Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.
Effects of breast cancer-associated gene 1 (BRCA1) missense mutations on the function of BRCA1 protein in DNA recombination have been little studied. In this report, we adapted a homology-directed recombination (HDR) assay to analyze the effects of BRCA1 mutations on this function. Using a HeLa-derived cell line with a genomically integrated recombination substrate, we expressed an endonuclease creating a double-stranded break in the substrate that the HDR assay scores by generation of green fluorescent protein-positive cells. By combining RNA interference (RNAi) that targets cellular BRCA1 mRNA with expression of RNAi-resistant BRCA1 mutants, we could effectively substitute selected point mutants to test these in the cellular recombination assay. We found that approximately 300 residues at both termini of the BRCA1 protein were essential for HDR. Whereas some mutations analyzed were neutral, mutations that altered any zinc-coordinating residue or generated M18T and T37R alterations were defective for recombination. This study established a robust assay system to analyze the function of BRCA1 in regulating homologous recombination, which is critical for its tumor suppressor function.
Ransburgh
Derek J R
DJ
Department of Biomedical Informatics, Ohio State University, Columbus, Ohio 43210, USA.
Chiba
Natsuko
N
Ishioka
Chikashi
C
Toland
Amanda Ewart
AE
Parvin
Jeffrey D
JD
D019313:BRCA1 Protein / Q000235:genetics* / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D034622:RNA Interference; D011995:Recombination, Genetic / Q000235:genetics*
D019313:BRCA1 Protein; D004273:DNA, Neoplasm; D049452:Green Fluorescent Proteins