Stepwise Activation of the ATR Signaling Pathway upon Increasing Replication Stress Impacts Fragile Site Integrity

<div><p>Breaks at common fragile sites (CFS) are a recognized source of genome instability in pre-neoplastic lesions, but how such checkpoint-proficient cells escape surveillance and continue cycling is unknown. Here we show, in lymphocytes and fibroblasts, that moderate replication stresses like those inducing breaks at CFSs trigger chromatin loading of sensors and mediators of the ATR pathway but fail to activate Chk1 or p53. Consistently, we found that cells depleted of ATR, but not of Chk1, accumulate single-stranded DNA upon Mre11-dependent resection of collapsed forks. Partial activation of the pathway under moderate stress thus takes steps against fork disassembly but tolerates S-phase progression and mitotic onset. We show that fork protection by ATR is crucial to CFS integrity, specifically in the cell type where a given site displays paucity in backup replication origins. Tolerance to mitotic entry with under-replicated CFSs therefore results in chromosome breaks, providing a pool of cells committed to further instability.</p></div

Moderate fork slowing is not associated with formation of ssDNA foci.

<p>(A) ssDNA detection scheme. Cells hemi-substituted throughout their genome after growth in the presence of CldU for about 1.5 cell generations were treated or not with aphidicolin or HU for different periods of time prior to fixation. CldU was immuno-detected without DNA denaturation, which only permits visualization of substituted and single-stranded regions. (B) Co-detection of ssDNA (red) and chromatin bound PCNA (green) by immunostaining of untreated cells (-) and cells treated as indicated. Nuclei were counterstained with DAPI. (C) Typical pattern of PCNA and CldU foci in cells treated with HU 1 mM during 1 h. (D) Typical pattern of PCNA and CldU foci in cells depleted of ATR and treated with 0.3 µM of aphidicolin for 4 h.</p

ATR-depletion enhances formation of ssDNA foci and fork asymmetry.

<p>(A) Percentage of S-phase cells with ssDNA foci in the indicated conditions of transfection and aphidicolin treatment. Mean ± s.e.m are presented. (B) Upper panel: Western blot analyses of total cell extracts 48 h post-transfection with a NONsi RNA or siRNAs specific to ATR or/and Mre11. Loading control: β-actin. Lower panel: percentage S-phase cells (mean ± s.e.m) displaying ssDNA foci in populations of cells depleted of ATR and treated or not with aphidicolin 0.3 µM. Mre11 status is indicated (siMre11 or treatment with Mirin: 100 µM). (C) Percentage of FISH signal co-localizing with ssDNA foci in cells depleted of ATR and treated with 0.3 µM aphidicolin. The immunostaining of ssDNA and PCNA was combined with FISH with BAC probes. Upper panel: JEFF lymphoblastoid cells, the BACs probe early replicating regions (875H7 and 456N14) and late replicating regions (321A23, 660J14, and 641C17 that corresponds to <i>FRA3B</i>, the major CFS in lymphocytes). Lower panel: MRC-5 fibroblasts, BAC 456N14 probe for an early replicating region, other BACs correspond to late replicating regions (875H7, 321A23, 641C17, and 660J14 which corresponds to the major CFS in fibroblast mapping at 3q13.3). (D) Distributions of fork asymmetry in cells transfected and treated as indicated. Forks travelling in the <i>FRA3B</i> locus are represented by red circles (<i>n</i> = 17) and in the bulk genome by black circles (untreated: NONsi, <i>n</i> = 128; siCHk1, <i>n</i> = 103; siATR, <i>n</i> = 147; with aphidicolin: NONsi, <i>n</i> = 121; siCHk1, <i>n</i> = 132; siATR, <i>n</i> = 123). Horizontal orange lines represent the medians of fork distributions. Medians and <i>P</i> values are indicated above the distributions (not significant, <i>ns</i>).</p

Moderate fork slowing triggers neither γ-H2AX foci nor ATM activation.

<p>(A) Western blot analysis of ATM-Ser1981 phosphorylation and γ-H2AX in exponentially growing cells, untreated (-) or treated as indicated. (B) Immunostaining of γ-H2AX together with chromatin-bound PCNA in untreated cells (-) and in cells treated with aphidicolin 0.6 µM for the indicated periods of time, or for 1 h with HU 1 mM.</p

Moderate fork slowing triggers chromatin loading of sensors and mediators of the ATR pathway.

<p>(A) Measurement of replication fork speed. Typical examples of combed DNA molecules displaying replications forks in control cells (Ctl) and cells treated with aphidicolin. Cells were pulse-labelled with IdU then CldU before DNA was purified and combed (see Methods). The analogues were revealed on stretched molecules as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003643#pgen.1003643-Anglana1" target="_blank">[44]</a> and fork speed was determined by measuring the length of IdU (red) and CldU (green) tracks. Blue: counterstaining of the DNA used to select unbroken IdU and CldU tracks (B) Mean fork speed (upper panel), kb per min ± standard error of the mean (s.e.m.), and chromatin recruitment of checkpoint proteins (lower panel). Mean fork speed and chromatin recruitment of ATR and RPA2 were studied after 4 h of treatment with the indicated aphidicolin concentrations or 1 h of treatment with HU 1 mM. Asterisks indicate that fork speed cannot be measured. (C) Western blot analysis of chromatin extracts from exponentially growing cells, untreated (-) or treated as indicated.</p

ATR depletion compromises genome stability.

<p>(A) Quantification of metaphases (M) with breaks in cells transfected as above and treated for 16 h with the indicated aphidicolin concentrations. Mean ± s.e.m are presented. (B) Correlations between replication fork speed and chromosome instability. The data presented in A and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003643#pgen-1003643-g005" target="_blank">Figure 5C</a> were used to plot the percentage of metaphase plates with chromosome breaks against fork speed in each condition of transfection and treatment. (C) Breaks occurring at <i>FRA3B</i> quantified as in C. (D) Correlations between replication speed and <i>FRA3B</i> instability evaluated as in B.</p

Moderate fork slowing does not trigger phosphorylation of ATR targets.

<p>(A) Western blot detection of Chk1-Ser317 and Ser345 phosphorylations in total extracts of cells treated 4 h with aphidicolin or 1 h with HU 1 mM (upper panel). Quantification of Chk1-Ser317P and -Ser345P from 3 independent experiments (lower panel). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003643#s2" target="_blank">Results</a> (mean ± s.e.m) are expressed as percentage of the level of phosphorylation found in cells treated with 1 mM HU. Lanes 1–6 and lanes 7–9 correspond to different blots. (B) Western blot detection of RPA2-Ser33 phosphorylation, p53 and p53-Ser15 phosphorylation in total extracts of cells treated for 4 h with aphidicolin or 1 h with HU 1 mM. Loading control: β-actin.</p

Spontaneous γH2AX and XRCC1 foci.

<p><b>A/γ</b>H2AX 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>B/</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.).</p

Impact of NAC or low O₂ levels on supernumerary centrosomes in mitotic cells (MEC).

<p><b>A/</b> 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>B/</b> Frequencies of mitotic cells with aberrant centrosome numbers. Left histograms: V79 cells and derivatives; right histograms: V-C8 cells and derivatives. <b>C/</b> 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.</p

Level of intracellular ROS.

<p><b>A/</b> endogenous ROS. NAC: 2mM. The mean value +/- s.d. was calculated from at least three independent experiments. <b>B/ After exposure to H</b>_<b>2</b><b>O</b>_<b>2</b>. The value +/- s.d. was calculated from at least three independent experiments.</p