The selected missense variants were cloned into a 3x FLAG-tagged full-length BRCA2 cDNA expression plasmid using the QuickChange site-directed mutagenesis kit XL (Stratagene) (13 (link)). Mutations were verified by DNA sequencing. Wild-type and mutant constructs were co-transfected with an I-Sce1–expressing pcBASce plasmid (13 (link)) into V-C8 cells containing the DR-GFP reporter plasmid. The BRCA2 deficient V-C8 hamster lung fibroblast cell line (XRCC11) displays chromosomal instability and abnormal centrosomes, reduced nuclear localization of the RAD51 protein that is central in the HDR processes, and sensitivity to cross-linking agents such as methyl methanesulfonate (MMS) (19 (link)). Cells expressing green fluorescent protein (GFP) following HDR-dependent repair of an I-Sce1 induced DNA double strand break in the DR-GFP reporter plasmid were quantified by fluorescent activated cell sorting (FACS) after 72 hrs. Transfection efficiency was evaluated by immunofluorescence (IF) based counting of GFP positive cells. Expression of wildtype and mutant BRCA2 was evaluated by immunoblotting with rabbit Anti-Flag antibody (1:1000 F7425 Sigma) of mouse Anti-Flag M2 (F1804 Sigma) immunoprecipitates from protein lysates.