Journal: EMBO Reports

Article Title: CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage

doi: 10.15252/embr.201948920

Figure Lengend Snippet: A Outline of experimental procedure. U2OS cells were either untreated or treated with 10 μM XL413 or with 4 mM HU in the presence or absence of XL413 for 24 h. Cells were EdU‐labelled for 30 min before harvesting. Cells that had been treated with both XL413 and HU for 24 h were washed with equilibrated media and retreated for a further 2 h. All further treatments included 4 mM HU, to prevent further DNA synthesis in either the absence or presence of CDC7 (XL413), MRE11 (Mirin) or ATR (AZD6738) inhibitors. B CSK soluble and chromatin‐enriched protein fractions were analysed by Western blotting with the indicated antibodies. Data are representative of two independent experiments. C, D Flow cytometry analysis to assess the levels of pS139 histone H2AX (c). Histograms show the mono‐parametric analysis of cell count against pS139 histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 histone H2AX intensity upon treatment (red lines) relative to appropriate experimental baseline controls (grey lines). Data are representative of two independent experiments. E Graphical concept: cells treated with HU and XL413 for 24 h would have stalled forks, which exist in a stable state due to inhibition of CDC7 and with an active ATR‐dependent origin firing checkpoint. Upon washing and retreating of cells in the presence of HU, the late origin checkpoint is maintained, in an ATR‐dependent manner, and accumulation of DNA damage (pS139 H2AX) can be monitored independently from origin firing in the absence or presence of CDC7 (XL413) or MRE11 (Mirin) inhibitor. AZD6738 was used as a control to investigate molecular events occurring upon loss of checkpoint caused by both fork collapse and loss of the inhibition of origin firing. The increase in chromatin binding of CDC45 was used as a surrogate marker for origin (ori) activation, pS139 histone H2AX was used to monitor fork stability, pS345 CHK1 was used to monitor the ATR‐checkpoint signalling, and pS40/41 MCM2 was used to monitor CDC7 kinase activity. Source data are available online for this figure.

Article Snippet: Primary antibodies diluted in blocking buffer (TBST, 5% milk) included RPA2 (1:1,000, NA19L; Calbiochem), Histone H2A (1:1,000, 07‐146; Upstate), MRE11 (1:1,000, NB100‐142; Novus), EXO1 (1:1,000, A302‐640A; Bethyl Laboratories), BRCA2 (1:2,000, A303‐434A; Bethyl Laboratories; 1:500, OP 95; EMD Millipore Ab‐1), Histone H3 (1:1,000, A300‐823A; Bethyl Laboratories), CDC7 (1:1,000, K0070‐3; MBL), CHK1 (1:1,000, SC‐8408; Santa Cruz Biotech) and CDC45 (1:5, a gift from Broderick et al ).

Techniques: DNA Synthesis, Western Blot, Flow Cytometry, Cell Counting, Inhibition, Binding Assay, Marker, Activation Assay, Activity Assay

Journal: EMBO Reports

Article Title: CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage

doi: 10.15252/embr.201948920

Figure Lengend Snippet: U2OS cells were labelled with EdU for 30 min; then, EdU was washed, and cells were further incubated for either 1 or 2 h in the presence of thymidine. At the indicated time points, cells were fixed and proteins binding to EdU‐labelled DNA captured by the DNA‐mediated chromatin pull‐down technique (DmChP). Graphical experimental outline is shown above the analysis by Western blot of relevant proteins in both input and captured materials. U2OS cells were labelled with EdU for 30 min and then treated with 4 mM HU for 2 h in the presence or absence of 10 μM XL413. Proteins binding to EdU‐labelled DNA captured by DmChP were then analysed by Western blot as above. As in panel B, but incubation with HU was extended to 24 h. Black arrow indicates MRE11 electrophoretic mobility shift. U2OS cells were either mock‐treated or treated with 10 μM XL413 for 30 min, at which point 4 mM HU was added and cells incubated for a further 24 h. Extracts prepared from HU‐treated cells were then incubated in the presence or absence of λ‐phosphatase. Proteins were analysed by Western blotting with anti‐MRE11 antibodies. Total protein stain (TPS) is used as a loading control. Black arrow indicates MRE11 electrophoretic mobility shift. U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 h. PCNA (green) and MRE11 (red) were detected by immunofluorescence. Insets I–II represent enlargements of selected region of the merged images. Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ˜70 randomly selected cells for each condition from four biological replicates and expressed as Pearson's correlation coefficient. Error bars represent SEM. Statistical significance was assessed by Student's t ‐test (* P ˂ 0.05). Source data are available online for this figure.

Article Snippet: Primary antibodies diluted in blocking buffer (TBST, 5% milk) included RPA2 (1:1,000, NA19L; Calbiochem), Histone H2A (1:1,000, 07‐146; Upstate), MRE11 (1:1,000, NB100‐142; Novus), EXO1 (1:1,000, A302‐640A; Bethyl Laboratories), BRCA2 (1:2,000, A303‐434A; Bethyl Laboratories; 1:500, OP 95; EMD Millipore Ab‐1), Histone H3 (1:1,000, A300‐823A; Bethyl Laboratories), CDC7 (1:1,000, K0070‐3; MBL), CHK1 (1:1,000, SC‐8408; Santa Cruz Biotech) and CDC45 (1:5, a gift from Broderick et al ).

Techniques: Incubation, Binding Assay, Western Blot, Electrophoretic Mobility Shift Assay, Staining, Immunofluorescence

Journal: EMBO Reports

Article Title: CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage

doi: 10.15252/embr.201948920

Figure Lengend Snippet: U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 h. RPA2 (green) and MRE11 (red) were detected by immunofluorescence. Insets I–II represent enlargements of selected region of the merged images. Quantification of RPA2 and MRE11 colocalization was assessed with ImageJ in ˜200 randomly selected cells for each condition from three biological replicates and expressed as Pearson's correlation coefficient. Error bars represent SEM. Statistical significance was assessed by Student's t ‐test (** P ˂ 0.01). Source data are available online for this figure.

Article Snippet: Primary antibodies diluted in blocking buffer (TBST, 5% milk) included RPA2 (1:1,000, NA19L; Calbiochem), Histone H2A (1:1,000, 07‐146; Upstate), MRE11 (1:1,000, NB100‐142; Novus), EXO1 (1:1,000, A302‐640A; Bethyl Laboratories), BRCA2 (1:2,000, A303‐434A; Bethyl Laboratories; 1:500, OP 95; EMD Millipore Ab‐1), Histone H3 (1:1,000, A300‐823A; Bethyl Laboratories), CDC7 (1:1,000, K0070‐3; MBL), CHK1 (1:1,000, SC‐8408; Santa Cruz Biotech) and CDC45 (1:5, a gift from Broderick et al ).

Techniques: Immunofluorescence

Journal: EMBO Reports

Article Title: CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage

doi: 10.15252/embr.201948920

Figure Lengend Snippet: MCF10A cells were either mock‐treated or treated with 10 μM XL413 and labelled with IdU (red) for 30 min, at which point 4 mM HU was added for 2 h. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413. A set of representative DNA fibres from each condition is shown, and the percentage of IdU (red)‐only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent SEM from three biological repeats, and statistical significance was assessed by the Student t ‐test (** P ˂ 0.01). U2OS cells were either mock‐treated or treated with 10 μM XL413, 50 μM Mirin or both and labelled with IdU (red) for 30 min, at which point 4 mM HU was added for 2 h. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413 and/or Mirin. A set of representative DNA fibres from each condition is shown, and the percentage of IdU (red)‐only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent SEM from four biological repeats, and statistical significance was assessed by the Student t ‐test (* P < 0.05, ** P ˂ 0.01). U2OS cells were either mock‐treated or pre‐treated for the indicated times with 10 μM XL413, 50 μM Mirin or both and labelled with CldU (red). Then, 50 nM CPT was added and cells labelled with of IdU (green) for a further 30 min. A set of representative DNA fibres from each condition is shown and the IdU/CldU tract length ratio is plotted. The box extends from the 25 th to the 75 th percentile with the line in the box representing the median. Whiskers indicate the 10–90 percentiles with data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analysed for each condition. P ‐values were calculated using one‐way ANOVA, Kruskal–Wallis test and Dunn's multiple comparison post‐test (ns, not significant; **** P < 0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment. U2OS cells were transfected with control (siLuc) or MRE11‐targeting siRNA (siMRE11). After 48 h, cells were either mock‐treated or pre‐treated with 10 μM XL413 and labelled with CldU (red) for 30 min. Where indicated, 50 nM CPT was added, and then, cells were labelled with IdU (green). A set of representative DNA fibres from each condition is shown, and the IdU/CldU tract length ratio is plotted. The box extends from the 25 th to the 75 th percentile with the line in the box representing the median. Whiskers indicate the 10–90 percentiles with data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analysed for each condition. P ‐values were calculated using one‐way ANOVA, Kruskal–Wallis test and Dunn's multiple comparison post‐test (ns, not significant; **** P < 0.0001) and are related to the experiment shown. Similar results were obtained in two other independent experiments. Source data are available online for this figure.

Article Snippet: Primary antibodies diluted in blocking buffer (TBST, 5% milk) included RPA2 (1:1,000, NA19L; Calbiochem), Histone H2A (1:1,000, 07‐146; Upstate), MRE11 (1:1,000, NB100‐142; Novus), EXO1 (1:1,000, A302‐640A; Bethyl Laboratories), BRCA2 (1:2,000, A303‐434A; Bethyl Laboratories; 1:500, OP 95; EMD Millipore Ab‐1), Histone H3 (1:1,000, A300‐823A; Bethyl Laboratories), CDC7 (1:1,000, K0070‐3; MBL), CHK1 (1:1,000, SC‐8408; Santa Cruz Biotech) and CDC45 (1:5, a gift from Broderick et al ).

Techniques: Transfection