Fig. 2
BRCA1 and BRCA2 protect reversed replication forks from MRE11- and EXO1-dependent degradation following HU treatment. a Representative electron micrograph of a reversed fork observed on genomic DNA upon HU treatment. Inset, magnified four-way junction at the reversed replication fork. D daugher strand, P parental strand, R reversed arm. b Frequency of fork reversal in BRCA1- and BRCA2-deficient U2OS cells treated with 4 mM HU for 5 h. Cells were transfected with control siRNA (siNEG) or EXO1 siRNA. Mirin (50 μM) was added concomitantly with HU, as indicated. 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 < 0.05; **P < 0.01; ***P < 0.001. c Frequency of fork reversal in BRCA2-proficient PEO4 and BRCA2-deficient PEO1 cells treated with 4 mM HU for 5 h. Mirin (50 μM) was added concomitantly with HU, as indicated. 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 < 0.05; ***P < 0.001. d Frequency of fork reversal in BRCA2-deficient U2OS cells treated with 4 mM HU for 0, 30, or 120 min. 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. e CldU/IdU tract ratio in BRCA2-deficient U2OS cells treated with 4 mM HU for 0, 30, or 120 min. Out of 3 repeats; n ≥ 250 tracts scored for each data set. Bars represent the median. Statistics: Mann–Whitney; ****P < 0.0001. f Plot the percentages of reversed forks as a function of mean values of the CldU/IdU ratios measured after treating BRCA2-deficient U2OS cells with 4 mM HU for 30 or 120 min. Errors, S.E.M.
