Analysis of DNA repair and recombination responses in mouse cells depleted for Brca2 by SiRNA.
The tumor suppressor BRCA2 is considered to play an important role in the maintenance of genome integrity through the repair of DNA lesions by homologous recombination. A mechanistic understanding of BRCA2 has been complicated by the embryonic lethality of mice bearing allelic knockouts of Brca2, and by variation in the DNA damage response in cells bearing BRCA2 deficiencies. It would be advantageous to develop approaches that avoid the cell lethality associated with complete inactivation of the gene, or the use of established tumor cell lines in which other genes in addition to BRCA2 may be mutant. In this study, SiRNA was used in stable transformation assays to knockdown Brca2 in mouse hybridoma cells by at least 75%. The Brca2-depleted cells were analyzed with respect to cell growth, sensitivity to DNA damaging agents (mitomycin C, methylmethane sulfonate, or ionizing radiation), intrachromosomal homologous recombination and gene targeting. Although the effect of Brca2-depletion on cell growth and sensitivity to DNA damaging agents was modest, the Brca2-depleted cells did show a significant shift in homologous recombination from gene conversion to single-strand annealing and a significant decrease in the efficiency of gene targeting. Both of these phenotypes are consistent with the proposed role of Brca2 in DNA repair and recombination.
Lee
Shauna A
SA
Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Baker
Mark D
MD
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D024682:BRCA2 Protein / Q000235:genetics* / Q000502:physiology*; D004249:DNA Damage; D004260:DNA Repair; D018390:Gene Targeting; D005821:Genetic Techniques; D016678:Genome; D006825:Hybridomas; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D009154:Mutation; D034741:RNA, Small Interfering / Q000378:metabolism*; D011839:Radiation, Ionizing; D011995:Recombination, Genetic
D051017:Apoptosis Regulatory Proteins; C118024:BLID protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D034741:RNA, Small Interfering; D016685:Mitomycin; D008741:Methyl Methanesulfonate