Similar Effects of Brca2 Truncation and Rad51 Paralog Deficiency on Immunoglobulin V Gene Diversification in DT40 Cells Support an Early Role for Rad51 Paralogs in Homologous Recombination

Abstract

BRCA2 is a tumor suppressor gene that is linked to hereditary breast and ovarian cancer. Although the Brca2 protein participates in homologous DNA recombination (HR), its precise role remains unclear. From chicken DT40 cells, we generated BRCA2 gene-deficient cells which harbor a truncation at the 3 end of the BRC3 repeat (brca2tr). Comparison of the characteristics of brca2tr cells with those of other HR-deficient DT40 clones revealed marked similarities with rad51 paralog mutants (rad51b, rad51c, rad51d, xrcc2, or xrcc3 cells). The phenotypic similarities include a shift from HR-mediated diversification to single-nucleotide substitutions in the immunoglobulin variable gene segment and the partial reversion of this shift by overexpression of Rad51. Although recent evidence supports at least Xrcc3 and Rad51C playing a role late in HR, our data suggest that Brca2 and the Rad51 paralogs may also contribute to HR at the same early step, with their loss resulting in the stimulation of an alternative, error-prone repair pathway.Individuals carrying germ line mutations of the BRCA2 gene have a high risk of developing breast and ovarian cancer (40,45,76). The Brca2 protein acts as a tumor suppressor, and its loss results in genome instability (1,30,35). Mice carrying a deletion of all eight BRC repeats die during early embryonic development, while mice carrying a truncation at the 3Ј end of the third BRC repeat (brca2tr mice) are partially viable (10,13,29,35). However, interpretation of their phenotype may be hampered by additional mutations acquired during the derivation of the line as a result of the genomic instability imposed by impaired Brca2 function.While yeast Rad51, Rad52, and Rad54 contribute to homologous DNA recombination (HR) to similar extents, vertebrate Rad51 plays a dominant role in HR (25,48,57). HR is initiated by polymerization of Rad51 on single-stranded DNA at sites of DNA damage, leading to formation of nucleoprotein filaments (3,33,50,72). The filaments undergo homologous pairing and strand exchange with other intact homologous DNA to form the Holliday junction recombination intermediates, followed by resolution of the two homologous DNAs. It has been suggested that Brca2 may regulate the Rad51 recombinase (30,36,67,70), because the brca2 mutation impairs ionizing radiation (IR)-induced subnuclear Rad51 focus formation (20,58,61,73), which is believed to represent the polymerization of Rad51 at sites of DNA damage (16,27,39,55). However, the biological significance of impaired Rad51 focus formation has not been clarified, because brca2tr cells exhibit only marginal IR sensitivity and thus retain a substantial fraction of the Rad51-dependent capability for repair of IR-induced double strand breaks (DSBs). Furthermore, in mammalian cells, there is neither a detailed phenotypic assay that can address the role of each molecule in the HR reaction nor a collection of HR mutant cell lines with which each phenotype can be accurately compared. Therefore, reverse genetic studies of mammalian cells have g...

Functional Interplay between BRCA2/FancD1 and FancC in DNA Repair

“…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).…”

“…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.…”

“…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).…”

Targeting Fanconi Anemia/BRCA2 Pathway Defects in Cancer: The Significance of Preclinical Pharmacogenomic Models

“…In hamster cells, the compound heterozygote BRCA2 line V-C8, derived from (random) chemical mutagenesis of V79 cells, harbors two different nonsense BRCA2 mutations (82). In chicken DT40 cells, the complete disruption of BRCA2 did not yield viable clones (90), whereas truncating mutations, which spared the first two BRC repeats, only decreased the cell growth rate in these cells (91). Engineered BRCA2 loss thus variably affects different species, presumably due to the different balance of cellular selection, reduction of fitness, and compensatory evolution, which operates after gene targeting.…”

A chemical compound that stimulates the human homologous recombination protein RAD51

“…For example, RAD51 overexpression in chicken DT40 cells can partially compensate for the loss of filament assembly mediator proteins, including RAD51 paralogues and other HR-related proteins (6)(7)(8). Furthermore, Fortin and Symington (9) have generated and characterized several gain-of-function mutations in yeast RAD51.…”