title: Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase.
abstract: 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.
authors.affiliation: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Kass
authors.forename: Hildur R
authors.initials: HR
authors.lastname: Helgadottir
authors.forename: Chun-Chin
authors.initials: CC
authors.lastname: Chen
authors.forename: Maria
authors.initials: M
authors.lastname: Barbera
authors.forename: Raymond
authors.initials: R
authors.lastname: Wang
authors.forename: Ulrica K
authors.initials: UK
authors.lastname: Westermark
authors.forename: Thomas
authors.initials: T
authors.lastname: Ludwig
authors.forename: Mary Ellen
authors.initials: ME
authors.lastname: Moynahan
authors.forename: Maria
authors.initials: M
authors.lastname: Jasin
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D019313:BRCA1 Protein / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D018274:Electroporation; D005347:Fibroblasts; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D023421:Models, Animal; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D059767:Recombinational DNA Repair / Q000502:physiology*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific