Homologous recombination assay
BRCA1 and BRCA2 are considered to mediate corrected repair of double-strand DNA
breaks by homologous recombination (HR). The plasmid, p-direct repeat (DR)–green
fluorescent protein (GFP; Addgene plasmid 26475), and the I-SceI expression
plasmid, cBAS (Addgene plasmid 26477), were both purchased from Addgene. The
pDR-GFP, originally designed by Professor M. Jasin, contains two GFP genes with
different inactivating mutations, and one is inactivated by an I-SceI recognition site1-3.
Double-strand DNA break develops in this I-SceI recognized GFP gene after
expressing the SceI endonucleases. If this break is repaired by HR by another
inactivated GFP as donor sequence, this broken GFP becomes functional.
To establish this assay, HCC 1937 cell, a BRCA1-/- cell line purchased from
American Type Culture Collection, was used for in vivo HR assay. The wild type
BRCA1 and T1691K were constructed into pcDNA3 vectors. On day 1, HCC 1937
cells (around 4 x 105) were cultured in a 24-well plate. On day 2, 1.5 μg of the
pcDNA3-BRCA1 (wt and T1691K) or pc-DNA3 vector alone, 0.75μg of the
pDR-GFP and 0.75μg of the pcBAS-SceI were co-transfected with 3μl of the
lipofectamine 2000 in each well. On day 5, transfectants were observed under the
fluorescence microscopy. The GFP expression represents the successful HR, which
indicates that the function of this BRCA1 (wt or mutant) is not compromised.
From the original photos shown above, functional GFP-cells can be identified in
the wild type BRCA1 transfectants, but no functional GFP-cells can be seen in the
T1691K tranfectants after 96 hr transfection. This result demonstrated that T1691K
was lack of DSB repair activity.
References
1.
Moynahan, M. E., Chiu, J. W., Koller, B. H., Jasin, M. Brca1 controls
homology-directed DNA repair. Mol. Cell. 4, 511-8 (1999).
2.
Pierce, A. J., Johnson, R. D., Thompson, L. H., Jasin, M. XRCC3 promotes
homology-directed repair of DNA damage in mammalian cells. Genes Dev. 13,
2633-8. (1999).
3.
Richardson, C., Moynahan, M. E., Jasin, M. Double-strand break repair by
interchromosomal recombination: suppression of chromosomal translocations.
Genes Dev. 12, 3831-42. (1998).