Journal Article

Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase

Kass, Elizabeth M., Chen, Chun-Chin, Westermark, Ulrica K., Helgadottir, Hildur R., Barbera, Maria, Ludwig, Thomas, Wang, Raymond, Moynahan, Mary Ellen, Jasin, Maria

2013

Overview

Homology-directed repair (HDR) is a critical pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Efficient HDR is thought to be crucial for maintenance of genomic integrity during organismal development and tumor suppression. However, most mammalian HDR studies have focused on transformed and immortalized cell lines. We report here the generation of a Direct Repeat (DR)-GFP reporter-based mouse model to study HDR in primary cell types derived from diverse lineages. Embryonic and adult fibroblasts from these mice as well as cells derived from mammary epithelium, ovary, and neonatal brain were observed to undergo HDR at I-SceI endonuclease-induced DSBs at similar frequencies. When the DR-GFP reporter was crossed into mice carrying a hypomorphic mutation in the breast cancer susceptibility gene Brca1 , a significant reduction in HDR was detected, showing that BRCA1 is critical for HDR in somatic cell types. Consistent with an HDR defect, Brca1 mutant mice are highly sensitive to the cross-linking agent mitomycin C. By contrast, loss of the DSB signaling ataxia telangiectasia-mutated (ATM) kinase did not significantly alter HDR levels, indicating that ATM is dispensable for HDR. Notably, chemical inhibition of ATM interfered with HDR. The DR-GFP mouse provides a powerful tool for dissecting the genetic requirements of HDR in a diverse array of somatic cell types in a normal, nontransformed cellular milieu.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: adults / Alleles / animal models / animal ovaries / Animals / ataxia (disorder) / Ataxia Telangiectasia Mutated Proteins / Biological Sciences / brain / BRCA1 Protein - metabolism / Breast cancer / breast neoplasms / Cell cycle / Cell Cycle Proteins - metabolism / Cell lines / Cells / cross-linking reagents / Deoxyribonucleases, Type II Site-Specific / DNA / DNA Breaks, Double-Stranded / DNA damage / DNA repair / DNA-Binding Proteins - metabolism / Electroporation / Epithelial cells / epithelium / Fibroblasts / Flow Cytometry / genes / Genetic recombination / Germ cells / Green Fluorescent Proteins - metabolism / Kinases / Mammals / Mice / mitomycin / Models, Animal / mutants / Mutation / Protein Serine-Threonine Kinases - metabolism / Recombinational DNA Repair - physiology / Repetitive Sequences, Nucleic Acid - genetics / Saccharomyces cerevisiae Proteins / Somatic cells / tumor suppressor proteins / Tumor Suppressor Proteins - metabolism