1. Fusion of PALB2(L21P) to the BRCT repeats of BRCA1 mediates the assembly of DNA damage foci by PALB2 and RAD51 in PALB2-deficient cells.
Fusion of PALB2(L21P) to the BRCT repeats of BRCA1 mediates the assembly of DNA damage foci by PALB2 and RAD51 in PALB2-deficient cells. (A) Expression of different forms of PALB2 (PB2) in EUFA1341 cells. EUFA1341 cells are deficient for endogenous PALB2. Actin signal is shown as a loading control. Each construct had an N-terminal Flag-HA epitope tag. (B) Examples of foci assembled by PALB2 or BRCT–PALB2(L21P), detected with anti-PALB2 antibodies, in EUFA1341 cells 16 h after exposure to 10 Gy IR. A cell in which BRCT–PALB2(L21P) strongly colocalized with γ-H2AX is indicated by the white arrow. Colocalization is evident from the yellow signal in the merged image. (C,D) Quantification of PALB2-related foci, detected with anti-PALB2 antibodies (C), or of RAD51 foci (D) in EUFA1341 cells that expressed the various forms of PALB2 or the vector alone. Results in untreated populations or 16 h after exposure to 15 Gy IR are shown. (E) Quantitative comparison of the assembly of PALB2(L21P) fused with the BRCT repeats of either BRCA1 or BARD1 into foci, as detected with anti-HA antibodies. Quantification of the assembly of RAD51 foci in EUFA1341 cells reconstituted with either mutant fusion protein or vector alone is also shown. Cells were fixed 16 h after exposure to 15 Gy IR. Results are shown relative to the assembly of foci in cells reconstituted with the BRCT[BRCA1]–PALB2(L21P) fusion protein (B1-BRCT Fu). (C–E) Each value represents the mean ± SD of three counts of 150 or more cells from the same coverslip.
Fan Zhang et al. J Cell Sci 2012;125:
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