TY - JOUR
T1 - Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination
AU - Ransburgh, Derek J.R.
AU - Chiba, Natsuko
AU - Ishioka, Chikashi
AU - Toland, Amanda Ewart
AU - Parvin, Jeffrey D.
PY - 2010/2/1
Y1 - 2010/2/1
N2 - 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 ∼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.
AB - 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 ∼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.
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U2 - 10.1158/0008-5472.CAN-09-2850
DO - 10.1158/0008-5472.CAN-09-2850
M3 - Article
C2 - 20103620
AN - SCOPUS:76249131820
VL - 70
SP - 988
EP - 995
JO - Cancer Research
JF - Cancer Research
SN - 0008-5472
IS - 3
ER -
