Fig. 5
MUS81 cleaves partially resected regressed forks with a ssDNA tail to promote fork rescue in BRCA2-deficient cells. D daughter strand, P parental strand, R reversed arm. a Left, representative electron micrograph of a reversed fork with a single-stranded regressed arm. Center, magnified four-way junction at the reversed replication fork with a single-stranded regressed arm. The black arrow points to the ssDNA region on the regressed arm. Right, schematic model of the substrate cleaved by MUS81. b Frequency of fork reversal and ssDNA composition of the reversed arms in BRCA2-deficient U2OS cells treated with 4 mM HU for 5 h. Cells were transfected with control siRNA (siNEG) or MUS81 siRNA. The percentage values are indicated on the top of the bar. “# RI” indicates the number of analyzed replication intermediates. Mean shown, n = 3. Errors, S.E.M. Statistics: unpaired t test; **P < 0.01; ***P < 0.001. c Percentage of forks with ssDNA at the fork junction in BRCA2-deficient U2OS cells treated with 4 mM HU for 5 h. Cells were transfected with control siRNA (siNEG) or MUS81 siRNA. The percentage values are indicated on the top of the bar. “# RI” indicates the number of analyzed replication intermediates. Mean shown, n = 3. Errors, S.E.M. Statistics: unpaired t test; **P < 0.01. d Quantification of restarting forks in BRCA2-deficient and -proficient U2OS cells with or without MUS81 siRNA knockdown. MUS81-depleted cells were complemented with wild-type (MUS81-WT) or catalytically dead (MUS81D338A-D339A) MUS81, when indicated. Out of 3 repeats, the percentage is established on at least 250 tracts scored for each data set. Mean shown. Errors, S.E.M. Statistics: unpaired t test; **P < 0.01; ***P < 0.001 e Cell viability assays 72 h upon treatment with the indicated doses of HU. U2OS cells were transfected with control siRNA (siNEG), BRCA2 siRNA, or MUS81 siRNA. Mean shown, n = 6. Errors, S.E.M. Statistics: two-way ANOVA, ***P > 0.001; ****P > 0.0001 (differences between BRCA2 siRNA and BRCA2/MUS81 siRNA)
