Functional Interplay between BRCA2/FancD1 and FancC in DNA Repair*

A rare hereditary disorder, Fanconi anemia (FA), is caused by mutations in an array of genes, which interact in a common FA pathway/network. These genes encode components of the FA “core” complex, a key factor FancD2, the familial breast cancer suppressor BRCA2/FancD1, and Brip1/FancJ helicase. Although BRCA2 is known to play a pivotal role in homologous recombination repair by regulating Rad51 recombinase, the precise functional relationship between BRCA2 and the other FA genes is unclear. Here we show that BRCA2-dependent chromatin loading of Rad51 after mitomycin C treatment was not compromised by disruption of FANCC or FANCD2. Rad51 and FancD2 form colocalizing subnuclear foci independently of each other. Furthermore, we created a conditional BRCA2 truncating mutation lacking the C-terminal conserved domain (CTD) (brca2ΔCTD), and disrupted the FANCC gene in this background. The fancc/brca2ΔCTD double mutant revealed an epistatic relationship between FANCC and BRCA2 CTD in terms of x-ray sensitivity. In contrast, levels of cisplatin sensitivity and mitomycin C-induced chromosomal aberrations were increased in fancc/brca2ΔCTD cells relative to either single mutant. Taken together, these results indicate that FA proteins work together with BRCA2/Rad51-mediated homologous recombination in double strand break repair, whereas the FA pathway plays a role that is independent of the CTD of BRCA2 in interstrand cross-link repair. These results provide insights into the functional interplay between the classical FA pathway and BRCA2.

Fanconi anemia (FA) 3 is a rare hereditary disorder characterized by bone marrow failure, genome instability, and an increased susceptibility to cancer (1)(2)(3). Cells isolated from FA patients display increased levels of spontaneous and DNA cross-link-induced chromosomal breakage, suggesting that the basic defect in FA could be related to DNA repair (4,5). Indeed, recent studies have indicated that FA cells are defective in two processes, both of which are implicated in restarting stalled replication forks: homologous recombination (HR) repair and translesion synthesis (TLS) (6 -9).
To date, 12 genetic FA subgroups have been identified on the basis of complementation analysis, and 11 of these have been cloned (FancA/B/C/D1/D2/E/F/G/J/L/M) (10,11). Among them, eight gene products interact with each other, forming the FA core complex (FancA/B/C/E/F/G/L/M). This complex is thought to be a multisubunit ubiquitin E3 ligase (12), which monoubiquitinates FancD2 protein upon DNA damage and during a normal unperturbed S phase (13,14). Monoubiquitination is both necessary and sufficient for FancD2 to be targeted to chromatin following DNA damage, form subnuclear foci, and participate in DNA repair (15,16).
Recent studies have revealed an increasingly complex picture of the FA pathway/network. It is likely that the core complex functions not only in the monoubiquitination of FancD2 but also in other roles required for the targeting of FancD2 to chromatin, as well as for DNA repair (16). Indeed, the FA core complex carries subunits that are required not only for monoubiquitinating FancD2 (E3 ligase FancL) but also for DNA modification (FancM) (17,18). FancM is a vertebrate homolog of the archaeal nuclease/helicase Hef (19), and, therefore, is likely to play a role in the processing of damaged DNA (20). The core complex also associates with BLM helicase (21) and regulates its subcellular localization (22). Thus the core complex seems to have multiple effector arms, which probably cooperate with FancD2 in DNA repair (16). However, the precise biochemical function of FancD2 is still unknown. Importantly, any patient-derived mutation found in the core complex components appears to disrupt the structural integrity of the complex and may cause complete or partial loss of function in effector molecules. It is currently unclear how the FA pathway couples with DNA repair pathways such as HR or TLS.
Besides the core complex components and FancD2, two additional FA proteins, FancD1 and FancJ, have been identified, both of which are intimately linked to familial breast cancer. In FANCD1 mutant cells, biallelic hypomorphic mutations of breast cancer susceptibility gene BRCA2 have been identified (23), whereas FANCJ was found to be the BRCA1-interacting helicase, Brip1 (also known as BACH1) (24 -27). Individuals carrying monoallelic BRCA1 or BRCA2 mutation are predis-posed to breast and ovarian cancer (28). FANCD1 or FANCJ mutant cells display normal FancD2 monoubiquitination following DNA damage (24), indicating that these genes function independently, or downstream, of the core complex-FancD2 ("classical FA") pathway. How BRCA2/FancD1 or the Brip1/ FancJ helicase functionally interact with the rest of the FA proteins is largely unknown. In this study, we have focused on the functional relationship between BRCA2 and the classical FA pathway.
A major cellular function of BRCA2 is to regulate the Rad51 recombinase, which catalyzes the initial strand invasion reaction in HR (28,29). Rad51 forms subnuclear foci upon DNA damage, and this could reflect Rad51 nucleoprotein filaments polymerized onto the single-stranded DNA ends generated by nucleolytic processing of DSB (29). This foci formation is severely impaired in BRCA2-deficient cells (28). Consistent with a connection between the classical FA pathway and BRCA2, the nuclear foci of FancD2 co-localize with both BRCA2 and Rad51 foci, and monoubiquitinated Fanc-D2 coimmunoprecipitates with BRCA2 from chromatin fractions (14,30).
To elucidate the functional hierarchy between FA genes and HR factors, analysis of Rad51 focus formation has been employed. However, this has yielded conflicting results to date. Although several groups have reported that Rad51 nuclear foci formation is significantly attenuated in FA cells (30 -32), other groups reported that DNA damage-induced Rad51 nuclear foci formation is unaffected in FANCD2-deficient PD20 cells and other human or mouse FA cells (33)(34)(35)(36)(37). Furthermore, siRNA treatment against BRCA2 does not increase the frequency of MMC-induced quadriradial chromosome formation in FANCA-deficient fibroblasts, whereas siRNA against BRCA1 does (38). On the other hand, another study found that depletion of BRCA2 or Rad51 by siRNA further sensitizes FANCD2deficient human cells against MMC (33).
To dissect the functional relationship between the classical FA pathway and BRCA2 more precisely, we analyzed MMCinduced Rad51chromatin loading and foci formation in cells with or without a functional FA pathway or BRCA2. We observed that BRCA2, but not FANCC or FANCD2, is indispensable for the loading of Rad51 to chromatin. We also found that FancD2 and Rad51 act independently of each other in terms of DNA damage-induced foci formation. To mimic the phenotype of FA-D1 patients (23), we generated a BRCA2 truncating mutation immediately downstream of BRC repeats, which resulted in deletion of the C-terminal domain (CTD) including the OB folds that bind single-stranded DNA (CTD truncation) (39). Based on this BRCA2 CTD truncation mutant, we created fancc/brca2⌬CTD double mutant cells. Phenotypic analysis of this set of mutant cells revealed that there is a complex interplay between the BRCA2-Rad51 and FancC-FancD2 pathways depending on the type of DNA damage.

EXPERIMENTAL PROCEDURES
Cells Lines and Gene Targeting-Wild type and various mutant chicken DT40 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% chicken serum, 2 mM L-glutamine, 50 M 2-mercaptoethanol, penicillin, and streptomycin in a 5% CO 2 incubator at 39.5°C. Generation of fancc (22), fancd2 (7), and brca2⌬BRC (40) cells has been described previously. In the brca2⌬BRC cell line, one allele was designed to produce chicken BRCA2 truncated at a point immediately downstream of the BRC3 repeat, whereas the second allele was rendered conditionally null by using inducible MerCreMer recombinase (40).
The generation of brca2⌬CTD and fancc/brca2⌬CTD cells is shown schematically in Fig. 3B. We constructed the BRCA2-CTD truncation vector by replacing the upper homology arm of the BRCA2-BRC truncation vector (40) with an ϳ1 kb genomic PCR fragment. The BRCA2-BRC truncation vector was kindly provided by Drs. Mitsuyoshi Yamazoe and Shunichi Takeda (Kyoto University). The genomic PCR fragment harbored a sequence change to introduce the truncating mutation (CTD truncation) downstream of the BRC8 repeat (codon 2151 was changed to stop codon). As with brca2⌬BRC cells, the BRCA2-CTD truncation was achieved in cells also harboring conditionally null allele of BRCA2 (a kind gift from Drs. Mitsuyoshi Yamazoe and Shunichi Takeda), resulting in BRCA2 ⌬CTD/con cells (Fig. 3B). Then FANCC gene disruption was achieved using the previously described FANCC targeting vector (22). The deletion of BRCA2 conditionally null allele was induced by the addition of 4-hydroxy-tamoxifen into both BRCA2 ⌬CTD/con and fancc/BRCA2 ⌬CTD/con and was further verified by subcloning. Genomic Southern blotting and RT-PCR analysis were done as described previously (22,40).
A GFP-FancD2 expression vector was made by subcloning the chicken FANCD2 cDNA into pEGFP-C1 (Clontech, Palo Alt, CA). A point mutation to remove the monoubiquitination site (K563R) was introduced using a QuikChange kit (Stratagene, La Jolla, CA). Transfections were done by electroporation as described previously (6).
Western Blotting Analysis-Anti-chicken N-terminal Brca2 and anti-FancD2 antisera were raised as described previously (7,40). Anti-Rad51 polyclonal antibody was kindly provided by Dr. Hitoshi Kurumizaka (Waseda University, Tokyo). Anti-histone H4 antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Cell fractionation into the soluble and chromatin fractions (16) and Western blotting (40) were performed as described previously.
Cell Analysis by Flow Cytometry-Cell growth and cell cycle distribution were analyzed using FACSCalibur flow cytometer (BD Biosciences) as described (6). Anti-BrdUrd antibody was purchased from BD Pharmingen. Mitotic index was determined as described previously (41). Briefly, washed cells were fixed in 70% ethanol, stained with anti-phosphorylated histone H3 antibody (Upstate Biotechnology Inc.) followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG, and analyzed with FACSCalibur.
Analysis of Chromosomal Aberrations-Chromosome analysis was performed as previously described (6). Briefly, colcemid (0.1 mg/ml) was added immediately following x-ray irradiation, and cells were harvested 3 h later. In some cases, cells were cultured in medium containing 2.5 or 5 ng/ml MMC for 24 h, and colcemid was added 2 h before harvesting. Scoring was performed, as described previously (6), on coded slides.

Disruption of FANCC or FANCD2 Does Not Significantly
Attenuate MMC-induced Chromatin Loading of Rad51-We have shown previously that MMC-induced Rad51 nuclear focus formation is not significantly impaired in fancg (6) or fancd2 (7) cells. We also found that the human fancd2 cell line PD20 and PD20 cells complemented with human FancD2 displayed similar levels of Rad51 foci following both x-ray and MMC treatments (supplemental Fig. S1). Given a number of contradictory results reported in the literature (30 -32), we wished to evaluate the efficiency of Rad51 relocalization after DNA damage in a more quantitative manner. To this end, we separated cell lysates of wild type, fancc, fancd2, and brca2⌬BRC chicken DT40 cells into detergent-soluble and -insoluble (chromatin) fractions, as described previously (16), and quantified the abundance of Rad51 in the chromatin fraction by Western blotting with an anti-Rad51 antibody.
In wild type cells, we observed a significant increase of Rad51 protein in the chromatin fraction in response to MMC treatment, which probably reflects nucleoprotein Rad51 filament formation (Fig. 1). Strikingly, we found similarly increased levels of chromatin-bound Rad51 protein in fancc or fancd2 cells (Fig. 1). In con-trast, we were not able to detect this induction in brca2⌬BRC cells, in which BRCA2 is not functional (Fig. 1). However, chromatin loading of FancD2, which is dependent on its monoubiquitination, was normal in brca2⌬BRC but was compromised in fancc cells, which is consistent with previous studies (16) (Fig. 1). These results strongly suggest that following DNA damage, Rad51 loading onto single-stranded DNA ends is regulated by BRCA2/FancD1 but not by monoubiquitinated FancD2.
DNA Damage-induced FancD2 or Rad51 Focus Formation Proceeds Independently of Each Other-Next, we examined Rad51 foci formation in relation to FancD2. To conveniently visualize FancD2 distribution in wild type or fancc cells, we expressed a chicken FancD2 protein fused to GFP in fancd2 DT40 cells (designated GFP-FancD2 WT cell). We also then deleted FANCC gene in those cells (GFP-FancD2 fancc cells). We also expressed GFP-FancD2 protein devoid of the monoubiquitination site by mutating lysine 563 to arginine in fancd2 cells (GFP-FancD2 KR cells). GFP-FancD2 WT protein was converted to L-form (monoubiquitinated form) following DNA damage, whereas in the other two mutant cell line, this conversion was not detectable. Consistently, only GFP-FancD2 WT cells showed cisplatin tolerance comparable with that of wild type DT40 cells. 4 In GFP-FancD2 WT cells, MMC or x-ray treatment induced robust Rad51 focus formation ( Fig. 2A), and the majority of these foci co-localized with GFP-FancD2 foci ( Fig. 2A and data not shown). Although GFP foci formation was completely defective in GFP-FancD2 KR or GFP-FancD2 fancc cells ( Fig.  2A), Rad51 foci formation occurred with similar kinetics, there appeared to be some reduction in the fraction of Rad51 focipositive cells following x-ray exposure (Fig. 2, A and B). In these two cell lines, Rad51 foci appeared to be slightly smaller and hence more difficult to visualize, which might explain the observed small reduction in percentage of cells with foci. Thus, we conclude that nuclear focus formation of Rad51 remains largely unaffected, irrespective of whether FancD2 protein can form foci or not. Conversely, we observed normal FancD2 focus formation in the absence of Rad51 foci formation caused by brca2⌬BRC disruption (Fig. 2C). These data indicate that Rad51 and FancD2 foci formation are independent events, despite the fact that these two proteins accumulated at the same DNA damage sites.
To exclude the possibility that Rad51 focus formation in FA cells in response to DNA damage is qualitatively different from those in wild type cells (6, 7), we examined whether these Rad51 foci are properly localized at sites of DNA damage. Following DSB induction, Histone H2AX is rapidly phosphorylated at its C terminus in a megabase-sized chromatin region surrounding the DNA damage site, and this leads to the formation of subnuclear foci (42), which serve as a marker for DNA damage sites. We therefore examined the co-localization of Rad51 foci with ␥H2AX foci in cells lacking FANCC or FANCD2. MMC treatment induced co-localizing nuclear foci of Rad51 and ␥H2AX in wild type cells, and this co-localization was also preserved in fancc or fancd2 cells (data not shown). Collectively, 4 N. Matsushita and M. Takata, manuscript in preparation. these results suggest that Rad51 protein is properly recruited to and loaded onto DNA damage sites in chromatin even in cells lacking FANCC or FANCD2.
fancc/brca2⌬CTD Double Mutant Cells Display Growth Defects Similar to brca2⌬CTD Cells-To examine the functional interplay between classical FA pathway and BRCA2/ FancD1, attempts at "epistasis-type" analysis have been carried out using BRCA2 siRNA knockdown in FA cells, but this has yielded conflicting results (33,38). To dissect the relationship more precisely, we employed a genetic approach. We first established BRCA2 conditional knock-out cells in which one BRCA2 allele expressed a truncated form of the protein that contained all of the BRC repeats but had lost the C-terminal conserved domain, whereas the other allele was conditionally null (referred to as BRCA2 ⌬CTD/con ) (Fig. 3A). In this cell line, both wild type and CTD truncated BRCA2 are expressed (data not shown). Cells harboring the ⌬CTD truncation may mimic the defective allele found in FA-D1 patients, because they often have a mutation that leads to truncation of the CTD region (23).
Following the above described gene manipulations, deletion of the conditional allele expressing wild type BRCA2 protein was achieved by the addition of 4-hydroxy-tamoxifen as described previously (40), producing mutant clones that expressed only truncated BRCA2 protein (Fig. 3C) (referred to as brca2⌬CTD). Using BRCA2 ⌬CTD/con conditional knock-out cells, we further disrupted the FANCC gene (referred to as fancc/BRCA2 ⌬CTD/con ), and isolated double mutant clones by 4-hydroxy-tamoxifen treatment (referred to as fancc/ brca2⌬CTD) (Fig. 3B). We confirmed each genotype by Southern blotting and RT-PCR analysis (Fig. 3D). FANCC gene disruption was also verified by the loss of FancD2 L-form using Western blotting with anti-FancD2 antibody (Fig. 3E). The CTD-truncated BRCA2 protein should contain intact copies of all BRC repeats, which interact with Rad51, and the truncated BRCA2 could be detected by Western blotting using N terminus-specific anti-BRCA2 antibody (Fig. 3C). However, DNA damage-induced Rad51 foci were abrogated in brca2⌬CTD or fancc/brca2⌬CTD cells (supplemental Fig. S2). A previous report has shown that brca2⌬BRC cells grow slowly, which is explained by the high rate of spontaneous cell death (40). We also found that brca2⌬CTD cells displayed slower growth than wild type cells (Fig. 4A) and higher levels (ϳ30%) of spontaneous cell death (Fig. 4B) than the BRCA2 ⌬CTD/con cells. In contrast, a milder growth defect was found in fancc/BRCA2 ⌬CTD/con cells, which was consistent with the known phenotype of fancc cells (22) (Fig. 4A). Interestingly, the growth rate and levels of spontaneous cell death were almost the same in fancc/brca2⌬CTD cells as in brca2⌬CTD cells (Fig. 4, A and B).
We next examined cell cycle distribution following BrdUrd pulse labeling. We observed that both brca2⌬CTD cells and fancc/brca2⌬CTD cells displayed a modest accumulation of cells in the G 1 and G 2 /M phases compared with BRCA2 ⌬CTD/con or fancc/BRCA2 ⌬CTD/con cells (Fig. 4C). The apparent increase of cells in the G 2 /M phase was shown to be caused by G 2 accumulation, because the proportion of M phase cells was found to be decreased as determined by flow cytometric analysis using a specific antibody against the phosphorylated form of histone H3 (Fig. 4B). These results indicate that both brca2⌬CTD and fancc/brca2⌬CTD cells exhibit higher levels of spontaneous G 2 arrest, which might be due to residual unrepaired DNA damage. Furthermore, we observed a similar (ϳ1 h) elongation of single cell cycle length in brca2⌬CTD and fancc/brca2⌬CTD cells when compared with their parental cells (data not shown). We concluded that the growth defect in brca2⌬CTD cells is caused by spontaneous cell death and aberrant cell cycle progression. In addition, FANCC gene disruption, even though it makes wild type cells grow more slowly, had no additional effect in brca2⌬CTD cells on their growth defects, consistent with the possibility that FANCC and BRCA2 cooperate in a common pathway for handling DNA damage that occurs spontaneously.
Functional Interplay between FANCC and BRCA2 in Cell Survival following DNA Damage-To examine the capacity of BRCA2 ⌬CTD/con , fancc/BRCA2 ⌬CTD/con , brca2⌬CTD, and fancc/brca2⌬CTD cells to repair different DNA lesions, we exposed these cell lines to various DNA damaging agents and measured colony-forming survival. Consistent with our previous report (22), fancc/BRCA2 ⌬CTD/con cells showed only a moderate level of sensitivity to x-rays or MMS (Fig. 5, A and B), whereas brca2⌬CTD cells were consistently more sensitive to x-rays or MMS (Fig. 5, A and B). Interestingly, the levels of sensitivity to x-ray or MMS were comparable in brca2⌬CTD cells and fancc/brca2⌬CTD cells (Fig. 5, A and B), indicating an epistatic relationship between the classical FA pathway and BRCA2 in repairing chromosomal DSBs. In sharp contrast, fancc/brca2⌬CTD double mutant cells showed a more severe level of sensitivity to cisplatin and MMC compared with either of the single mutants (Fig. 5, C and D). This additive sensitivity indicates that BRCA2 CTD and FANCC have nonoverlapping function in repairing ICL lesions on chromosome.
Unrepaired DNA lesions are often represented in metaphase chromosome spreads as chromosomal aberrations. Hence, we scored several types of chromosomal aberrations in these cells after treatment with MMC or x-rays. X-ray treatment did not induce higher levels of aberrations in fancc/brca2⌬CTD cells compared with brca2⌬CTD cells (Fig. 6A), although in both cases this frequency was elevated compared with cells expressing functional BRCA2. In contrast, not only were high levels of chromosomal aberrations induced in brca2⌬CTD cells using very low concentrations of MMC, but also fancc/brca2⌬CTD cells displayed a higher level of aberrations than brca2⌬CTD single mutant cells (Fig. 6B). These results confirmed the conclusions from the epistasis analysis between FANCC and BRCA2 suggested by the colony survival data, also indicating that the functional interplay between these molecules depends on the particular type of DNA damage involved.

DISCUSSION
In response to ionizing radiation, the BRCA2/FancD1 protein is loaded onto chromatin and associates specifically with monoubiquitinated FancD2 (30). BRCA2 also interacts with FancG (43) as well as FancE (30). Given that BRCA2 is one of the FA genes, these data indicate a close functional relationship between the classical FA pathway and BRCA2. It has been hypothesized that BRCA2/FANCD1 functions down- The appearance of the shorter ϳ140-bp FANCC RT-PCR band was due to anomalous splicing resulting from gene targeting followed by excision of the resistance marker cassette. This resulted in a frameshift, leading to production of short truncated protein (residues 1-55 plus six amino acids) as described (22). E, Western blot analysis of chicken FancD2. Total cell lysates from indicated cell lines were immunoblotted (IB) with anti-chicken FancD2 antibody. Cells were either untreated (lanes 1, 3, 5, and 7) or treated with 500 ng/ml MMC for 6 h (lanes 2, 4, 6, and 8).
stream of monoubiquitinated FANCD2, because FancD2 is normally monoubiquitinated and activated in FA-D1 cells. To verify this proposal, the formation of Rad51 foci has been examined as an indication of BRCA2 function in the absence of classical FA proteins. However, the results of such studies are contradictory, and it has not been possible to draw a definite conclusion.
In our present study, we employed biochemical fractionation of DT40 FA cells and examined the MMC-induced chromatin loading of Rad51, which probably reflects relocalization of Rad51 to DNA damage sites. We found that Rad51 chromatin loading occurs normally following MMC treatment in fancc or fancd2 cells but not in brca2⌬BRC cells. Using cells expressing GFP-tagged FancD2 protein, we also show that Rad51 and FancD2 form co-localizing foci with similar kinetics in response to DNA damage. However, Rad51 and FancD2 focus formation are inde-pendent events, suggesting that the classical FA pathway functions parallel to the BRCA2-Rad51 pathway. Alternatively, it is possible that the FA pathway functions at late stage of HR, downstream of BRCA2-Rad51. Recent identification of the Brip1 helicase as FANCJ (25)(26)(27)44) may support this latter possibility, because Brip1 may function downstream of FancD2 (24) and preferentially unwind certain recombination intermediates such as D-loops (45). In agreement with this hypothesis, Rad51 foci form normally in BRIP1/FANCJ-deficient cells (26).
We also examined the phenotype of fancc/brca2⌬CTD double mutant cells relative to either single mutant. We found that FANCC and the CTD of BRCA2 function together in an epistatic manner in cell proliferation as well as in repairing DSBs induced by x-rays, whereas they have distinct and nonoverlapping functions in repairing ICLs. The ICL repair pathway in vertebrates is not very well understood; however, it likely employs a complex network of DNA repair machineries including nucleotide excision repair (NER), HR, TLS, and FA pathways (46,47). In our current understanding, ICL repair proceeds in several steps: (i) incisions mediated by endonuclease XPF/ ERCC1 to uncouple the ICL, (ii) DSB formation by a mechanism probably related to replication, (iii) translesion DNA synthesis past the unhooked ICL lesion, (iv) a second incision to remove the remaining mono-adduct, and (v) repair of the ICL-associated DSB by HR. Recent studies have indicated that FANCC has overlapping roles with both HR and the TLS polymerases Rev1 and Rev3 in repairing ICLs (8,22,48). In support of a role for the FA pathway in TLS, we observed significantly decreased levels of hypermutation in Ig V in fancd2 DT40 mutant cells (7), and this hypermutation is known to depend on the presence of Rev1 (49). In contrast, BRCA2 is known to participate only in the early phases of HR, and DT40 ⌬BRC mutant cells display increased Ig hypermutation with loss of gene conversion (40). Thus in the repair of DSBs induced by x-rays, FANCC and the BRCA2 CTD function together in HR. On the other hand, the TLS function of FANCC might be required for repairing ICLs, providing an explanation for the distinct roles of FANCC and BRCA2 CTD in this type of DNA repair. BRCA2 and FANCC also appeared to have an epistatic function during normal cell cycle progression, suggesting that they are mainly handling DSBs in the context of the restart of stalled replication forks. These data suggest that the relationship between the classical FA pathway and the function of BRCA2 is not necessarily a simple, linear pathway, depending on types of DNA damage. BRCA2 knock-out mice display embryonic lethality (50), and only hypomorphic mutant mice can survive. This also seems to be true for human subjects (23). In contrast, fancc, fancg, fanca, and fancd2 mice are able to grow largely unaffected (1,2). Interestingly, fancd2 mice display overlapping features with BRCA2 hypomorphic mice, such as epithelial carcinogenesis or germ cell defects (37). However, a number of previous studies have suggested distinct functional defects in FA-D1 patients. First, FA-D1 patients are much more susceptible to cancer, including early onset leukemia and solid tumor, than other subtypes of FA (51,52). Second, a recent study provided evidence that HR defects in BRCA mutant human cells are much larger in size than in classical FA cells (fancc, fancg, and fancd2) (9). Furthermore, these FA genes, but not BRCA2, are necessary for one type of HR repair, i.e. single-strand annealing pathway (9). Thus, the similarity of the symptoms between FA-D1 patients and other  FA patients (e.g. bone marrow failure) could be caused by shared defects in HR repair.
In conclusion, we have reexamined chromatin loading and focus formation of Rad51 in FA mutant cell lines and confirmed that these early phase events in HR repair were compromised in BRCA2 BRC⌬ or CTD⌬ mutants but not in classical FA mutants. However, genetic analysis indicated that the BRCA2 CTD and FANCC function in the common DSB repair pathway mediated by HR. In contrast, we found that in dealing with ICLs, the BRCA2 CTD and FANCC have nonoverlapping functions. How the FA pathway connects with HR or TLS pathway is still unknown. Whether the recently identified FancM or FancJ serves as the link should be clarified in the near future.