Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

Abstract

Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.

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. Level of intracellular ROS.

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. dNTP pool measurements.

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.