Robust homology-directed repair within mouse mammary tissue is not specifically affected by Brca2 mutation.

The mammary gland undergoes significant proliferative stages after birth, but little is known about how the developmental changes impact DNA double-strand break (DSB) repair. Mutations in multiple genes involved in homology-directed repair (HDR), considered a particularly accurate pathway for repairing DSBs, are linked to breast cancer susceptibility, including BRCA2. Using reporter mice that express an inducible endonuclease, we find that HDR is particularly robust in mammary tissue during puberty and pregnancy, accounting for 34-40% of detected repair events, more than in other tissues examined. Brca2 hypomorphic mutation leads to HDR defects in mammary epithelium during puberty and pregnancy, including in different epithelial lineages. Notably, a similar dependence on Brca2 is observed in other proliferative tissues, including small intestine epithelium. Our results suggest that the greater reliance on HDR in the proliferating mammary gland, rather than a specific dependence on BRCA2, may increase its susceptibility to tumorigenesis incurred by BRCA2 mutation.

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

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.

RAD51 can inhibit PDGF-B-induced gliomagenesis and genomic instability.

Faithful replication and DNA repair are vital for maintenance of genome integrity. RAD51 is a central protein in homologous recombination repair and during replication, when it protects and restarts stalled replication forks. Aberrant RAD51 expression occurs in glioma, and high expression has been shown to correlate with prolonged survival. Furthermore, genes involved in DNA damage response (DDR) are mutated or deleted in human glioblastomas, corroborating the importance of proper DNA repair to suppress gliomagenesis. We have analyzed DDR and genomic instability in PDGF-B-induced gliomas and investigated the role of RAD51 in glioma development. We show that PDGF-B-induced gliomas display genomic instability and that co-expression of RAD51 can suppress PDGF-B-induced tumorigenesis and prolong survival. Expression of RAD51 inhibited proliferation and genomic instability of tumor cells independent of Arf status. Our results demonstrate that the RAD51 pathway can prevent glioma initiation and maintain genome integrity of induced tumors, suggesting reactivation of the RAD51 pathway as a potential therapeutic avenue.

Assaying double-strand break repair pathway choice in mammalian cells using a targeted endonuclease or the RAG recombinase.

DNA damage repair is essential for the maintenance of genetic integrity in all organisms. Unrepaired or imprecisely repaired DNA can lead to mutagenesis, cell death, or malignant transformation. DNA damage in the form of double-strand breaks (DSBs) can occur as a result of both exogenous insults, such as ionizing radiation and drug therapies, and normal metabolic processes including V(D)J recombination. Mammalian cells have multiple pathways for repairing DSBs, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). This chapter describes the use of reporter substrates for assaying the contributions of these pathways to DSB repair in mammalian cells, in particular murine embryonic stem cells. The individual contributions of NHEJ, HR, and SSA can be quantified using fluorescence and PCR-based assays after the precise introduction of DSBs either by the I-SceI endonuclease or by the RAG recombinase. These reporters can be used to assess the effects of genetic background, dominant-negative constructs, or physiological conditions on DSB repair in a wide variety of mammalian cells.