Abnormal migration behavior linked to Rac1 signaling contributes to primordial germ cell exhaustion in Fanconi anemia pathway-deficient Fancg-/- embryos.

Fanconi anemia (FA) is a rare human genetic disorder characterized by bone marrow failure, predisposition to cancer and developmental defects including hypogonadism. Reproductive defects leading to germ cell aplasia are the most consistent phenotypes seen in FA mouse models. We examined the role of the nuclear FA core complex gene Fancg in the development of primordial germ cells (PGCs), the embryonic precursors of adult gametes, during fetal development. PGC maintenance was severely impaired in Fancg-/- embryos. We observed a defect in the number of PGCs starting at E9.5 and a strong attrition at E11.5 and E13.5. Remarkably, we observed a mosaic pattern reflecting a portion of testicular cords devoid of PGCs in E13.5 fetal gonads. Our in vitro and in vivo data highlight a potential role of Fancg in the proliferation and in the intrinsic cell motility abilities of PGCs. The random migratory process is abnormally activated in Fancg-/- PGCs, altering the migration of cells. Increased cell death and PGC attrition observed in E11.5 Fancg-/- embryos are features consistent with delayed migration of PGCs along the migratory pathway to the genital ridges. Moreover, we show that an inhibitor of RAC1 mitigates the abnormal migratory pattern observed in Fancg-/- PGCs.

Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression.

Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.

Associations of complementation group, ALDH2 genotype, and clonal abnormalities with hematological outcome in Japanese patients with Fanconi anemia.

Fanconi anemia (FA) is a genetically and clinically heterogeneous disorder that predisposes patients to bone marrow failure (BMF), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). To study which genetic and phenotypic factors predict clinical outcomes for Japanese FA patients, we examined the FA genes, bone marrow karyotype, and aldehyde dehydrogenase-2 (ALDH2) genotype; variants of which are associated with accelerated progression of BMF in FA. In 88 patients, we found morphologic MDS/AML in 33 patients, including refractory cytopenia in 16, refractory anemia with excess blasts (RAEB) in 7, and AML in 10. The major mutated FA genes observed in this study were FANCA (n = 52) and FANCG (n = 23). The distribution of the ALDH2 variant alleles did not differ significantly between patients with mutations in FANCA and FANCG. However, patients with FANCG mutations had inferior BMF-free survival and received hematopoietic stem cell transplantation (HSCT) at a younger age than those with FANCA mutations. In FANCA, patients with the c.2546delC mutation (n = 24) related to poorer MDS/AML-free survival and a younger age at HSCT than those without this mutation. All patients with RAEB/AML had an abnormal karyotype and poorer prognosis after HSCT; specifically, the presence of a structurally complex karyotype with a monosomy (n = 6) was associated with dismal prognosis. In conclusion, the best practice for a clinician may be to integrate the morphological, cytogenetic, and genetic data to optimize HSCT timing in Japanese FA patients.

Studies in an Early Development Window Unveils a Severe HSC Defect in both Murine and Human Fanconi Anemia.

Fanconi anemia (FA) causes bone marrow failure early during childhood, and recent studies indicate that a hematopoietic defect could begin in utero. We performed a unique kinetics study of hematopoiesis in Fancg-/- mouse embryos, between the early embryonic day 11.5 (E11.5) to E12.5 developmental window (when the highest level of hematopoietic stem cells [HSC] amplification takes place) and E14.5. This study reveals a deep HSC defect with exhaustion of proliferative and self-renewal capacities very early during development, together with severe FA clinical and biological manifestations, which are mitigated at E14.5 due to compensatory mechanisms that help to ensure survival of Fancg-/- embryos. It also reports that a deep HSC defect is also observed during human FA development, and that human FA fetal liver (FL) HSCs present a transcriptome profile similar to that of mouse E12.5 Fancg-/- FL HSCs. Altogether, our results highlight that early mouse FL could represent a good alternative model for studying Fanconi pathology.

FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange.

FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.

Map of synthetic rescue interactions for the Fanconi anemia DNA repair pathway identifies USP48.

Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities, and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). Here, we identify the deubiquitylating enzyme USP48 as synthetic viable for FA-gene deficiencies by performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2). Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA.

Bone Marrow Mesenchymal Stem Cells Carrying FANCD2 Mutation Differ from the Other Fanconi Anemia Complementation Groups in Terms of TGF-ß1 Production.

Transforming growth factor beta (TGF-ß) secretion from cells in the bone marrow (BM) niche affects hematopoietic stem cell (HSC) fate and has a cardinal role in HSC quiescence. BM mesenchymal stem cells (BM-MSCs), a component of the BM niche, may produce abnormal levels of TGF-ß in Fanconi anemia (FA) and may play a role in bone marrow failure. Here, we molecularly and cellularly characterized FA BM-MSCs by addressing their immunophenotype, proliferation- and differentiation- capacity, reactive oxygen species (ROS) production, senescence activity as well as expression and secretion levels of TGF-ß isoforms. In ten FA patients, mutations were detected in FANCA (n = 7), FANCG (n = 1) and FANCD2 (n = 2) genes. The immunophenotype, with the exception of CD29, and differentiation capacity of FA BM-MSCs were similar to healthy donors. FA BM-MSCs showed decreased proliferation, increased ROS level and an arrest in G2 following DEB treatment. ß-galactosidase staining indicated elevated senescence of FANCD2-deficient cells. FA BM-MSCs displayed TGF-ß1 mRNA levels similar to donor BM-MSCs, and was not affected by DEB treatment. However, secretion of TGF-ß was absent in FA-D2 BM-MSCs. Absence of TGF-ß secretion may be related to early onset of senescence of the FANCD2-deficient BM-MSCs. The proliferative response of FA-D2 BM-MSCs to rTGF-ß1 was not different from FANCA-deficient and donor cells and raises the possibility that rTGF-ß1 may reverse the senescence of the FANCD2-deficient BM-MSCs which needs to be investigated further.

Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway.

Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia.

A systems biology approach for elucidating the interaction of curcumin with Fanconi anemia FANC G protein and the key disease targets of leukemia.

Fanconi anemia (FA) is an autosomal recessive disorder with a high risk of malignancies including acute myeloid leukemia and squamous cell carcinoma. There is a constant search out of new potential therapeutic molecule to combat this disorder. In most cases, patients with FA develop haematological malignancies with acute myeloid leukemia and acute lymphoblastic leukemia. Identifying drugs which can efficiently block the pathways of both these disorders can be an ideal and novel strategy to treat FA. The curcumin, a natural compound obtained from turmeric is an interesting therapeutic molecule as it has been reported in the literature to combat both FA as well as leukemia. However, its complete mechanism is not elucidated. Herein, a systems biology approach for elucidating the therapeutic potential of curcumin against FA and leukemia is investigated by analyzing the computational molecular interactions of curcumin ligand with FANC G of FA and seven other key disease targets of leukemia. The proteins namely DOT1L, farnesyl transferase (FDPS), histone decetylase (EP3000), Polo-like kinase (PLK-2), aurora-like kinase (AUKRB), tyrosine kinase (ABL1), and retinoic acid receptor alpha (RARA) were chosen as disease targets for leukemia and modeled structure of FANC G protein as the disease target for FA. The docking investigations showed that curcumin had a very high binding affinity of -8.1 kcal/mol with FANC G protein. The key disease targets of leukemia namely tyrosine kinase (ABL1), aurora-like kinase (AUKRB), and polo-like kinase (PLK-2) showed that they had the comparable binding affinities of -9.7 k cal/mol, -8.7 k cal/mol, and -8.6 k cal/mol, respectively with curcumin. Further, the percentage similarity scores obtained from PAM50 using EMBOSS MATCHER was shown to provide a clue to understand the structural relationships to an extent and to predict the binding affinity. This investigation shows that curcumin effectively interacts with the disease targets of both FA and leukemia.

AMP-activated protein kinase is involved in the activation of the Fanconi anemia/BRCA pathway in response to DNA interstrand crosslinks.

Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway.

K63-linked ubiquitination of FANCG is required for its association with the Rap80-BRCA1 complex to modulate homologous recombination repair of DNA interstand crosslinks.

DNA interstrand crosslinks (ICLs) are extremely deleterious lesions that are repaired by homologous recombination (HR) through coordination of Fanconi anemia (FA) proteins and breast cancer susceptibility gene 1 (BRCA1) product, but the exact role these proteins have remains unclear. Here we report that FANCG was modified by the addition of lysine63-linked polyubiquitin chains (K63Ub) in response to DNA damage. We show that FANCG K63Ub was dispensable for monoubiquitination of FANCD2, but was required for FANCG to interact with the Rap80-BRCA1 (receptor-associated protein 80-BRCA1) complex for subsequent modulation of HR repair of ICLs induced by mitomycin C. Mutation of three lysine residues within FANCG to arginine (K182, K258 and K347, 3KR) reduced FANCG K63Ub modification, as well as its interaction with the Rap80-BRCA1 complex, and therefore impeded HR repair. In addition, we demonstrated that K63Ub-modified FANCG was deubiquitinated by BRCC36 complex in vitro and in vivo. Inhibition of BRCC36 resulted in increased K63Ub modification of FANCG. Taken together, our results identify a new role of FANCG in HR repair of ICL through K63Ub-mediated interaction with the Rap80-BRCA1 complex.

Fanca deficiency reduces A/T transitions in somatic hypermutation and alters class switch recombination junctions in mouse B cells.

Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca(-/-) mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation.

Areca nut induces miR-23a and inhibits repair of DNA double-strand breaks by targeting FANCG.

Previous investigations have shown that areca nut extracts (ANE) or arecoline (ARE) causes DNA damage, which in turn contributes to oral cell carcinogenesis. To understand the role of microRNA (miRNA) in ANE-associated carcinogenesis, miRNA expression profile was examined in ANE-treated normal human oral fibroblasts. Among the miRNAs changed by ANE exposure, we found that ANE-induced miR-23a overexpression was correlated with an increase of γ-H2AX, a DNA damage marker. In addition, DNA double-strand breaks (DSB) repair that was determined by an in vivo plasmid-based assay was reduced in ANE-treated or miR-23a-overexpressed cells, suggesting the role of miR-23a in DSB repair. FANCG is one of Fanconi anemia susceptibility genes that participate in DSB repair pathway to prevent chromosomal aberrations. FANCG was predicted as a candidate target of miR-23a by TargetScan algorithm. This was confirmed by ectopic overexpression or knockdown of miR-23a. The correlation between miR-23a overexpression and areca nut-chewing habit could also be found in oral cancer patients. Finally, we showed that ANE-induced/ARE-induced miRNAs were significantly associated with the functional categories of "genetic disorders" and "cancer" using network-based analyses. In conclusion, our data showed for the first time that ANE-induced miR-23a was correlated with a reduced FANCG expression and DSB repair, which might contribute to ANE-associated human malignancies.

RAD51D- and FANCG-dependent base substitution mutagenesis at the ATP1A1 locus in mammalian cells.

Elaborate processes act at the DNA replication fork to minimize the generation of chromatid discontinuity when lesions are encountered. To prevent collapse of stalled replication forks, mutagenic translesion synthesis (TLS) polymerases are recruited temporarily to bypass DNA lesions. When a replication-associated (one-ended) double-strand break occurs, homologous recombination repair (HRR) can restore chromatid continuity in what has traditionally been regarded as an "error-free" process. Our previous mutagenesis studies show an important role for HRR in preventing deletions and rearrangements that would otherwise result from error-prone nonhomologous end joining (NHEJ) after fork breakage. An analogous, but distinct, role in minimizing mutations is attributed to the proteins defective in the cancer predisposition disease Fanconi anemia (FA). Cells from FA patients and model systems show an increased proportion of gene-disrupting deletions at the hprt locus as well as decreased mutation rates in the hprt assay, suggesting a role for the FANC proteins in promoting TLS, HRR, and possibly also NHEJ. It remains unclear whether HRR, like the FANC pathway, impacts the rate of base substitution mutagenesis. Therefore, we measured, in isogenic rad51d and fancg CHO mutants, mutation rates at the Na(+)/K(+)-ATPase alpha-subunit (ATP1A1) locus using ouabain resistance, which specifically detects base substitution mutations. Surprisingly, we found that the spontaneous mutation rate was reduced approximately 2.5-fold in rad51d knockout cells, an even greater extent than observed in fancg cells, when compared with parental and isogenic gene-complemented control lines. A approximately 2-fold reduction in induced mutations in rad51d cells was seen after treatment with the DNA alkylating agent ethylnitrosurea while a lesser reduction occurred in fancg cells. Should the model ATP1A1 locus be representative of the genome, we conclude that at least 50% of base substitution mutations in this mammalian system arise through error-prone polymerase(s) acting during HRR-mediated restart of broken replication forks.

Mesenchymal stem/progenitor cells promote the reconstitution of exogenous hematopoietic stem cells in Fancg-/- mice in vivo.

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow failure and complex congenital anomalies. Although mutations in FA genes result in a characteristic phenotype in the hematopoietic stem/progenitor cells (HSPCs), little is known about the consequences of a nonfunctional FA pathway in other stem/progenitor cell compartments. Given the intense functional interactions between HSPCs and the mesenchymal microenvironment, we investigated the FA pathway on the cellular functions of murine mesenchymal stem/progenitor cells (MSPCs) and their interactions with HSPCs in vitro and in vivo. Here, we show that loss of the murine homologue of FANCG (Fancg) results in a defect in MSPC proliferation and in their ability to support the adhesion and engraftment of murine syngeneic HSPCs in vitro or in vivo. Transplantation of wild-type (WT) but not Fancg(-/-) MSPCs into the tibiae of Fancg(-/-) recipient mice enhances the HSPC engraftment kinetics, the BM cellularity, and the number of progenitors per tibia of WT HSPCs injected into lethally irradiated Fancg(-/-) recipients. Collectively, these data show that FA proteins are required in the BM microenvironment to maintain normal hematopoiesis and provide genetic and quantitative evidence that adoptive transfer of WT MSPCs enhances hematopoietic stem cell engraftment.

Identification, developmental expression and regulation of the Xenopus ortholog of human FANCG/XRCC9.

Fanconi anemia (FA) is associated with variable developmental abnormalities, bone marrow failure and cancer susceptibility. FANCG/XRCC9 is member of the FA core complex, a group of proteins that control the monoubiquitylation of FANCD2, an event that plays a critical role in maintaining genomic stability. Here we report the identification of the Xenopus laevis ortholog of human FANCG (xFANCG), its expression during development, and its molecular interactions with a partner protein, xFANCA. The xFANCG protein sequence is 47% similar to its human ortholog, with highest conservation in the two putative N-terminal leucine zippers and the tetratricopeptide repeat (TPR) motifs. xFANCG is maternally and zygotically transcribed. Prior to the midblastula stage, a single xFANCG transcript is observed but two additional alternatively spliced mRNAs are detected after the midblastula transition. One of the variants is predicted to encode a novel isoform of xFANCG lacking exon 2. The mutual association between FANCG and FANCA required for their nuclear import is conserved in Xenopus egg extracts. Our data demonstrate that interactions between FANCA and FANCG occur at the earliest stage of vertebrate development and raise the possibility that functionally different isoforms of xFANCG may play a role in early development.

Defective mitochondrial peroxiredoxin-3 results in sensitivity to oxidative stress in Fanconi anemia.

Cells from patients with Fanconi anemia (FA), an inherited disorder that includes bone marrow failure and cancer predisposition, have increased sensitivity to oxidative stress through an unknown mechanism. We demonstrate that the FA group G (FANCG) protein is found in mitochondria. Wild-type but not G546R mutant FANCG physically interacts with the mitochondrial peroxidase peroxiredoxin-3 (PRDX3). PRDX3 is deregulated in FA cells, including cleavage by a calpainlike cysteine protease and mislocalization. FA-G cells demonstrate distorted mitochondrial structures, and mitochondrial extracts have a sevenfold decrease in thioredoxin-dependent peroxidase activity. Transient overexpression of PRDX3 suppresses the sensitivity of FA-G cells to H2O2, and decreased PRDX3 expression increases sensitivity to mitomycin C. Cells from the FA-A and -C subtypes also have PRDX3 cleavage and decreased peroxidase activity. This study demonstrates a role for the FA proteins in mitochondria witsh sensitivity to oxidative stress resulting from diminished peroxidase activity. These defects may lead to apoptosis and the accumulation of oxidative DNA damage in bone marrow precursors.

Evidence for subcomplexes in the Fanconi anemia pathway.

Fanconi anemia (FA) is a genomic instability disorder, clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. Cells derived from patients with FA display a marked sensitivity to DNA cross-linking agents, such as mitomycin C (MMC). This observation has led to the hypothesis that the proteins defective in FA are involved in the sensing or repair of interstrand cross-link lesions of the DNA. A nuclear complex consisting of a majority of the FA proteins plays a crucial role in this process and is required for the monoubiquitination of a downstream target, FANCD2. Two new FA genes, FANCB and FANCL, have recently been identified, and their discovery has allowed a more detailed study into the molecular architecture of the FA pathway. We demonstrate a direct interaction between FANCB and FANCL and that a complex of these proteins binds FANCA. The interaction between FANCA and FANCL is dependent on FANCB, FANCG, and FANCM, but independent of FANCC, FANCE, and FANCF. These findings provide a framework for the protein interactions that occur "upstream" in the FA pathway and suggest that besides the FA core complex different subcomplexes exist that may have specific functions other than the monoubiquitination of FANCD2.

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2.

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.