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Seiki Hirano,1
Masamichi Ishiai,1
Kenichi Morishima,2
Hiroyuki Kitao,1
Keiko Namikoshi,1
Masayo Kimura,1
Nobuko Matsushita,1
Hiroshi Arakawa,3
Jean-Marie Buerstedde,3
Kenshi Komatsu,2
Larry H. Thompson,4 and
Minoru Takata1*
Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki, Okayama,1 Department of Genome Repair Dynamics, Radiation Biology Centre, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto, Japan,2 GSF, Institute for Molecular Radiobiology, Neuherberg, Munich, Germany,3 Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California4
Received 14 May 2004/ Returned for modification 4 June 2004/ Accepted 8 October 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eleven genes have been identified by complementation analysis (FancA/B/C/D1/D2/E/F/G/I/J/L) (12, 16, 25, 30, 32). Many of the FA proteins interact with each other in the nucleus, forming a multiprotein complex (the core complex) containing FancA/C/E/F/G/L, BLM helicase, and other unidentified components (12, 16, 25, 32, 33). During S phase or after DNA damage, FancD2 protein is monoubiquitinated on a specific lysine residue (K561 in the human protein) (15, 54). This event is apparently mediated by FancL/PHF9 E3 ligase but also requires the integrity of the core complex (12, 32, 58). This monoubiquitination appears to be essential for FancD2 function, since FancD2 K561R mutant protein cannot complement the MMC sensitivity of human FANCD2-deficient cells (15). Interestingly, FancD2 is phosphorylated at serine 222 by ATM protein kinase after ionizing irradiation (IR) (55). This phosphorylation is required for the IR-induced S-phase checkpoint but not for MMC tolerance, whereas the monoubiquitination is dispensable for the S-phase checkpoint (54).
Several lines of evidence indicate that the FA pathway influences homologous recombination (HR)-mediated DNA repair. The monoubiquitinated FancD2 (FancD2-L form) is targeted to nuclear foci that colocalize with HR proteins Rad51 and BRCA1 (15, 54). BRCA1, a tumor suppressor for familial breast cancer that is important for HR (reviewed in references 46 and 59), colocalizes with FancD2 after DNA damage (15, 58). Other FA proteins are reported to overlap or interact with HR proteins (24, 36, 50). The discovery that FANCD1 gene is identical to BRCA2, another tumor suppressor gene involved in familial breast cancer, provides a strong link between HR and the FA "pathway" (22). BRCA2 promotes nuclear localization and nucleoprotein filament formation of Rad51 (13, 46, 59) and physically interacts with FancD2 (23, 61). Finally, we have recently reported mild HR defects in FANCG-deficient cells derived from DT40 (66), a chicken B-cell line that is highly proficient in gene targeting (6).
FancD2 and its putative activator FancL are found even in Caenorhabditis elegans and Drosophila (32, 57), whereas other FA proteins exist only in vertebrates, with the exception of FancD1/BRCA2, which is conserved in Ustilago maydis (28). Notably, The Fancd2-null mouse has a more severe phenotype (21) than knockouts of Fanca (63), Fancc (9), and Fancg (29, 67). However, FancD2's precise biochemical function remains enigmatic.
Recently, three mechanisms of immunoglobulin (Ig) diversification (Ig gene conversion, class switch recombination, and somatic hypermutation) were shown to be dependent on activation-induced deaminase (AID) (2, 18, 20, 35). Avian species utilize gene conversion (templated substitutions by pseudogene donor sequences located upstream of the Ig gene), and DT40 cells recapitulate this process during growth in culture (1, 2, 5). Since Ig gene conversion is mediated by HR proteins, including Rad54 (4) and Rad51 paralogs (43, 51), DT40 provides a system to define a role for FancD2 in this process.
We report here the generation and phenotypic characterization of FANCD2-deficient DT40 cells (6). Compared to FANCG mutant (66), FANCD2 deficiency was associated with a severe phenotype. We find that FancD2 is required for efficient gene targeting, HR-directed repair of chromosomal double-strand breaks (DSBs), and gene conversion at the Ig light-chain locus without increased frequency of nontemplated point mutations. In contrast, spontaneous sister chromatid exchange (SCE) is elevated in FANCD2-deficient cells. Since SCE is thought to be mediated by HR (49), this phenotype indicates that not all HR processes are affected similarly. We propose that there are subpathways of HR in mitotic cells, which are dependent or independent of FancD2 protein.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chicken FANCD2 cloning and gene targeting.
An expressed sequence tag clone containing only C-terminal 
200-bp chicken FANCD2 cDNA was obtained by searching the chicken expressed sequence tag database and then used for cDNA library (Clontech, Palo Alto, Calif.) screening. The library was also screened by using human FANCD2 probe that was obtained by reverse transcription-PCR from human cDNA. Full-length chicken FANCD2 was PCR amplified from DT40 cDNA and then inserted into pcDNA3 vector (Invitrogen, Carlsbad, Calif.). The sequence of the cloned cDNA was compared to that from direct sequencing of reverse transcription-PCR products; three PCR-associated errors were corrected by site-directed mutagenesis (Stratagene, La Jolla, Calif.). The human Rad51 expression vector has been described (34).
Genomic clones of FANCD2 were isolated by library screening (Stratagene). Targeting vectors were designed to replace a genomic segment containing exons with a resistance marker gene cassette. As a result, chicken FancD2 amino acids 554 to 653, which contain the conserved monoubiquitination site (K563 in chicken protein), were deleted.
FANCD2 disruption was achieved in three lines of DT40: wild type, DT40 carrying the recombination substrate SCneo (14, 27), and DT40Cre1. DT40Cre1 cells express transfected v-myb and MerCreMer (mutated estrogen receptor fused to Cre recombinase, which is translocated from cytoplasm to nucleus upon addition of 4-hydroxy tamoxifen) (2, 68). We disrupted FANCD2 loci in this cell line by sequential targeting by using single targeting vector (FANCD2-bsr) and MerCreMer-mediated recycling of the drug resistance marker. FANCC disruption will be described elsewhere (S. Hirano et al., unpublished data).
Sensitivity of cells to X-rays, UV, MMC, and cisplatin. Colony formation was assayed in medium containing 1.4% methylcellulose. Serially diluted cells were irradiated with 4-MV X-rays (Linear Accelerator; Mitsubishi Electric, Inc., Tokyo, Japan) as described previously (66). Irradiation of synchronized cells was done after nocodazole arrest (52). UV irradiation was performed on cells suspended in phosphate-buffered saline. Cells were exposed for 1 h to MMC (Kyowa-Hakkou, Tokyo, Japan) or continuously to cisplatin (Nihon-Kayaku, Tokyo, Japan).
Western blot analysis and Rad51 focus assay. Anti-chicken FancD2 serum was raised by injecting recombinant chicken FancD2 protein (amino acids 3 to 250) into rabbits. Anti-Rad51 serum was kindly provided by Akira Shinohara (Osaka University). Whole-cell extracts were separated with sodium dodecyl sulfate-6% polyacrylamide gel electrophoresis and then transferred to a membrane. Detection was done with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway, N.J.) by using ECL Western blotting detection reagents (Amersham Biosciences). To visualize subnuclear focus formation of Rad51, Cytospin slides were fixed and stained with the antiserum. Images were captured by using TCS-NT laser scanning confocal microscopy (Leica Microsystems, Bannockburn, Ill.) and processed with Adobe Photoshop software. Cells with more than four brightly fluorescing foci were scored as positive. At least 100 morphologically intact cells were examined at each time point.
Analysis of chromosomal aberrations and SCE. Chromosome analysis was done as previously described (51, 52). For SCE analysis, Colcemid (0.1 µg/ml) was added for the last 2 h of this incubation before harvest, and scoring was performed as previously described on coded slides (51).
Measurement of HR-mediated repair of DSB induced by I-SceI expression. I-SceI expression vector was transiently transfected by electroporation, and cells were selected in 96-well culture plates containing 2.0 mg of G418/ml. Surviving colonies were counted after 14 days. To determine the nature of repair events, genomic DNA was extracted from expanded colonies, restricted with KpnI and SacI double digestion, and examined by Southern blotting with a neo fragment as a probe (66). To determine cell survival after I-SceI expression, cells were transiently transfected with enhanced green fluorescent protein (EGFP)-I-SceI expression vector. After 16 h, EGFP-positive cells were clone sorted into 96-well culture plates by using FACSAria cell sorter (BD Biosciences, San Jose, Calif.) and cultured without G418. The EGFP-I-SceI expression vector was made by subcloning I-SceI coding sequence to pEGFP-C1 (Clontech).
Measurement of gene conversion events at Ig locus.
The rate of generation of surface IgM (sIgM) loss or gain variants was monitored by flow cytometric analysis of cells that had been kept in culture for the indicated time after limiting dilution and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-chicken IgM (Bethyl, Inc., Montgomery, Tex.). The percent sIgM-positive or -negative cells was calculated for live cells based on propidium iodide (PI) staining and light scatter. Cells were sorted after staining with anti-chicken IgM-FITC and anti-FITC magnetic beads (Miltenyi Biotech, Auburn, Calif.). PCR amplification and sequencing of the rearranged V
gene was done as described previously (43). Nucleotide changes were aligned with the pseudo V database (kindly provided by Julian Sale) and then classified into a gene conversion, nontemplated point mutation, or ambiguous category according to the criteria described (43). The tract length of gene conversion was defined as number of the nucleotides that were changed from the original sequence. Statistical analysis was done by using StatView software (SAS Institute, Inc., Cary, N.C.).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The full-length cDNA (DDBJ accession number AB162932) encodes a 1,439-amino-acid protein, which is very similar in size to the 1,451 amino acids in human FancD2 protein. Chicken and human FancD2 are 55% identical and 70% similar. Both the monoubiquitination (15) residue (K561) and the ATM phosphorylation (55) residue (S222) are conserved in chicken FancD2.
We also obtained chicken FANCD2 genomic clones by library screening and designed FANCD2 disruption vectors that should abolish the exon containing the putative monoubiquitination site (K563 in chicken sequence) (Fig. 1A). Gene targeting events were verified by Southern, Northern, and Western blotting analysis (Fig. 1B, C, and D). Of note, our anti-FancD2 sera detected two forms of FancD2, FancD2-S and FancD2-L (Fig. 1D). FancD2-L, presumed to be a monoubiquitinated form, was absent in DT40 fancc mutant as in human cells (Fig. 1D).
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Gene targeting is defective in fancd2 cells. Mechanisms of targeted integration of homologous DNA are poorly understood, but it is known that this process requires HR factors. To examine whether FancD2 is involved in targeted integration, we measured the gene targeting efficiency at several loci. After transfection, cells were selected with appropriate drugs, and individual colonies were examined by Southern blot analysis. All four targeting vectors showed a clearly decreased ratio of targeted to random integration events in fancd2 cells compared to the wild type (Table 1). Furthermore, chicken FANCD2 expression clearly increased the targeting efficiency using three of the four targeting vectors (Table 1). The lack of increase at the FANCG locus may be a statistical anomaly.
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8 h incubation with nocodazole, followed by release into drug-free medium (14, 52). In G1 to early S phase (4 h after the release) or in late S to G2 phase (9 h), cells were X-irradiated and seeded onto plates containing medium supplemented with methylcellulose. Ku70 cells showed extreme IR sensitivity in G1 to early S but were more resistant than wild type in late S to G2 (Fig. 3B) as previously described (14, 52). In contrast, fancd2 cells were more sensitive than wild type in late S to G2, whereas they were only marginally sensitive in G1 to early S phase (Fig. 3B). Because breaks and gaps in metaphase chromosomes probably represent unrepaired DSBs, we also counted these aberrations in four consecutive 3-h time intervals starting immediately after the IR (2-Gy) exposure. Cells that entered M phase 0 to 3 h after IR would have been irradiated in S or G2 phase. Only in this first interval did fancd2 cells exhibit highly increased aberrations compared to the wild type (Fig. 3A). These results, along with the survival data of Fig. 3B, suggest that FancD2 contributes to HR repair of DSBs produced directly by X-rays.
HR-directed repair of I-SceI-induced DSBs is reduced in fancd2 cells.
To measure HR capacity by using the artificial recombination substrate SCneo (27), we disrupted FANCD2 alleles in DT40 cells carrying SCneo at OVALBUMIN locus (14). In addition, mutant cells were transfected with chicken FANCD2 cDNA, and two complemented clones were established. After DSB induction in a nonfunctional neo segment in SCneo by transient expression of I-SceI, G418 selection was applied for 2 weeks until the colonies were clearly visible. Cells should be G418 resistant only if the DSB was repaired correctly by HR with the upstream neo fragment as a template (Fig. 4A). Significantly, the number of G418-resistant colonies was reduced 40-fold in fancd2 cells compared to the wild type and FANCD2-complemented fancd2, whereas transfection with control DNA (pBluescript) yielded a similar number of colonies in all genotypes (Fig. 4B). We isolated neo-resistant clones from wild-type or fancd2 cells after I-SceI expression and determined the plating efficiency in the presence of G418. The mean plating efficiencies of wild-type (three clones) and fancd2 (three clones) cells were 52 and 34%, respectively. Correcting for the reduced plating efficiency of fancd2 cells compared to that of wild type, the HR deficiency determined by this assay was calculated to be 26-fold.
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22%) containing wild type, and 36 of 384 wells (9%) containing fancd2 cells. Given the difference in the plating efficiency, this result indicates that the overall DSB repair efficiency (including NHEJ activity) in fancd2 cells was not drastically compromised. Next, we examined the structure of the repaired SCneo substrate by Southern blot analysis of genomic DNA from randomly picked G418-resistant colonies. Most (79%) HR repair events in fancd2 cells were short-tract gene conversions (STGC) as in the wild type (85%, as described in reference 66) (Fig. 4C). The remaining colonies underwent either long-tract gene conversion (LTGC) or unequal SCE events with expansion of two to three neo repeats in wild-type (15%) and fancd2 (17%) cells. These two types of events could not be distinguished, since daughter cells having a short neo fragment originating from SCE would be killed by G418. Although the DSB repair with unequal SCE has been reported to be very rare in the SCneo substrate in hamster cells (26), we could not exclude the possibility that there were some changes in LTGC/SCE ratio in fancd2 cells. Taken together, these results indicate that overall gene conversion frequencies were reduced in fancd2 cells.
In addition, it seems noteworthy that, in five clones (4%) from fancd2 cells, aberrant repair products were found (Fig. 4C and data not shown). The sizes of these products were variable and larger than that of STGC but incorrect for LTGC events. No such products were observed in wild-type colonies, suggesting a qualitative HR defect in fancd2 cells. We isolated genomic DNA from three of such clones and tried to determine the structure of repair products by genomic Southern blotting and PCR, followed by sequencing. In two of the clones, the repair products were compatible with premature termination of repair DNA synthesis, followed by ligation with the other broken ends without homology (HR/NHEJ mechanisms) (26). In another clone, the product was associated with an extensive expansion of neo segments, which might be created by repeated cycles of repair (LTGC) and DSB induction (see data in the supplemental material).
Ig gene conversion is defective in fancd2 cells.
To examine the role of FancD2 in a more physiological context, we measured Ig gene conversion in fancd2 cells. DT40 cells continue to diversify the Ig V locus through gene conversion that depends on the HR machinery using upstream pseudogene segments as a donor template (1, 4, 5). Since our fancd2 cells were derived from an sIgM-positive subline of DT40, we first measured the sIgM conversion rate from positive to negative. Subclones were expanded for 3 weeks and then analyzed. The subclones derived from fancd2 cells contained fewer (3-fold reduction) sIgM-negative revertants than subclones from wild type (Fig. 5A). Since the loss of sIgM in wild type was shown to be mostly due to gene conversion (43), these data suggest that FANCD2 disruption likely reduces gene conversion events at Ig loci.
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) (Fig. 5C) (2). Thus, we prepared FANCD2 disruptants in DT40Cre1 cells. These mutants are cisplatin sensitive like mutants derived from wild-type DT40 (data not shown). Similarly to the data shown in Fig. 2C, the length of the cell cycle measured by BrdU pulse-labeling did not differ significantly between the wild-type and the fancd2 DT40Cre1 (8 h in both cases). Subclones of the mutant and wild-type DT40Cre1 were expanded for 4 weeks and assayed for sIgM expression by flow cytometry. Strikingly, the percentage of sIgM-positive cells in subclones from fancd2 cells was remarkably reduced (3.5-fold) compared to wild-type and heterozygous FANCD2+/– cells (Fig. 5B). FANCD2-transfected fancd2 cells showed only very partial complementation (Fig. 5B), suggesting that fine regulation of expression levels by endogenous cis elements might be necessary for normal activity in this assay. Consistent with this idea, gene conversion defects in rad54 cells also show partial correction by chicken Rad54 cDNA expression (4).
To verify that the configuration of the V gene was the same in DT40Cre1 and its derivatives, we carried out nucleotide sequencing. As shown in Fig. 5C, we found two single-nucleotide deletions (of ambiguous origin) in each derivative of DT40Cre1. Thus, the drastic reduction of gene conversion events in fancd2 cells is not due to these mutations, since the FANCD2+/– cells carrying the same mutations showed nearly normal levels of gene conversion.
Ig V gene conversion is associated with a decrease in nontemplated mutations in fancd2 cells.
The subclones of FANCD2+/– and fancd2 cells derived from DT40Cre1 that were kept in culture for 4 weeks were pooled (unsorted populations), and the V nucleotide sequences were determined. In fancd2 cells, only 8 V sequences among 299 sequences had gene conversion events (2.7%), whereas FANCD2+/– cells had 48 conversion events among 279 sequences (17.1%) (Fig. 5D and E). Thus, our estimate for the degree of reduction of Ig gene conversion events conferred by FANCD2 disruption is 6.3-fold.
Nontemplated single nucleotide substitution in the Ig V gene is another important mechanism for diversification, which is analogous to somatic hypermutation in germinal center B cells (20, 43). RAD51 paralog mutants (e.g., XRCC2, XRCC3, and RAD51B) exhibit a striking shift from gene conversion to nontemplated substitutions (43). Thus, we looked at the nontemplated changes in the sequence data from DT40Cre1 FANCD2+/– and fancd2 cells. Relatively few of these events were noted in cells with both genotypes, but they appeared to be present at a reduced level in fancd2 cells (2%; 6 events in 299 sequences) compared to FANCD2+/– cells (7.2%; 20 events in 279 sequences) (PM class in Fig. 5D and E). This difference was statistically significant (P = 0.0028 [Mann-Whitney U test]).
We next looked at the nature of gene conversion events in the fancd2 background. Sequencing analysis of the V in sorted sIgM-positive subpopulations revealed that the tract length of gene conversion was not different in the absence of FancD2 (mean ± the standard deviation [SD]; 11.8 ± 13.7 bp in FANCD2+/–, n = 43 versus 15.6 ± 23.9 bp in FANCD2–/–, n = 38). In addition, we could not detect any aberrant events, such as nucleotide changes surrounding gene conversion tracts that may suggest compromised fidelity of the gene conversion events in fancd2 cells.
Spontaneous SCEs are elevated, and Rad51 focus formation is normal in fancd2 cells.
Unexpectedly, we found that spontaneous SCEs were 2-fold higher in fancd2 cells than in wild-type and in complemented fancd2 cells (Fig. 6A and B). In contrast, after MMC treatment (50 ng/ml), the levels of SCEs were similar in fancd2 cells compared to the wild type. With increasing concentrations of MMC, fancd2 cells showed only a minor increase compared to the more pronounced increase in the wild type (Fig. 6C). Inefficient S-phase checkpoints in response to ICL in human FA cells (7, 42) might be relevant to this phenotype.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we provide genetic evidence for the involvement of vertebrate FancD2 in a variety of processes that require HR. First, fancd2 cells are more IR sensitive than wild type to killing in late S to G2 phase when DSB repair predominantly takes place through HR. In contrast, fancd2 cells have only slight sensitivity in G1 to early S phase when NHEJ predominates (52). Consistent with this result are our data showing that fancd2 cells display extensive chromosomal aberrations in the 0-to-3-h interval after IR, which corresponds to irradiation in late S or G2 phase. Second, HR repair efficiency of chromosomal DSBs created by I-SceI endonuclease is reduced in fancd2 cells. Physical analysis revealed some aberrant repair products that were not observed in wild-type cells. Third, Ig gene conversion events are greatly reduced in fancd2 cells. Ig gene conversion is known to depend on AID (2, 18) and the HR machinery (4, 43, 51), although the AID-mediated initiating DNA lesion in V gene is not necessarily a DSB (1, 38). Fourth, the efficiency of targeted integration events at multiple loci is drastically reduced in fancd2 cells. Previous studies showed that mutants of many HR genes display this phenotype (4, 49, 51, 53, 56, 65). Interestingly, we found that DNA damage-induced focus formation of Rad51, a central molecule of HR, is not impaired in the absence of FancD2, suggesting that FancD2 acts either independently of Rad51 or in later steps after the focus formation.
FANCD2 is dispensable for spontaneous SCEs. A portion of the HR-mediated restart events at stalled forks results in crossing over when two Holliday junctions are resolved, leading to the cytologically detectable SCE. We found that spontaneous SCE was clearly elevated in fancd2 cells. A similar elevation of SCEs was noted in fancc DT40 cells as well (Hirano et al., unpublished). Although SCEs are known to depend on HR proteins, including Rad51 (49), Rad54 (49), the five Rad51 paralogs (51, 53), and Nbs1 (56), FancD2 and FancC are clearly dispensable for spontaneous SCE in DT40 cells. Taken together, we suggest a role of FancD2 might be restricted to a certain subpathway of HR. For example, mitotic HR events in general are thought to proceed via processes that avoid crossing over such as through synthesis-dependent strand annealing (11, 17, 26, 41, 62). It is possible that FancD2 might be required for synthesis-dependent strand annealing but not for HR that accompanies double Holliday junction formation such as SCE.
Of note, we recently found that the elevated spontaneous SCE in fancc or fancd2 cells could be explained by partially impaired relocalization of BLM helicase (Hirano et al., submitted). Bloom syndrome is characterized by chromosomal instability and greatly elevated (10-fold) spontaneous SCE (8). Notably, the BLM helicase, which suppresses crossing over via the topoisomerase III-dependent "dissolution" of double Holliday junctions (19, 64), is associated with the core complex of FANCA/C/E/F/G (33).
Roles of FANCD2 in Ig gene conversion and hypermutation. AID initiates gene conversion or hypermutation (nontemplated nucleotide substitutions) in chicken Ig V gene probably by creating a common DNA intermediate (3, 20, 37). The processed initiating events eventually trigger either HR (leading to gene conversion) or TLS (leading to hypermutation) (4, 43, 51). Interestingly, defective gene conversion in DT40 mutants lacking XRCC2, XRCC3, and RAD51B is associated with drastically increased hypermutation (43). On the other hand, the decrease in gene conversion in rad54 cells does not accompany a significant change in hypermutation, probably because the lesions in those cells may be committed to HR and are no longer available for the TLS pathway (43, 47).
We found that defective gene conversion in fancd2 cells is associated with decreased nontemplated mutations (Fig. 5D). Thus, it seems likely that FancD2 promotes TLS and spontaneous mutagenesis. The well-documented hypomutability of human FA cells at the hprt locus is quite consistent with this hypothesis (39, 40). Fancd2 cells share extremely high sensitivity to MMC or cisplatin with TLS mutants such as rev1 or rev3 cells (47, 48). Elucidation of the precise biochemical function of FancD2 will be required to clarify this issue.
In summary, the current study has established a role for FancD2 in a variety of HR-mediated DNA repair processes. However, the overall phenotype of fancd2 cells clearly differs significantly from that of mutants of Brca2, Rad51, Rad51 paralogs, Rad54, and Nbs1, which consistently show reduced spontaneous SCEs. Given that spontaneous SCE is not decreased in fancd2 cells, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over.
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ACKNOWLEDGMENTS |
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sequence database and suggestions for analyzing gene conversion and hypermutation events; Akira Shinohara for anti-Rad51 antibody; and Maria Jasin for providing the SCneo and I-SceI expression vector. This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (M.T. and N.M.). Financial support was also provided by Kawasaki Medical School (Project Research Grants No. 13-211, 14-203, and 15-201A), the Naito Foundation, the Takeda Foundation, and the Sagawa Foundation for Promotion of Cancer Research. A portion of the study was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under contract no. W-7405-Eng-48, with funding from NIH/NCI grant CA89405 (L.H.T.).
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: Division of Hematology/Oncology, Mount Sinai School of Medicine, New York, NY 10029.
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