Table 1.
Cell lines and derivatives.
Fig 1.
Impact of NAC or low O2 levels on the genome-wide replication fork speed.
A/ Examples of combed DNA fibers with replication tracts: IdU (green), CldU (red) and the scheme of the experiment (upper panels). B/ RF speed distribution in V79 cells and derivatives (left) and V-C8 cells and derivatives (right) after exposure to NAC (3 mM). C/ RF speed distribution in V79 cells and derivatives (left) and V-C8 cells and derivatives (right) that were maintained at 20% versus 3% O2. The median and p-values are indicated in the histogram (*P < 0.05; **P < 0.01; ***P < 0.001). The median values are represented as horizontal black lines. ns: not significant. Approximately 100–120 fibers were scored per condition.
Fig 2.
Impact of NAC or low O2 levels on supernumerary centrosomes in mitotic cells (MEC).
A/ Examples of labeled centrosomes in mitotic cells (see chromosomal DAPI staining in lower panels). Left photograph: normal centrosome number (= 2); right photographs: aberrant centrosome numbers (≠2) leading to metaphase alterations (see DNA labeling in lower panels). Scale bar, 10 μm. B/ Frequencies of mitotic cells with aberrant centrosome numbers. Left histograms: V79 cells and derivatives; right histograms: V-C8 cells and derivatives. C/ Impacts of a low level of O2 (3%) on MECs. The mean value +/- s.d. was calculated from at least three independent experiments. In total, 150 mitoses were scored for each experiment and condition.
Fig 3.
A/ endogenous ROS. NAC: 2mM. The mean value +/- s.d. was calculated from at least three independent experiments. B/ After exposure to H2O2. The value +/- s.d. was calculated from at least three independent experiments.
Fig 4.
Impact of H2O2 on the genome-wide replication speed.
A/ Examples of molecular combing fibers. B/ RF speed distribution in V79 cells and derivatives (left) and V-C8 cells and derivatives (right) after exposure to H2O2 (5 μM). The median values are represented as horizontal black lines. p-value: *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. Approximately 100–120 fibers were scored per condition. C/ Impact of H2O2 on MECs in V79 cells and derivatives (left) and V-C8 cells and derivatives (right). The mean value +/- s.d. was calculated from at least three independent experiments. In total, 150 mitoses were scored for each experiment and condition. D/ The effects of the addition of dNs on the replication fork speed after exposure to H2O2. The replication fork speed distribution in V79 cells and derivatives (left) and V-C8 cells and derivatives (right) is presented. The numbers correspond to the median replication speed. The median (black lines) and p-values are indicated in the histogram (*P < 0.05; **P < 0.01; ***P < 0.001). ns: not significant. 100 to 145 fibers were scored per condition.
Fig 5.
A/ dNTP concentrations after exposure to different doses of HU. B/ dNTP concentrations after exposure to H2O2. C/ dNTP concentrations in unchallenged WT and HR- cells. D/ Impact of NAC (2 mM, 48 h) on dNTP concentrations in WT and HR- cells. The experiments were performed in triplicate. NT: not treated.
Fig 6.
Spontaneous γH2AX and XRCC1 foci.
A/γH2AX foci. Left panels: example of γH2AX foci. The nuclei are counterstained with DAPI (blue). Right panels: normalized frequency of cells with >10 spontaneous foci. B/ XRCC1 foci. Left panel: example of XRCC1 foci. The nuclei are counterstained with DAPI (blue). Right panel: normalized frequency of cells with >10 spontaneous foci. At least 200 cells were counted. NAC: 2 mM, 48 h. The data were obtained from three independent experiments (error bars: s.e.m.).
Fig 7.
Impact of H2O2 on HR frequency.
A/ HR was measured in three different cell lines (U2OS, RKO and RG37, which is an SV40 immortalized human fibroblast [56]) bearing an intrachromosomal substrate (DR-GFP) that monitors HR [50]. Left panel: HR substrate (DR-GFP); two inactive GFP genes organized into direct repeats are integrated into the cells’ genomes. The 5’ GFP cassette is inactivated because of a mutational insertion (white scare). The 3’ GFP cassette is inactivated because of deletions in both the 5’ and 3’ sequences. HR between the two GFP genes can generate a functional GFP gene through gene conversion without crossing over. Recombinant cells are thus GFP-positive (GFP+) and can be monitored by FACS [50]. Right panel: experimental scheme. B/ Induction of HR events by H2O2 in the RG37 (left panel), RKO (middle panel) and U2OS (right panel) cell lines. The values correspond to the induced number of recombinant cells (GFP+): the number of GFP+ cells in 2x105 cells exposed to H2O2 subtracted from the number of GFP+ cell clones among 2x105 untreated cells. The values correspond to at least three independent experiments. C/ Impact of supplying dNs on H2O2-induced HR. The values correspond to at least three independent experiments.
Fig 8.
Impact of endogenous versus exogenous OS on replication.
A/ In WT cells, endogenous OS can produce spontaneous DNA damage, replisome alterations and dNTP pool restriction. However, the cells have reached a steady state that allows the delivery of dNTPs to DNA repair without affecting replication. B/ An exogenous OS stress or HR defect increases the level of endogenous ROS, generating additional DNA damage (1) and altering replisome proteins (2) and the dNTP pool (3). In addition, the accumulation of DNA damage diverts dNTPs at the expense of replication. All of these processes affect replication dynamics, leading to chromosome segregation defects. Supplying dNs rescues both DNA repair and replication.