the fanconi anemia protein, fancg, binds to the ercc1-xpf endonuclease via its tetratricopeptide...

pubs.acs.org/Biochemistry Published on Web 06/02/2010 r 2010 American Chemical Society

5560 Biochemistry 2010, 49, 5560–5569

DOI: 10.1021/bi100584c

The Fanconi Anemia Protein, FANCG, Binds to the ERCC1-XPF Endonuclease via ItsTetratricopeptide Repeats and the Central Domain of ERCC1†

Chuan Wang and Muriel W. Lambert*

Department of Pathology and Laboratory Medicine, New Jersey Medical School and Graduate School of Biomedical Sciences,University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103

Received April 16, 2010; Revised Manuscript Received May 28, 2010

ABSTRACT: There is evidence that Fanconi anemia (FA) proteins play an important role in the repair of DNAinterstrand cross-links (ICLs), but the precise mechanism by which this occurs is not clear. One of the criticalsteps in the ICL repair process involves unhooking of the cross-link from DNA by incisions on one strand oneither side of the ICL and its subsequent removal. The ERCC1-XPF endonuclease is involved in thisunhooking step and in the removal of the cross-link.We have previously shown that several of the FAproteinsare needed to produce incisions created by ERCC1-XPF at sites of ICLs. To more clearly establish a linkbetween FA proteins and the incision step(s) mediated by ERCC1-XPF, we undertook yeast two-hybridanalysis to determine whether FANCA, FANCC, FANCF, and FANCG directly interact with ERCC1 andXPF and, if so, to determine the sites of interaction. One of these FA proteins, FANCG, was found to have astrong affinity for ERCC1 and a moderate affinity for XPF. FANCG has been shown to contain seventetratricopeptide repeat (TPR) motifs, which are motifs that mediate protein-protein interactions. Mappingthe sites of interaction of FANCG with ERCC1, using site-directed mutagenesis, demonstrated that TPRs 1,3, 5, and 6 are needed for binding of FANCG to ERCC1. ERCC1, in turn, was shown to interact withFANCG via its central domain, which is different from the region of ERCC1 that binds to XPF. This bindingbetween FANCG and the ERCC1-XPF endonuclease, combined with our previous studies which show thatFANCG is involved in the incision step mediated by ERCC1-XPF, establishes a link between an FA proteinand the critical unhooking step of the ICL repair process.

Fanconi anemia (FA)1 is a genetic disorder characterized bygenomic instability, bone marrow failure, diverse congenitalabnormalities, an increased incidence of cancer, and a markedcellular hypersensitivity to DNA interstrand cross-linkingagents (1-5). This hypersensitivity correlates with a defect in theability to repair cross-links produced by these agents (5-12).Thirteen FA complementation groups have been identified (FA-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, and -N) (5, 13-15).Proteins encoded by eight of the FA genes form a nuclear corecomplex (FANCA, FANCB, FANCC, FANCE, FANCF,FANCG, FANCL, and FANCM) (4, 16, 17). There is evidencethat the core and the other FANC proteins are involved in DNAinterstrand cross-link (ICL) repair, but the precise mechanism bywhich this occurs is not clear (5, 10, 12, 18-22). ICLs representimportant blocks to DNA replication and are of particularsignificance in FA cells where there is a defect in replication-dependent ICL repair (4, 12, 22-24). Repair of DNA interstrandcross-links at stalled replication forks has been proposed to

involve a number of steps, including creation of a double-strandbreak (DSB) at the site of the stalled replication fork, unhookingof the cross-link, translesion synthesis, excision of the mono-adduct by nucleotide excision repair (NER), and homologousrecombination (HR) (10, 12, 23-25). FA proteins have beenimplicated in one or more of these steps (5, 10, 12, 18-22, 26).

The initial unhooking of the cross-link is a critical step in theICL repair process. ERCC1-XPF is a heterodimeric complex thatis a structure-specific endonuclease that has been shown to createincisions at the site of a DNA ICL (9, 11, 27). It has beenproposed that it plays an important role in the unhooking step inthe cross-link repair process (28-32). It may also potentially beinvolved in subsequent steps in which the cross-link is excisedfrom the DNA and in the completion of homologous recombina-tion (31, 32). Whether there are other proteins that may interactwith ERCC1-XPF and are involved in its role in the repair pro-cess is not known. We have shown that the structural proteinnon-erythroidR spectrin (RIISp) is involved in the repair ofDNAICLs (19, 20, 26, 33). RIISp and three FANC proteins that arecomponents of the FA core complex, FANCA, FANCC, andFANCG, are important for the production of incisions createdby ERCC1-XPF at the site of a DNA ICL (11, 19, 26). Anti-bodies against RIISp and these FANC proteins inhibit produc-tion of these incisions (11). There is a deficiency in these incisionsin FA-A, FA-C, and FA-G cells after they are exposed to aDNAinterstrand cross-linking agent, which correlates with reducedlevels ofRIISp in these cells, and this deficiency is corrected whenlevels of the corresponding FANC protein have been restored bytransfection of these cells with the appropriate FA cDNA (11).

†This work was supported by National Heart, Lung and BloodInstitute (National Institutes of Health) Grant RO1 HL054860 (toM.W.L.).*To whom correspondence should be addressed: Department of

Pathology and Laboratory Medicine, UMDNJ, New Jersey MedicalSchool, 185 S. Orange Ave., Newark, NJ 07103. Phone: (973) 972-4405.Fax: (973) 972-7293. E-mail: [email protected].

1Abbreviations: FA, Fanconi anemia; ICL, interstrand cross-link;NER, nucleotide excision repair; HR, homologous recombination; RIISp,non-erythroidR spectrin;TPR, tetratricopeptide repeat;BD,DNA-bindingdomain; AD, activation domain; HnH2, helix-hairpin-helix domain;DSB, double-strand break; MMC, mitomycin C; SH3, src-homology 3;WD, wild type.

Article Biochemistry, Vol. 49, No. 26, 2010 5561

RIISp is also important in the formation of ERCC1-XPF nuclearfoci after DNA ICL damage: knockdown of RIISp in normalcells by siRNA leads to the loss of formation of ERCC1-XPFfoci and in FA-A cells, where there are reduced levels of RIISp,there is decreased level of formation of these foci (20, 33). It isthus possible that the interaction of these FANC proteins withRIISp and ERCC1-XPF is necessary for the incisions producedby ERCC1-XPF in the cross-link repair process.

These studies were undertaken to examine whether there isdirect physical interaction among RIISp, several members of theFA core complex, and either of the two proteins in the ERCC1-XPF heteroduplex and, if so, to determine the sites of interaction.Importantly, if a direct interaction could be demonstrated betw-een any of the FA proteins examined and ERCC1 and/or XPF,this would more clearly establish a link between FA proteins andthe incision step(s) in the ICL repair process. These studiesexamined the ability of RIISp, FANCA, FANCC, FANCF, andFANCG to bind directly to ERCC1 and XPF using yeast two-hybrid analysis. The results demonstrated that one of the FANCproteins, FANCG, had a strong affinity for ERCC1 and amoderate affinity for XPF. FANCG contains seven tetratrico-peptide repeat (TPR) motifs, which have been shown to mediateprotein-protein interactions and are thought to be important inthe assembly of multiprotein complexes (34-39). Mapping thesites of interaction of FANCG with ERCC1 demonstrated thatTPR motifs 1, 3, 5, and 6 are needed for binding of FANCG toERCC1. In contrast, FANCA, FANCC, and FANCF, whichdid not contain these motifs, did not bind directly to ERCC1.RIISp also did not bind directly to ERCC1. ERCC1, in turn, wasshown to interact with FANCG via its central domain. This isdifferent from the region of ERCC1 that binds to XPF, whichis the C-terminal HhH2 domain (40-42). These studies thus de-monstrate that there is binding between a FA protein, FANCG,and the ERCC1-XPF endonuclease. These results, combinedwith our previous studies (11, 19), provide evidence that there isa link between at least one of the proteins in the FA core complexand the incision step(s) in the ICL repair process.

EXPERIMENTAL PROCEDURES

Bacterial and Yeast Strains. Escherichia coli strain DH5R(Invitrogen) was used in the construction and propagation of allyeast two-hybrid plasmid constructs. E. coli was grown in LuriaBroth at 37 �C. Saccharomyces cerevisiae strain EGY48 (MATRtrp1 his3 ura3 leu2:6 LexAop-Leu2) (OriGene Technologies) wasgrown in liquid YPD (Q-Biogene) or YPD agar plates at 30 �C.Yeast Two-Hybrid Vectors and Expression Constructs.

All yeast expression constructs were created in the DupLexAyeast two-hybrid system (OriGene Technologies). Constructscontaining the LexADNA-binding domain (BD) were subclonedinto the pEG202 vector, and constructs containing the B42transcriptional activation domain (AD) were subcloned intothe pJG4-5 vector as previously described (43).

Four overlapping regions of RIISp, R1 (residues 1-887), R2(residues 864-1239), R3 (residues 1089-1766), and R4 (residues1661-2477), which had been constructed and cloned into pJG4-5, were utilized in these studies (43). FANCC, FANCF, andFANCG cDNAs were created as previously described (43).Constructs of a N-terminal fragment and a C-terminal fragmentof FANCG, residues 1-293 (FANCG1-293) and residues261-622 (FANCG261-622), respectively, were created as pre-viously described (43). The FANCG fragment, residues 261-355(FANCG261-355), was created by introducing a stop codon at

residue 355 using FANCG261-622 as a template. FANCA cDNAwas amplified by PCR from pREP4 (a gift from H. Joenje, VrijeUniversiteit Medical Center, Amsterdam, The Netherlands)using Accuprime Pfx DNA polymerse supermix (Invitrogen),and EcoRI and NotI restriction sites were added to the 50 and 30

ends of the cDNA. The forward primer was 50-GCGCGAATT-CATGTCCGACTCGTGGGTCCC-30 and the reverse primer50-GCGCGCGGCCGCTCAGAAGAGATGAGGCTCCT-30.Then FANCA cDNAwas then inserted into the pEG202 vector.The sequences for all of the these constructs were verified bysequencing (Molecular Resource Facility, New Jersey MedicalSchool, University of Medicine and Dentistry of New Jersey).

Full-length human XPF cDNA was excised using the restric-tion enzymes NcoI and NotI from a cER4-40 cosmid (a giftfrom M. P. Thelen, Lawrence Livermore National Laboratory,Livermore, CA) and inserted into the corresponding sites ofpEG202. To make XPF in-frame with LexA, this construct wasfirst digested with BamHI, treated with the Klenow fragment ofDNA polymerase I (New England Biolab), and circularized byligation. The final sequence of the in-frame pEG202-XPF con-struct was confirmed by sequencing. The pJG4-5-XPF constructwas created by excising the XPF cDNA from pEG202 andsubcloning it into the pJG4-5 vector.

Four regions of ERCC1were constructed by PCRamplificationof sequences fromhuman full-length ERCC1 cDNA in the pOTB7vector (American Type Culture Collection) using Accuprime PfxDNA polymerase supermix. Primers (Operon) used to amplifyeach of these four regions are listed in Table S1 of the SupportingInformation. The regions ofERCC1amplifiedwere theN-terminaldomain (residues 1-99), the central domain with the C-terminaltandom helix-hairpin-helix (HhH2) domain (residues 88-297),the ERCC1 central domain without N-terminal residues 120-220,and the ERCC1 C-terminal HhH2 domain (residues 214-297).The constructs were then subcloned into the pCR4-TOPO vectorusing the TOPO TA cloning kit (Invitrogen). After sequenceverification, the inserts as well as full-length ERCC1 were sub-cloned into yeast two-hybrid vector pEG202 or pJG4-5.Site-Directed Mutagenesis. To evaluate the importance

of each of the seven TPR motifs in FANCG to its binding toERCC1, specific point mutations were separately created inthese motifs in full-length FANCG. For these studies, FANCGwas subcloned into the pENTR3C vector (Invitrogen). SevenFANCG TPR mutants were created with a PCR mutagenesisstrategy based on the protocol used in the ExSite PCR-based site-directed mutagenesis kit (Stratagene): TPR1 (G216Q), TPR2(G253Q), TPR3 (A351Q), TPR5 (G460Q), TPR6 (G521Q),TPR7a (A567Q), and TPR7b (R563E). The primers used for thisare listed in Table S2 of the Supporting Information. Aftersequence verification, each FANCG mutant cDNA was sub-cloned into the yeast two-hybrid pJG4-5 vector as previouslydescribed (43). FANCG containing a mutation in TPR4 was notexamined in these studies because of difficulties in cloning thecDNA of FANCG with a mutation in TPR4 into the yeast two-hybrid vector. However, this TPR may not be as important forFANCG function as the other TRP motifs since FANCG con-taining a TPR4mutation has been shown to fully complement thecross-link sensitivity of FANCG deficient cells (38).Yeast Two-Hybrid Analysis. The DupLEX-A yeast two-

hybrid system (OriGene Technologies) was used for yeast two-hybrid analysis. Yeast strain EGY48 was transformed with theAD vector (pEG202), BD vector (pJG4-5), and reporter vectorsusing a PEG/single-stranded DNA/lithium acetate procedure as

5562 Biochemistry, Vol. 49, No. 26, 2010 Wang and Lambert

previously described (43). Six colonies from each of these yeasttransformations were randomly selected and inoculated in rowsonto YNB (X-gal-His-Trp-urea) plates and grown for 2-5 daysto screen for activation of the LacZ reporter gene. Activation ofLacZ results in production of β-galactosidase, which cleavesX-gal and produces a blue color. Positive interactions betweentheADandBD fusion proteins were documented by the presenceof blue colonies on the plates. Each experiment was repeated fourto six times.Co-Immunoprecipitation of Proteins.Normal human lym-

phoblastoid cells (GM 3299) (Coriell Institute for MedicalResearch) were grown in suspension culture in RMPI 1640medium. Nuclear extracts of the cells were prepared accordingto the method of Yamashita et al. (44). Co-immunoprecipitationof FANCG and ERCC1 was conducted as previously des-cribed (26, 45). Briefly, anti-ERCC1 and rabbit IgG, which wasused as a control, were bound to protein-G-coated agarose beads(Sigma Aldrich Corp.) and the beads washed in binding buffer[25mMTris-HCl (pH 7.3), 150mMNaCl, 1%TritonX-100, andprotease inhibitor mixture (Roche Molecular Biochemicals)],blocked with 1% BSA (Sigma Aldrich Corp.), and washed inbinding buffer as described previously (26, 45). The agarose bead/antibody suspension was incubated with the protein extract asdescribed previously (26, 45). For reciprocal IPS, anti-FANCGand preimmune serum, which served as a control, were bound toprotein A-coated agarose beads (Sigma) and the samples pro-cessed as described above. The IPswere subjected to SDS-PAGEand the proteins analyzed by Western blot analysis as previouslydescribed (19, 26, 45). For the primary antibodies, an affinity-purified antibody against FANCG was generated by BethylLaboratories (19) and anti-ERCC1 (FL297, Santa Cruz Biotech-nology Inc.) was used. The specificity of this particular antibodyfor ERCC1 has been validated by Bhagwat et al. (46).

RESULTS

RIISp Does Not Directly Interact with ERCC1 or XPF.Whether RIISp interacted directly with ERCC1 or XPF wasexamined by yeast two-hybrid analysis. Because of its large size,four overlapping regions of RIISp were constructed. No directinteractionwas detected between any of the four regions of RIISp(R1-R4) and either ERCC1 or XPF (Figure 1). We have previ-ously shown that each of these four regions of RIISp is expressedin the yeast cells, indicating that this lack of interaction was notdue to the lack of expression of the hybrid proteins in the experi-mental system (43). We have previously shown that region 2 ofRIISp binds to FANCG (43), and this interaction and the knowninteraction of ERCC1 and XPF (40-42) were used as positivecontrols. Direct binding between these two groups of proteinswas evidenced by the presence of blue colonies, indicating that theyeast two-hybrid system was functional (Figure 1).FANCG Interacts Directly with ERCC1 and XPF.Yeast

two-hybrid analysis was conducted to determine whether the FAcore complex proteins, FANCA,FANCC,FANCG, andFANCF,interacted directly with either XPF or ERCC1. The interaction ofFANCAwithFANCGand that ofXPFwithERCC1were used aspositive controls since these proteins have been shown to directlyinteract with each other (40-42, 47). Direct binding between thesetwo groups of proteins was evidenced by the presence of bluecolonies (Figure 2A). Very low levels of autoactivation wereobserved for FANCA, as seen by the presence of pale blue colonies.Interaction between FANCA and either XPF or ERCC1 was notdetected, as seen by the presence of white colonies, which indicated

that the reporter gene had not been activated and that there was nopositive interaction between the fusion proteins (Figure 2A). Directinteraction was also not detected between FANCC and either XPFor ERCC1 (Figure 2B). A very weak interaction was detectedbetween FANCF and ERCC1, but this interaction was not con-sistently observed, and no interaction was observed betweenFANCF and XPF (Figure 2B). Analysis of the binding reactionsshowed, however, that FANCG displayed very strong binding toERCC1, as evidenced by activation of the reporter gene and thedevelopment of an intense blue color in the colonies (Figure 2B).FANCG also bound to XPF, but to a lesser extent (Figure 2B). Allof these results are from a single experiment in which six randomlychosen colonies were examined. Each of these binding experiments,as well as those in the subsequent studies, was repeated four to sixtimes with similar results. The results of these protein interactionsare summarized in Figure 2C. Immunoblot analysis of proteinextracts of the transformed yeast cells showed that FANCA,FANCC, and FANCF were expressed in the yeast cells, indicatingthat the lack of interaction of these proteins with XPF or ERCC1was not due to the lack of expression of these proteins in thesecells (43).

The interaction of FANCG with ERCC1 was examinedfurther to determine the region in FANCG that binds to ERCC1.For this, three overlapping fragments ofFANCGwere constructed

FIGURE 1: Nodirect interactionwas detectedbetween any of the fourregions of RIISp and either ERCC1 or XPF. RIISp was divided intofour overlapping regions, R1 (residues 1-887), R2 (residues 864-1239), R3 (residues 1089-1766), and R4 (residues 1661-2477),which were subcloned into yeast two-hybrid vectors. The LexAfusion proteins were co-expressed in yeast with the B42-XPF orB42-ERCC1 construct. Six randomly selected colonies from each ofthese yeast transformations were replica plated in a row to platescontaining X-gal to test for activity of the reporter gene, β-galacto-sidase. Positive interaction between fusion proteins is evidenced bythe presence of blue colonies. White colonies indicate no positiveprotein interactions. The interaction between ERCC1 and XPF andthat between R2 of RIISp and FANCG were used as positivecontrols.


and expressed as fusion proteins in yeast two-hybrid experi-ments: FANCG1-291, FANCG261-355, and FANCG261-622. TheFANCG261-622 fragment showed some direct binding to ERCC1,while both the FANCG1-291 and FANCG261-355 fragments failedto show positive interaction with ERCC1 (Figure 2A,B). Auto-activation was not observed with these FANCG fragments. TheFANCG261-622 fragment contained more than half of theFANCG protein from the carboxyl terminal, indicating that thisis an important region of binding to ERCC1 (Figure 3B).However, compared to full-length FANCG, FANCG261-622

bound to ERCC1 with much less intensity (Figure 3A,B). Thissuggested that residues outside of this fragment may also beneeded for binding to ERCC1 and/or that the overall structure ofFANCG is important in this interaction.The TPR Motifs in FANCG Are Essential for Its Bind-

ing to ERCC1. Since it has been shown that FANCG containsseven TPR motifs, and a number of these have been demon-strated to be important in FANCG’s interactions with otherproteins (38, 39), studies were conducted to determine whetherone ormore of these TPRmotifs could be important inmediatingthe binding of FANCG to ERCC1 (Figure 4A). Single-aminoacid substitutions were created at selected residues for each motifby site-directed mutagenesis. In each of these seven TPR motifs,the eighth amino acid is strongly conserved and is usually analanine or a glycine (38). In TPR1, -2, -3, -5, and -6, the eighthamino acid was replaced by the hydrophilic residue glutamine(Tables S2 and S3 of the Supporting Information). Since inTPR7, the eighth residue is neither alanine nor glycine, the highly

FIGURE 2: Yeast two-hybrid analysis of interaction of ERCC1 andXPF with FA proteins. (A) The LexA-FANCA fusion protein wasco-expressed in yeast with an empty vector (-) or the B42-XPF orB42-ERCC1 construct. Six randomly selected colonies from each ofthese yeast transformations were replica plated in a row to platescontaining X-gal to test for activity of the reporter gene, β-galacto-sidase. Positive interaction between fusion proteins is evidenced bythe presence of blue colonies, which indicates that the reporter genehas been activated. White colonies indicate no positive proteininteractions. The interaction between FANCA and FANCG andthat between XPF and ERCC1 were used as positive controls. (B)The LexA-ERCC1 or LexA-XPF fusion protein was co-expressedin yeast with the B42-FANCC, B42-FANCG, and B42-FANCFconstructs. Protein interaction was assessed as described above. (C)Summary of the results of yeast two-hybrid analysis of the interactionof ERCC1 and XPF with FANCA, FANCC, FANCF, andFANCG: (þþ) very good binding, (þ) binding, and (-) no binding.

FIGURE 3: Full-length FANCG but not its fragments binds toERCC1. (A) Full-length and deletion constructs of FANCG(residues 1-293, residues 261-622, and residues 261-355) werecreated and subcloned into yeast two-hybrid vectors. LexA-ERCC1fusion proteins were co-expressed in yeast with the B42-FANCG full-length or deletion construct. Six colonies were selected and replicaplated in rows as shown in Figure 1. Blue colonies indicate positiveinteraction between fusion proteins. White colonies indicate no posi-tive protein interactions. (B) Summary of the results of yeast two-hybrid analysis of the interaction of ERCC1 with full-length FANCGand deletion constructs of FANCG: (þþ) very good binding, (þ)binding, and (-) no binding.


conserved alanine at the 20th position was chosen and replacedwith glutamine to create TPR7a (Tables S2 and S3). In addition,another mutant TPR7was created, TPR7b, in which the arginineat position 16, which is also conserved between species (38), wassubstituted with a glutamic acid (Tables S2 and S3). The cDNAsof these FANCG TPR mutants were subcloned into the pJG4-5vector, and Western blot analysis showed that all of theseFANCG TPRmutants were expressed in yeast cells (Figure 4B).

The interaction of FANCG with ERCC1 was severely dis-rupted by mutations in TPR1, -3, -5, and -6 in FANCG, as seenby the absence of reporter gene activation (Figure 4C). However,the mutations in TPR7a and TPR7b had no significant effect onthe interaction of FANCGwith ERCC1. Both of these FANCGmutants maintained their ability to interact with ERCC1 asdemonstrated by a level of reporter gene activation that was asstrong as that of wild-typeFANCG (Figure 4C). Themutation inthe TPR2 mutant resulted in a moderate decrease in the level ofinteraction of FANCG with ERCC1 (Figure 4C). Each of thesebinding experiments was repeated four to six times with similarresults, and the results of these protein interactions are summar-ized in Figure 4D. This indicates that TPR1, -3, -5, and -6 arecritical for the binding of FANCG to ERCC1. TPR2 may havesome importance as well. However, TPR7 does not appear to beneeded for the interaction. Figure 4A shows the localization ofeach of the TPR motifs in the three FANCG regions examinedfor binding to ERCC1 (Figure 4C,D). It can be seen that the

FANCG261-622 fragment, which showed some binding toERCC1, contains TPR3-7. On the basis of these findings, thisbinding could be contributed by TPR3, -5, and -6. The presenceof TPR1, alongwithTPR2, is not sufficient, however, for bindingof the FANCG1-191 fragment to ERCC1 (Figure 4C,D). How-ever, TPR1 and possibly TPR2 are probably contributing to thebinding of FANCG to ERCC1 since full-length FANCG hadmuch stronger binding to ERCC1 than did any of the FANCGfragments. These studies demonstrate the importance of TPR1,-3, -5, and -6 in mediating FANCG’s interaction with ERCC1.The possibility also exists that the overall TPR structure, ratherthan a subset of these motifs, is required for efficient ERCC1binding.The Central Domain of ERCC1 Binds to FANCG. Since

strong binding was observed between FANCG and ERCC1, thedomains inERCC1 that interactedwithFANCGwere examined.ERCC1 contains three domains (Figure 5A): the N-terminaldomain; the central domain, which contains the sites of interac-tion with XPA; and the C-terminal HhH2 domain, which isthe domain that binds toXPF (40-42, 48-51). For these studies,ERCC1 was divided into four overlapping regions. The firstregion contained the N-terminal domain (residues 1-99). Thesecond region contained the central and C-terminal HhH2

domains (residues 88-297). The third region contained thecentral domain without its N-terminal residues (residues 120-220). The fourth region contained theC-terminal (HhH2) domain

FIGURE 4: Specific TPRmotifs in FANCG are essential for its binding to ERCC1. (A) Diagram showing the TPRmotifs in full-length FANCGand the FANCG fragments. (B)Mutations were created in TPR1, -2, -3, -5, -6, and -7 in FANCG. Yeast strain EGY48 was transfected with thepJG4-5 vector containing the TPR mutants of FANCG and wild-type (WT) FANCG. The fusion proteins were detected with an anti-HAantibody in Western blot analysis of yeast cell extracts. (C) Yeast two-hybrid analysis of the interaction of wild-type FANCG and FANCG-containing mutant TPR motifs with ERCC1. LexA-ERCC1 fusion proteins were co-expressed in yeast with the B42-FANCG WT or B42-FANCG TPR mutants. Six colonies were selected and replica plated in rows as shown in Figure 1. Blue colonies indicate positive interactionbetween fusion proteins. White colonies indicate no positive protein interactions. (D) Summary of the results of yeast two-hybrid analysis of theinteraction of wild-type FANCG and FANCG-containing TPR mutations with ERCC1: (þþ) very good binding, (þ) binding, and (-) nobinding.


(residues 214-297) (Figure 5A). The cDNAs corresponding tothese regions were subcloned into the yeast two-hybrid vectors.

The regions of ERCC1 that contained the central and HhH2

domains, ERCC188-297, and the central domain without itsN-terminus, ERCC1120-220, showed strong binding to FANCG(Figure 5B). The N-terminal domain of ERCC1, ERCC11-96,and the HhH2 domain, ERCC1214-297, did not bind to FANCG(Figure 5B). The results of four or five experiments are summar-ized in Figure 5C. These results indicate that ERCC1 interactswith FANCG through its central domain, which is different thanthe domain that binds to XPF.FANCG Co-Immunoprecipitates with ERCC1 in Human

Cells. We further assessed the interaction of FANCG andERCC1 by conducting co-immunoprecipitation experiments.Immunoprecipitation of normal human cell extracts with anti-ERCC1 and immunoblotting with either anti-ERCC1 or anti-FANCG showed that FANCG co-immunoprecipitated withERCC1 (Figure 6A). Similarly, when co-immunoprecipiationwas conducted using anti-FANCG, immunoblotting with anti-FANCG or anti-ERCC1 showed that ERCC1 co-immunopre-cipitated with FANCG (Figure 6B). These studies indicate thatFANCG and ERCC1 interact with each other in human cells.

DISCUSSION

Though there is evidence that FA proteins are involved in therepair of DNA interstrand cross-links (ICLs), the exact rolethey play is not yet clear (5, 10-12, 18-21). Our laboratory hasshown that several of the FA proteins are important in theincision step(s) of the repair process in which ERCC1-XPFincises DNA at the site of a ICL: FANCA colocalizes with XPFandRIISp at cross-link-induced nuclear foci (20); antibodies againstFANCA, FANCC, and FANCG inhibit incisions produced by

ERCC1-XPF at the site of a psoralen interstrand cross-link (11); inFA-A,FA-C, andFA-Gcells, there is a deficiency in the productionof incisions by ERCC1-XPF at the site of aDNA interstrand cross-link (9, 11); and in corrected FA-A, FA-C, and FA-G cells, whichexpress the FANCA,FANCC, andFANCGproteins, respectively,levels of incisions produced by ERCC1-XPF at sites of cross-linksare restored to normal (9, 11). ERCC1-XPF, which is known to beinvolved in nucleotide excision repair (NER) (40, 41, 52-54), hasalso been shown to play a role in the repair of DNA interstrandcross-links and is believed to be important in the repair of thesecross-links at stalled replication forks (9, 11, 27-31). In FA cells,there is a defect inDNArepair that occurs atDNAreplication forksstalled at sites of interstrand cross-links (4, 12, 21-23). Of theproposed steps in the repair of DNA interstrand cross-links atstalled replication forks, one of the most critical involves theincisions that are produced at the site of the cross-link and resultin its unhooking. For the cross-link to become unhooked from onestrand ofDNA, incisions must bemade on the 30 and 50 sides of thelesion. The structure-specific nuclease, Mus81-Em1, is thought tofacilitate formation of the 30 incision, producing a double-strandbreak (DSB) (10, 12, 18, 55, 56). ERCC1-XPF is then proposed toincise the DNA on the 50 side of the cross-link leading to itsunhooking (12, 18, 29). Though our studies indicate that several ofthe FA proteins are important in this step, the precise role that FAproteins play in this process is not known. To elucidate this, weexamined in this study whether there is a direct association betweenseveral of theFANCcore complex proteins, in particular, FANCA,FANCC, FANCF, or FANCG, and proteins involved in theunhooking step of the repair process, specifically either ERCC1or XPF.

The results showed, using yeast two-hybrid analysis, that thereis a strong interaction between one of these FAproteins, FANCG,

FIGURE 5: Central domain of ERCC1 binding to FANCG. (A)Diagram of the three domains of ERCC1 and the binding of XPA, FANCG, andXPF to specific domains of ERCC1. ERCC1 was divided into four overlapping regions: a region containing the N-terminal domain, a regioncontaining the central andHhH2domains, a region containing the central domainwithout itsN-terminus (-Nt), and a region containing theHhH2

domain. (B) Yeast two-hybrid analysis of the interaction of full-length ERCC1 and the four regions of ERCC1 with FANCG. LexA-ERCC1fusion protein and LexA fusion proteins of the four regions of ERCC1 were co-expressed in yeast with the B42-FANCG construct. Six colonieswere selected and replica plated in rows as shown in Figure 1. Blue colonies indicate positive interaction between fusion proteins. White coloniesindicate no positive protein interactions. (C) Summary of the results of yeast two-hybrid analysis of the interaction of full-length ERCC1 and thefour regions of ERCC1 with FANCG: (þþ) very good binding, (þ) binding, and (-) no binding.


and ERCC1. Full-length FANCG was needed for this interac-tion. Overlapping N- and C-terminal fragments of FANCGshowed no or only weak binding to ERCC1 compared to thebinding of full-length FANCG. This indicates that sites in bothfragments are critical for this interaction or that the overallstructure of FANCG is important. There was also interactionbetween FANCG and XPF; however, it was not as strong. Thatthe interaction between FANCG and ERCC1 occurs in vivo wasconfirmed by studies that showed that FANCG and ERCC1 co-immunoprecipitated from human cell extracts.

FANCG contains seven TPRmotifs, which are 34-amino acidhelix-turn-helix motifs that are involved in mediating protein-protein interactions (34-39). TheseTRPmotifs have been shownto be important in the interaction of FANCG with severaldifferent proteins (39). TPR5 and TPR6 are required for theinteraction of FANCG with XRCC3, FANCA, and the N-ter-minus of BRCA2; TPR1 and TPR2 are involved in the interac-tions of FANCG with the C-terminus of BRCA2, and TPR1,TPR2, TPR5, and TPR6 are important in the binding ofFANCG to FANCF (39).

Investigation in this study of whether any of the TPR motifswere responsible for the binding between FANCG and ERCC1showed that mutations in consensus position 8 of TPR1, TPR3,TPR5, and TPR6 in FANCG resulted in a loss of binding ofFANCG to ERCC1. This demonstrated that these TPR motifsare critical for this interaction. Of interest, TPR3, -5, and -6 are inthe C-terminal fragment of FANCGwhich showed some bindingto ERCC1. TPR1 is in the N-terminal fragment of FANCG

which did not bind to ERCC1. However, since TPR1 is neededfor interaction of FANCG with ERCC1, this indicates that thecombined action of TPR1 with TPR3, -5, and -6 is needed forbinding of FANCG to ERCC1. Missense mutations at position8 in TPR1, TPR5, and TPR6 in FANCG have been shown tocause failure of these mutant proteins to correct the cellularsensitivity of FA-A cells to a DNA interstrand cross-linkingagent, mitomycin C (MMC) (38). These findings, combined withours, which show that TPR1, -3, -5, and -6 are involved in thebinding of FANCG to ERCC1, and our previous finding thatFANCG is needed for the incisions produced by ERCC1-XPF atthe site of a cross-link (11), indicate that binding of FANCG toERCC1 is critical for the repair response after exposure to across-linking agent.

Studies of which of the three domains in ERCC1 bind toFANCG demonstrated that the central domain of ERCC1,without residues 96-119, binds to FANCG. It has been shownthat residues 96-119 are necessary for interaction of ERCC1with XPA (47, 57, 58). Recent studies indicate that residueAsn110 within this region is required for NER and that residueTyr145 is also important for interaction of ERCC1 with XPAand for NER (51). However, though these sites of interaction ofERCC1 with XPA have been shown to be important for NER,they do not appear to be involved in ICL repair (51). Since theseresults combined with our previous studies show that residues120-220 but not residues 96-119 in ERCC1 are needed forbinding of FANCG to ERCC1, and that FANCG is necessaryfor the production of incisions by ERCC1-XPF at sites of DNAinterstrand cross-links (11), this suggests that the sites on ERCC1that are involved inNERare different from those involved in ICLrepair. These studies thus indicate that ERCC1, in addition tosites for binding of XPA in its central domain and of XPF in itsC-terminalHhH2 domain, contains a region in its central domainfor binding of FANCG (Figure 5A).

Recent studies indicate that ERCC1-XPF is involved in theunhooking of the cross-link at the site of a stalled replication forkand that this process is not required for FA pathway activationbut is required for the localization of monoubiquitinatedFANCD2 on chromatin (30). Since we have shown that associa-tion of ERCC1 with XPF is necessary for the ability of XPF toincise DNA at sites of cross-links (59) and that FANCG is alsoneeded for these incisions (11), it is possible that the binding ofFANCG toERCC1 is of key importance for this unhooking step.

In addition to being a component of the FA core complex,studies indicate that FANCG can have a role that is independentof this complex (60). It forms a complex with FANCD1/BRCA2,FANCD2, and XRCC3 and is proposed to play a role in homo-logous recombination repair that occurs duringDNA interstrandcross-link repair (60). These studies, combined with our previousfindings, indicate that FANCG may form another complex orinteract directly with proteins involved in the unhooking step ofthe cross-link repair process, ERCC1-XPF and RIISp. We haveshown that FANCG, in addition to binding to ERCC1 viaTPR1, -3, -5, and -6, interacts with the Src-homology 3 (SH3)domain ofRIISp via amotif that binds to SH3 domains (43). Thismotif, residues 380-388 in FANCG, is located between TPR3and -4. SH3 domains are modular domains that play a rolein protein-protein interactions and formation of protein net-works (61-64). Thus, FANCG has two different types of motifsthat have been shown to be important in protein-proteininteractions and assembly of complex protein networks. It hasTPR motifs, several of which interact with ERCC1, and it has a

FIGURE 6: FANCG co-immunoprecipitates with ERCC1. (A) Nor-mal human cell extracts were co-immunoprecipated with anti-ERCC1. Interaction of FANCG with ERCC1 was examined byWestern blot analysis using anti-FANCGand anti-ERCC1. The IgGheavy chain was used as a loading control. (B) Normal human cellextractswere co-immunoprecipitatedwith anti-FANCG. Interactionof FANCG with ERCC1 was examined by Western blot analysisusing anti-FANCG and anti-ERCC1.


SH3 bindingmotif that interacts withRIISp.We have shown thatthis interaction of FANCG with RIISp is important in theERCC1-XPF-mediated unhooking step in the repair of DNAinterstrand cross-links. On the basis of our studies as well asprevious work in our laboratory (19, 20, 26, 33), we haveproposed a model for the involvement of RIISp in the repair ofDNA interstrand cross-links (Figure 7). At a replication forkstalled at the site of a cross-link, a nuclease such as Mus81-Eme1cleaves the DNA on the 30 side of the ICL which leads to theproduction of aDSB (12, 18, 30, 65).RIISp binds at the site of theDNA interstrand cross-link and acts as a scaffold to aid in therecruitment of repair proteins to the site of damage. FANCGis either recruited to bind to RIISp or bound to the SH3domain of RIISp at the time it binds to the cross-linked DNA.Because these studies indicate that the interaction of RIISpwith ERCC1 or XPF is indirect, we further propose that

RIISp, which we have shown binds directly to DNA containingan interstrand cross-link (19) and to FANCG (43), recruitsERCC1-XPF to sites of damage via FANCG. Thus, theinteraction of FANCG with ERCC1-XPF would be criticalin the recruitment of this protein complex to the site of thecross-link. This is analogous to the recruitment of ERCC1-XPF to sites of damage in NER byXPA (53, 54). ERCC1-XPFthen incises the DNA on the 50 side of the cross-link, andunhooking of the cross-link takes place. Subsequent steps inthe repair process would then occur which involve localizationof monoubiquitinated FANCD2 and additional FA proteinsto chromatin, translesion synthesis, NER to excise the mono-adduct, HR, and repair of the DSB. Thus, the demonstratedbinding of FANCG to ERCC1 establishes a link between FAproteins and the critical unhooking step of the cross-link repairprocess.

FIGURE 7: Model showing the interaction of RIISp, FANCG, and ERCC1-XPF in the repair of a DNA interstrand cross-link at an arrestedreplication fork. (1) ADNA replication fork is arrested at the site of an ICL. (2)Mus81-Eme1 cleavesDNAat the stalled replication fork on the 30side of the ICL producing aDSB. (3)RIISp binds to theDNAat the site of the ICL, andFANCGbinds to the SH3 domain ofRIISp. (4) ERCC1-XPF is recruited to the ICLbyFANCG,which is bound toRIISp. (5) ERCC1-XPF incises theDNAon the 50 side of the ICL, thus unhooking thecross-link. (6)Monoubiquitinated FANCD2 localizes to the chromatin with other FA proteins. (7) Translesion synthesis with a polymerase suchas pol ζ fills in the gap resulting from the ICL unhooking. (8) NER with ERCC1-XPF and other NER proteins leads to excision of themonoadduct. (9) Thehomologous recombinationmachinery is recruited, and theDSB is repaired; theDNAreplication fork is re-established.Thisis based on our results and on models presented by Thompson and Hinz (12) and Bhagwat et al. (31).


This direct interaction of FANCG with ERCC1/XPF mayalso be important in any other function attributed to either ofthese proteins, such as the recombination step of the interstrandcross-link repair process or the second set of incisions to releasethe cross-link from the DNA during the proposed NER step ofcross-link repair. Further studies will be needed to examine thesequestions.

SUPPORTING INFORMATION AVAILABLE

A table showing the primers used to amplify cDNA for thedomains of ERCC1, a table showing the primers used to amplifythe cDNAs for TPR motifs mutated in FANCG, and a tableshowing the wild-type and mutated sequences of the TPR motifsin FANCG. This material is available free of charge via theInternet at http://pubs.acs.org.

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the fanconi anemia protein, fancg, binds to the ercc1-xpf endonuclease via its tetratricopeptide...

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