Fig. 6
POLD3 is required to restart resected forks in BRCA2-deficient cells. a Quantification of restarting forks in BRCA2-deficient and -proficient U2OS cells with or without POLD3 siRNA knockdown. Out of 2 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. b Size distribution of CldU tract length in U2OS cells transfected with control siRNA (siNEG), POLD3 siRNA, or BRCA2 siRNA before IdU and CldU labeling. Out of 3 repeats; n ≥ 150 tracts scored for each data set. Bars represent the median. Statistics: Mann–Whitney; ****P < 0.0001. c Neutral Comet assay monitoring DSB formation upon POLD3 depletion in BRCA2-deficient cells. Cells were transfected with control siRNA (siNEG), BRCA2 siRNA, MUS81 siRNA, or POLD3 siRNA. Out of 2 repeats; n ≥ 200 comets scored for each data set. Whiskers the 10th and 90th percentiles. ***P < 0.001: ****P < 0.0001 (Mann–Whitney test). d Left, representative images of MUS81 and POLD3 foci observed upon HU treatment. Scale bar, 10 μm. U2OS cells were transfected with control siRNA (siNEG), BRCA1 siRNA, or BRCA2 siRNA and processed for immunofluorescence. Right, quantitation of MUS81 and POLD3 foci. Out of 3 repeats; n ≥ 150 cells scored for each data set. Mean shown. Errors, S.E.M. Statistics: unpaired t test; *P < 0.05; **P < 0.01. e Top, roles of BRCA2, CtIP/MRE11/EXO1 and MUS81/POLD3 in reversed fork protection, degradation and cleavage/restart, respectively. Genotoxic agents lead to fork uncoupling and reversal. Reversed forks are stabilized by BRCA2, allowing accurate fork restart and genome stability. BRCA1 shares a similar function in reversed fork stabilization. RAD51 loss prevents fork reversal and compromises fork restart and cell viability. BRCA2 loss leads to CtIP/MRE11/EXO1-mediated degradation of the reversed replication forks. MUS81 cleaves the partially resected reversed forks with a ssDNA flap to grant POLD3-dependent fork restart and cell survival. Bottom, the MUS81 endonuclease cleaves the resected reversed forks with a ssDNA flap and lead to the subsequent formation of a D-loop structure that would in turn initiate POLD3-dependent DNA synthesis
