Abstract
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.
Highlights
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
We show that homology-directed repair (HDR) of the I-SceI-induced DSB is robust in mammary epithelium during the proliferative stages of puberty and pregnancy, comprising one-third or more of total DSB repair events, and is frequent in the small intestine epithelium
The direct repeat-green fluorescent protein (DR-GFP) reporter consists of two defective GFP genes; a DSB introduced into the upstream gene by the I-SceI endonuclease and repaired by HDR with the downstream gene gives rise to GFP þ cells
Summary
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. Quantitative in vivo measurements in tissues to accurately assess the contribution of each pathway to DSB repair have been lacking In their capacity as genomic caretakers, many HDR genes are breast tumour suppressors[5,6], including BRCA1 and BRCA2, suggesting the importance of this DNA repair pathway for tissues or tissue compartments that proliferate postnatally and may be at oncogenic risk when breaches occur to this pathway. We develop such a system where a defined lesion—a DSB—is efficiently introduced into the genome, allowing us to examine HDR within proliferative tissues In this system, we couple mice containing the widely used reporter for HDR, direct repeat-green fluorescent protein (DR-GFP)[16], with those developed to contain doxycycline-inducible I-SceI endonuclease for DSB formation. Mutation of Brca[2] impacts HDR in different mammary epithelial cell lineages, consistent with the heterogeneous nature of BRCA2-deficient breast tumours[18]
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