Fanconi anemia FANCG protein in mitigating radiation- and enzyme-induced DNA double-strand breaks by homologous recombination in vertebrate cells

Abstract

The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased approximately 9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.

FIG. 1.

Amino acid sequence comparison between chicken, human, mouse, and hamster FANCG proteins. Residues that are identical across four species or three species are indicated by black or grey shading, respectively. Locations of previously reported putative leucine zipper motifs (7, 32) are underlined. A line with arrowheads indicates amino acids that are missing in a variant transcript (see text).

FIG. 2.

Targeted disruption of the chicken FANCG locus. (A) Schematic representation of a partial restriction map of the chicken FANCG locus, the gene disruption vector, and the configuration of the targeted locus. Black boxes indicate the positions of exons that were disrupted. Relevant restriction enzymes sites: E, EcoRI; B, BamHI; Xh, XhoI; Xb, XbaI. (B) Southern blot analysis of wild-type (WT) and fancg mutant cells. EcoRI-digested genomic DNA was hybridized with the probes indicated in panel A. (C) RT-PCR analysis of chicken FANCG mRNA expression in wild-type and fancg mutant cells. Isolation of total RNA, cDNA synthesis, and PCR were carried out as described in Materials and Methods.

FIG. 3.

Proliferation characteristics of wild-type (WT) and FANCG-deficient cells. (A) Cells were counted by flow cytometry, using fixed numbers of plastic beads as standards. The growth curves shown represent results from at least four independent experiments with two fancg clones. (B) Cell cycle distribution analysis as measured by BrdUrd incorporation and PI staining (DNA content). Cells were pulse-labeled for 10 min with BrdUrd and stained with anti-BrdUrd monoclonal antibody as described in Materials and Methods. The upper, lower left, and right lower regions identify cells in S, G₁, and G₂-M phase, respectively. Numbers are the percentages of cells in each region.

FIG. 4.

Colony survival assay after genotoxic treatments. (A to D) The fractions of the surviving colonies after treatment compared to nontreated controls of the same genotype are shown as percent survivals. Error bars indicate standard deviations from at least three experiments. (A) MMC; (B) X rays; (C and D) cisplatin (CDDP). (E) Western blot analysis of Rad51 expression in fancg mutant cells transfected with human Rad51 expression vector. Positions of endogenous chicken Rad51 and transgene-derived human Rad51 are indicated.

FIG. 5.

Analysis of HR-mediated repair of I-SceI-induced DSBs. (A) Recombination frequencies in integrated SCneo substrate in wild-type (WT) (black bars) and fancg (hatched bars) cells. Means ± standard deviations from at least six separate experiments are shown. Cells carrying the recombination substrate were transiently transfected with the indicated plasmids and then selected in the presence of G418. (B) Structures of the SCneo construct and expected HR repair products. K, KpnI; N, NcoI; S, SacI. STGC or LTGC/SCE events could be identified by Southern blotting with KpnI- and SacI-restricted genomic DNA and the indicated neo probe. (C) Representative Southern blot analyses of G418-resistant clones from wild-type (eight clones) and fancg (eight clones) cells. The expected sizes of STGC (∼5 kb) and LTGC/SCE (∼8.5 kb) events are indicated. (D) Number of G418-resistant clones with STGC or LTGC/SCE events. One clone from fancg cells exhibited a larger band than expected (shown in panel C), but this was counted as LTGC events.

FIG. 6.

IR-induced chromosome aberrations in wild-type and fancg mutant cells. Cells were X irradiated (2 Gy) and sampled at 3-h intervals. One hundred cells were scored at each time point except for the 0- to 3-h interval, where 200 cells were scored. Error bars represent 95% confidence intervals. The percentage of abnormal cells at each time point is also shown. Cells harboring more than two aberrations were not detected after IR in both wild-type and fancg cells.

FIG. 7.

Subnuclear focus formation of Rad51 in response to MMC treatment. (A to D) Wild-type (A and B) and fancg (C and D) cells were analyzed 5 h after treatment with MMC (500 ng/ml for 1 h) (B and D) or were untreated (A and C). (E) Kinetics of Rad51 focus formation after the same MMC exposure. Cells with more than four distinct and bright foci were counted as positive. WT, wild type.