J. Med. Genet. 44 (2007) 1–9.

35] S.K. Sharan, M. Morimatsu, U. Albrecht, D.S. Lim, E. Regel, C. Dinh, A. Sands, G.Eichele, P. Hasty, A. Bradley, Embryonic lethality and radiation hypersensitivitymediated by Rad51 in mice lacking Brca2, Nature 386 (1997) 804–810.

36] N.C. Cheng, H.J. van der Vrugt, M.A. van der Valk, A.B. Oostra, P. Krimpenfort, Y.de Vries, H. Joenje, A. Berns, F. Arwert, Mice with a targeted disruption of theFanconi anemia homolog Fanca, Hum. Mol. Genet. 9 (2000) 1805–1811.

37] J.C. Wong, N. Alon, C. Mckerlie, J.R. Huang, M.S. Meyn, M. Buchwald, Targeteddisruption of exons 1 to 6 of the Fanconi Anemia group A gene leads to growthretardation, strain-specific microphthalmia, meiotic defects and primordialgerm cell hypoplasia, Hum. Mol. Genet. 12 (2003) 2063–2076.

38] M. Chen, D.J. Tomkins, W. Auerbach, C. McKerlie, H. Youssoufian, L. Liu, O. Gan,M. Carreau, A. Auerbach, T. Groves, C.J. Guidos, M.H. Freedman, J. Cross, D.H.Percy, J.E. Dick, A.L. Joyner, M. Buchwald, Inactivation of Fac in mice producesinducible chromosomal instability and reduced fertility reminiscent of Fanconianaemia, Nat. Genet. 12 (1996) 448–451.

39] M.A. Whitney, G. Royle, M.J. Low, M.A. Kelly, M.K. Axthelm, C. Reifsteck, S.

Olson, R.E. Braun, M.C. Heinrich, R.K. Rathbun, G.C. Bagby, M. Grompe, Germcell defects and hematopoietic hypersensitivity to gamma-interferon in micewith a targeted disruption of the Fanconi anemia C gene, Blood 88 (1996) 49–58.

40] S. Houghtaling, C. Timmers, M. Noll, M.J. Finegold, S.N. Jones, M.S. Meyn,M. Grompe, Epithelial cancer in Fanconi anemia complementation group D2(Fancd2) knockout mice, Genes Dev. 17 (2003) 2021–2035.

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41] Y. Yang, Y. Kuang, R. Montes De Oca, T. Hays, L. Moreau, N. Lu, B. Seed, A.D.D’Andrea, Targeted disruption of the murine Fanconi anemia gene, Fancg/Xrcc9,Blood 98 (2001) 3435–3440.

42] M. Koomen, N.C. Cheng, H.J. van de Vrugt, B.C. Godthelp, M.A. van der Valk, A.B.Oostra, M.Z. Zdzienicka, H. Joenje, F. Arwert, Reduced fertility and hypersensi-tivity to mitomycin C characterize Fancg/Xrcc9 null mice, Hum. Mol. Genet. 11(2002) 273–281.

43] S. Hussain, J.B. Wilson, A.L. Medhurst, J. Hejna, E. Witt, S. Ananth, A. Davies, J.Y.Masson, R. Moses, S.C. West, J.P. de Winter, A. Ashworth, N.J. Jones, C.G. Mathew,Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways,Hum. Mol. Genet. 13 (2004) 1241–1248.

44] X. Wang, P.R. Andreassen, A.D. D’Andrea, Functional interaction of monoubiq-uitinated FANCD2 and BRCA2/FANCD1 in chromatin, Mol. Cell Biol. 24 (2004)5850–5862.

45] R.D. Kennedy, C.C. Chen, P. Stuckert, E.M. Archila, M.A. De la Vega, L.A. Moreau,A. Shimamura, A.D. D’Andrea, Fanconi anemia pathway-deficient tumor cellsare hypersensitive to inhibition of ataxia telangiectasia mutated, J. Clin. Invest.117 (2007) 1440–1449.

46] A.R. Meetei, S. Sechi, M. Wallisch, D. Yang, M.K. Young, H. Joenje, M.E. Hoatlin,W. Wang, A multiprotein nuclear complex connects Fanconi anemia and Bloomsyndrome, Mol. Cell Biol. 23 (2003) 3417–3426.

47] A.R. Meetei, J.P. de Winter, A.L. Medhurst, M. Wallisch, Q. Waisfisz, H.J. van deVrugt, A.B. Oostra, Z. Yan, C. Ling, C.E. Bishop, M.E. Hoatlin, H. Joenje, W. Wang,A novel ubiquitin ligase is deficient in Fanconi anemia, Nat. Genet. 35 (2003)165–170.

48] A.R. Meetei, M. Levitus, Y. Xue, A.L. Medhurst, M. Zwaan, C. Ling, M.A. Rooimans,P. Bier, M. Hoatlin, G. Pals, J.P. de Winter, W. Wang, H. Joenje, X-linked inher-itance of Fanconi anemia complementation group B, Nat. Genet. 36 (2004)1219–1224.

49] A.R. Meetei, A.L. Medhurst, C. Ling, Y. Sue, T.R. Singh, P. Bier, J. Steltenpool, S.Stone, I. Dokal, C.G. Mathew, M. Hoatlin, H. Joenje, J.P. de Winter, W. Wang,A human ortholog of archaeal DNA repair protein Hef is defective in Fanconianemia complementation group M, Nat. Genet. 37 (2005) 958–963.

50] K. Gari, C. Décaillet, A.Z. Stasiak, A. Stasiak, A. Constantinou, The Fanconi ane-mia protein FANCM can promote branch migration of Holliday junctions andreplication forks, Mol. Cell 29 (2008) 141–148.

51] K. Gari, C. Décaillet, M. Delannoy, L. Wu, Constantinou, Remodeling of DNAreplication structures by the branch point translocase FANCM, Proc. Natl. Acad.Sci. U.S.A. 105 (2008) 16107–16112.

52] G. Mosedale, W. Niedzwiedz, A. Alpi, F. Perrina, J.B. Pereira-Leal, M. Johnson, F.Langevin, P. Pace, K.J. Patel, The vertebrate Hef ortholog is a component of theFanconi anemia tumor-suppressor pathway, Nat. Struct. Mol. Biol. 12 (2005)763–771.

53] J.M. Kim, Y. Kee, A. Gurtan, A.D. D’Andrea, Cell cycle-dependent chromatin load-ing of the Fanconi anemia core complex by FANCM/FAAP24, Blood 111 (2008)5215–5222.

54] Y. Xue, Y. Li, R. Guo, C. Ling, W. Wang, FANCM of the Fanconi anemia core complexis required for both monoubiquitination and DNA repair, Hum. Mol. Genet. 17(2008) 1641–1652.

55] A. Ciccia, C. Ling, R. Coulthard, Z. Yan, Y. Xue, A.R. Meetei, E.H. Laghmani, H.Joenje, N. McDonald, J.P. de Winter, W. Wang, S.C. West, Identification of FAAP24,a Fanconi anemia core complex protein that interacts with FANCM, Mol. Cell 25(2007) 331–343.

56] C. Ling, M. Ishiai, A.M. Ali, A.L. Medhurst, K. Neveling, R. Kalb, Z. Yan, Y. Xue,A.B. Oostra, A.D. Auerbach, M.E. Hoatlin, D. Schindler, H. Joenje, J.P. de Winter,M. Takata, A.R. Meetei, W. Weidong, FAAP100 is essential for activation of theFanconi anemia-associated DNA damage response pathway, EMBO J. 26 (2007)2104–2114.

57] L.J. Niedernhofer, A.S. Lalai, J.H. Hoeijmakers, Fanconi anemia (cross)linked toDNA repair, Cell 123 (2005) 1191–1198.

58] M. Levitus, Q. Waisfisz, B.C. Godthelp, Y. de Vries, S. Hussain, W.W. Wiegant,E. Elghalbzouri-Maghrani, J. Steltenpool, M.A. Rooimans, G. Pals, F. Arwert, C.G.Mathew, M.Z. Zdzienicka, K. Hiom, J.P. de Winter, H. Joenje, The DNA helicaseBRIP1 is defective in Fanconi anemia complementation group J, Nat. Genet. 37(2005) 934–935.

59] O. Levran, C. Attwooll, R.T. Henry, K.L. Milton, K. Neveling, P. Rio, S.D. Batish,R. Kalb, E. Velleuer, S. Barral, J. Ott, J. Petrini, D. Schindler, H. Hanenberg, A.D.Auerbach, The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia,Nat. Genet. 37 (2005) 931–933.

60] R. Litman, M. Peng, Z. Jin, F. Zhang, S. Powell, P.R. Andreassen, S.B. Cantor, BACH1is critical for homologous recombination and appears to be the Fanconi anemiagene product FANCJ, Cancer Cell 8 (2005) 255–265.

61] W.L. Bridge, C.J. Vandenberg, R.J. Franklin, K. Hiom, The BRIP1 helicase func-tions independently of BRCA1 in the Fanconi anemia pathway for DNA crosslinkrepair, Nat. Genet. 37 (2005) 953–957.

62] S.B. Cantor, D.W. Bell, S. Ganesan, E.M. Kass, R. Drapkin, S. Grossman, D.C.Wahrer, D.C. Sgroi, W.S. Lane, D.A. Haber, D.M. Livingston, BACH1, a novelhelicase-like protein, interacts directly with BRCA1 and contributes to its DNArepair function, Cell 105 (2001) 149–160.

63] S. Cantor, R. Drapkin, F. Zhang, Y. Lin, S. Pamidi, D.M. Livingston, The BRCA1-

associated protein BACH1 is a DNA helicase targeted by clinically relevantinactivating mutations, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 2357–2362.

64] R. Gupta, S. Sharma, J.A. Sommers, M.K. Kenny, S.B. Cantor, R.M. Brosh Jr., FANCJ(BACH1) helicase forms DNA damage inducible foci with replication protein Aand interacts physically and functionally with the single-stranded DNA-bindingprotein, Blood 110 (2007) 2390–2398.

[

Research 668 (2009) 11–19

65] M. Peng, R. Litman, J. Xie, S. Sharma, R.M. Brosh Jr., S.B. Cantor, TheFANCJ/MutLalpha interaction is required for correction of the cross-linkresponse in FA-J cells, EMBO J. 26 (2007) 3238–3249.

66] E. Kruisselbrink, V. Guryev, K. Brouwer, D.B. Pontier, E. Cuppen, M. Tijsterman,Mutagenic capacity of endogenous G4 DNA underlies genome instability inFANCJ-defective C. elegans, Curr. Biol. 18 (2008) 900–905.

67] J.L. Youds, L.J. Barber, J.D. Ward, S.J. Collis, N.J. O’Neil, S.J. Boulton, A.M. Rose,DOG-1 is the Caenorhabditis elegans BRIP1/FANCJ homologue and functions ininterstrand cross-link repair, Mol. Cell. Biol. 28 (2008) 1470–1479.

68] Y. Wu, K. Shin-Ya, R.M. Brosh Jr., FANCJ helicase defective in Fanconia anemiaand breast cancer unwinds G-quadruplex DNA to defend genomic stability, Mol.Cell. Biol. 28 (2008) 4116–4128.

69] B. Xia, Q. Sheng, K. Nakanishi, A. Ohashi, J. Wu, N. Christ, X. Liu, M. Jasin, F.J.Couch, D.M. Livingston, Control of BRCA2 cellular and clinical functions by anuclear partner, PALB2, Mol. Cell 22 (2006) 719–729.

70] B. Xia, J.C. Dorsman, N. Ameziane, Y. de Vries, M.A. Rooimans, Q. Sheng, G. Pals, A.Errami, E. Gluckman, J. Llera, W. Wang, D.M. Livingston, H. Joenje, J.P. de Winter,Fanconi anemia is associated with a defect in the BRCA2 partner PALB2, Nat.Genet. 39 (2007) 159–161.

71] S. Reid, D. Schindler, H. Hanenberg, K. Barker, S. Hanks, R. Kalb, K. Neveling, P.Kelly, S. Seal, M. Freud, M. Wurm, S.D. Batish, F.P. Lach, S. Yetgin, H. Neitzel, H.Ariffin, M. Tischkowitz, C.G. Mathew, A.D. Auerbach, N. Rahman, Biallelic muta-tions in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhoodcancer, Nat. Genet. 39 (2007) 162–164.

72] W. Sakai, E.M. Swisher, B.Y. Karlan, M.K. Agarwal, J. Higgins, C. Friedman, E. Vil-legas, C. Jacquemont, D.J. Farrugia, F.J. Couch, N. Urban, T. Taniguchi, Secondarymutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers,Nature 451 (2008) 1116–1120.

73] J.C. Dorsman, M. Levitus, D. Rockx, M.A. Rooimans, A.B. Oostra, A. Haitjema,S.T. Bakker, J. Steltenpool, D. Schuler, S. Mohan, D. Schindler, F. Arwert, G.Pals, C.G. Mathew, Q. Waisfisz, J.P. de Winter, H. Joenje, Identification of theFanconi anemia complementation group I gene, FANCI, Cell. Oncol. 29 (2007)211–218.

[74] A. Smogorzewska, S. Matsuoka, P. Vinciguerra, E.R. McDonald III, K.E. Hurov, J.Luo, B.A. Ballif, S.P. Gygi, K. Hofmann, A.D. D’Andrea, S.J. Elledge, Identificationof the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNArepair, Cell 129 (2007) 289–301.

75] A.E. Sims, E. Spiteri, R.J. Sims III, A.G. Arita, F.P. Lach, T. Landers, M. Wurm, M.Freund, K. Neveling, H. Hanenberg, A.D. Auerbach, T.T. Huang, FANCI the secondmonoubiquitinated member of the Fanconi anemia pathway, Nat. Struct. Mol.Biol. 14 (2007) 564–567.

76] M. Ishiai, H. Kitao, A. Smogorzewska, J. Tomida, A. Kinomura, E. Uchida, A. Saberi,E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, S. Tashiro, S.J. Elledge, M. Takata,FANCI phosphorylation functions as a molecular switch to turn on the Fanconianemia pathway, Nat. Struct. Mol. Biol. 15 (2008) 1138–1146.

77] M. Levitus, H. Joenje, J.P. de Winter, The Fanconi anemia pathway of genomicmaintenance, Cell. Oncol. 28 (2006) 3–29.

78] N. Ameziane, A. Errami, F. Léveillé, C. Fontaine, Y. Waterham, R.M.L. Spaen-donk, J.P. de Winter, G. Pals, Joenje, genetic subtyping of Fanconi anemia bycomprehensive mutation screening, Hum. Mutat. 29 (2008) 159–166.

79] Q. Waisfisz, J.P. de Winter, F.A. Kruyt, J. de Groot, L. van der Weel, L.M. Dijkmans,Y. Zhi, F. Arwert, R.J. Scheper, H. Youssoufian, M.E. Hoatlin, H. Joenje, A physicalcomplex of the Fanconi anemia proteins FANCG/XRCC9 and FANCA, Proc. Natl.Acad. Sci. U.S.A. 96 (1999) 10320–10325.

80] I. Garcia-Higuera, Y. Kuang, J. Denham, A.D. D’Andrea, The Fanconi anemiaproteins FANCA and FANCG stabilize each other and promote the nuclear accu-mulation of the Fanconi anemia complex, Blood 96 (2000) 3224–3230.

81] D. Adachi, T. Oda, H. Yagasaki, K. Nakasato, T. Taniguchi, A.D. D’Andrea, S. Asano,T. Yamashita, Heterogeneous activation of the Fanconi anemia pathway bypatient-derived FANCA mutants, Hum. Mol. Genet. 11 (2002) 3125–3134.

82] T. Yamashita, G.M. Kupfer, D. Näf, A. Suliman, H. Joenje, S. Asano, A.D. D’Andrea,The Fanconi anemia pathway requires FAA phosphorylation and FAA/FACnuclear accumulation, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 13085–13090.

83] A.L. Medhurst, E.H. Laghmani, J. Steltenpool, M. Ferrer, C. Fontaine, J. de Groot,M.A. Rooimans, R.J. Scheper, A.R. Meetei, W. Wang, H. Joenje, J.P. de Winter,Evidence for subcomplexes in the Fanconi anemia pathway, Blood 108 (2006)2072–2080.

84] S.M. Gordon, M. Buchwald, Fanconi anemia protein complex: mapping proteininteractions in the yeast 2- and 3-hybrid systems, Blood 102 (2003) 136–141.

85] A.L. Medhurst, P.A.J. Huber, Q. Waisfisz, J.P. de Winter, C.G. Mathew, Directinteractions of the five known Fanconi anaemia proteins suggest a commonfunctional pathway, Hum. Mol. Genet. 10 (2001) 423–429.

86] T. Taniguchi, A.D. D’Andrea, The Fanconi anemia protein, FANCE, promotes thenuclear accumulation of FANCC, Blood 100 (2002) 2457–2462.

87] F. Léveillé, M. Ferrer, A.L. Medhurst, E.H. Laghmani, M.A. Rooimans, P. Bier, J.Steltenpool, T.A. Titus, J.H. Postlethwait, M.E. Hoatlin, H. Joenje, J.P. Winter, Thenuclear accumulation of the Fanconi anemia protein FANCE depends on FANCC,DNA Repair 5 (2006) 556–565.

88] F. Léveillé, E. Blom, A.L. Medhurst, P. Bier, E.H. Laghmani, M. Johnson, M.A.Rooimans, A. Sobeck, Q. Waisfisz, F. Arwert, K.J. Patel, M.E. Hoatlin, H. Joenje,

J.P. de Winter, The Fanconi anemia gene product FANCF is a flexible adaptorprotein, J. Biol. Chem. 279 (2004) 39421–39430.

89] A. Alpi, F. Langevin, G. Mosedale, Y.J. Machida, A. Dutta, K.J. Patel, UBE2T, theFanconi anemia core complex, and FANCD2 are recruited independently tochromatin: a basis for the regulation of FANCD2 monoubiquitination, Mol. CellBiol. 27 (2007) 8421–8430.

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J.P. de Winter, H. Joenje / Mu

90] F. Qiao, A. Moss, G.M. Kupfer, Fanconi anemia proteins localize to chromatinand the nuclear matrix in a DNA damage- and cell cycle-regulated manner, J.Biol. Chem. 26 (2001) 23391–23396.

91] Y.J. Machida, Y. Machida, Y. Chen, A.M. Gurtan, G.M. Kupfer, A.D. D’Andrea, A.Dutta, UBE2T is the E2 in the Fanconi anemia pathway and undergoes negativeautoregulation, Mol. Cell 23 (2006) 589–596.

92] X.Z. Wang, R.D. Kennedy, K. Ray, P. Struckert, T. Ellenberger, A.D. D’Andrea, Chk1-mediated phosphorylation of FANCE is required for the Fanconi anemia/BRCApathway, Mol. Cell Biol. 27 (2007) 3098–3108.

93] S.M. Nijman, T.T. Huang, A.M. Dirac, T.R. Brummelkamp, R.M. Kerkhoven, A.D.D’Andrea, R. Bernards, The deubiquitinating enzyme USP1 regulates the Fanconianemia pathway, Mol. Cell 17 (2005) 331–339.

94] V.H. Oestergaard, F. Langevin, H.J. Kuiken, P. Pace, W. Niedzwiedz, L.J. Simpson,M. Ohzeki, M. Takata, J.E. Sale, K.J. Patel, Deubiquitination of FANCD2 is requiredfor DNA crosslink repair (USP1), Mol. Cell 28 (2007) 798–809.

[

Research 668 (2009) 11–19 19

95] K. Hanada, M. Buzowska, M. Modesti, A. Maas, C. Wyman, J. Essers, R. Kanaar,The structure-specific endonuclease Mus81-Eme1 promotes conversion ofinterstrand DNA crosslinks into double-strands breaks, EMBO J. 25 (2006)4921–4932.

96] I. Kuraoka, W.R. Kobertz, R.R. Ariza, M. Biggerstaff, J.M. Eismann, R.D.Wood, Repair of an interstrand DNA cross-link initiated by ERCC1-XPFrepair/recombination nuclease, J. Biol. Chem. 275 (2000) 26632–26636.

97] W. Niedzwiedz, G. Mosedale, M. Johnson, C.Y. Ong, P. Pace, K.J. Patel, The Fanconi

DNA repair, Mol. Cell 15 (2004) 607–620.98] M. Takata, M.S. Sasaki, S. Tachiiri, T. Fukushima, E. Sonoda, D. Schild, L.H. Thomp-

son, S. Takeda, Chromosome instability and defective recombinational repairin knockout mutants of the five Rad51 paralogs, Mol. Cell Biol. 21 (2001)2858–2866.