Abstract
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) (brca2DeltaCTD), and disrupted the FANCC gene in this background. The fancc/brca2DeltaCTD 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/brca2DeltaCTD 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.
Highlights
Increased susceptibility to cancer [1,2,3]
We 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
We separated cell lysates of wild type, fancc, fancd2, and brca2⌬BRC chicken DT40 cells into detergent-soluble and -insoluble fractions, as described previously [16], and quantified the abundance of Rad51 in the chromatin fraction by Western blotting with an anti-Rad51 antibody
Summary
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% CO2 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]. As with brca2⌬BRC cells, the BRCA2CTD truncation was achieved in cells harboring conditionally null allele of BRCA2 (a kind gift from Drs Mitsuyoshi Yamazoe and Shunichi Takeda), resulting in BRCA2⌬CTD/con cells (Fig. 3B). Western Blotting Analysis—Anti-chicken N-terminal Brca and anti-FancD2 antisera were raised as described previously [7, 40]. 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. As described previously [6], on coded slides
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
