Transcription–replication conflicts underlie sensitivity to PARP inhibitors – Nature

Cell culture and drug treatments

The human cervical cell line HeLa and the human osteosarcoma cell line U2OS were purchased from the American Type Culture Collection (ATCC) (CCL-2 and HTB-96 respectively) and were grown under standard conditions (37 °C and 5% CO₂) in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, catalogue no. 11960) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, catalogue no. 10500) and penicillin–streptomycin–glutamine (Invitrogen, catalogue no. 10378-016). The hTERT-RPE1 retinal pigment epithelial cells (CRL4000, ATCC), were cultured in DMEM and Ham’s F-12 (Invitrogen, catalogue no. 12634-010), supplemented with 10% FBS and penicillin–streptomycin–glutamine. DLD1 BRCA2^+/+ (ATCC, CCL-221) and DLD1 BRCA2^−/− (Horizon, catalogue no. HD 105-007) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Scientific, catalogue no. 11875093) supplemented with 10% FBS and penicillin–streptomycin–glutamine. HeLa cells expressing FLAG-tagged RNase H1 in a doxycycline (DOX)-dependent manner⁵³ were grown in DMEM supplemented with 10% Tet system-approved FBS (Biowest, catalogue no. S181T) and antibiotics. Expression of FLAG-RNase H1 was induced with addition of 2 μg ml⁻¹ DOX (Sigma-Aldrich, catalogue no. D9891) in the medium for 18 h. U2OS T-Rex cells expressing catalytically inactive GFP-RNaseH1^D201N in a DOX-dependent manner⁵⁴ were grown in DMEM supplemented with 10% Tet system-approved FBS, 1 μg ml⁻¹ puromycin (Sigma, catalogue no. P8833) and 50 μg ml⁻¹ hygromycin B (Thermo Scientific, catalogue no. 10687010). Expression of catalytically inactive GFP-RNaseH1^D201N was induced by addition of 1 ng ml⁻¹ DOX for 18 h. The human non-small cell lung carcinoma H1299 cells, expressing a short-hairpin RNA against BRCA2 in a DOX-dependent manner⁵⁵, were grown in DMEM supplemented with 10% Tet system-approved FBS and antibiotics. Expression of shBRCA2 was induced by addition of 2 μg ml⁻¹ DOX in the medium. The human PEO1 and PEO4 ovarian cancer cell lines⁴⁰ (provided by I. Labidi-Galy) were grown in RPMI 1640 supplemented with 2 mM sodium pyruvate and antibiotics; the human colorectal carcinoma HCT116 cells (ATCC, catalogue no. CCL-247) were grown in McCoy’s 5A medium (Thermo Scientific, catalogue no. 16600082) containing 10% FBS and antibiotics; the human OVSAHO ovarian cancer cell line was purchased from Sigma-Aldrich (catalogue no. SCC294) and was grown in RPMI 1640 medium supplemented with 10% FBS and antibiotics. All cell lines were routinely tested for the absence of mycoplasma contamination using the MycoGenie Rapid MycoPlasma Detection Kit (AssayGenie, catalogue no. MORV001) and found negative. Drugs and chemical compounds used in this study were purchased from the following sources: thymidine (Sigma-Aldrich catalogue no. T1895), EdU (Thermo Fisher Scientific, catalogue no. A10044), 5-ethynyl uridine (EU) (Jena Biosciences, catalogue no. CLK-N002-10), camptothecin (Sigma-Aldrich, catalogue no. C9911), 5,6-dichlorbenzimidazol 1-β-d-ribofuranosid (DRB; Sigma-Aldrich, catalogue no. D1916), cordycepin (Tocris, catalogue no. 2294), triptolide (Tocris, catalogue no. 3253), olaparib (Selleckchem, catalogue no. S1060), talazoparib (Selleckchem, catalogue no. S7048), veliparib (Selleckchem, catalogue no. S1004), saruparib (Selleckchem, catalogue no. S9875), hydrogen peroxide (Sigma-Aldrich, catalogue no. H3410), RO-3306 (Sigma-Aldrich, catalogue no. SML0569), TMZ (Sigma-Aldrich, catalogue no. T2577), nocodazole (Tocris, catalogue no. 1228) and PARGi (Tocris, catalogue no. 7006). DRB, cordycepin and triptolide were used at concentrations of 75, 50 and 1 μΜ, respectively.

PARP1 and PARP2 biochemical assay

PARP1 and PARP2 activity in presence of increasing concentrations of PARP inhibitors was measured using PARP1 and PARP2 colorimetric assay kits (BPS Bioscience, catalogue no. 80580-1) according to the manufacturer’s instructions. The assays were performed in triplicate. PARP inhibitors were dispensed with an acoustic liquid dispenser (Gen5-Acoustic Transfer System; EDC Biosystems). The final concentration of the PARP inhibitors ranged from 0.2 to 100 nM using twofold dilution steps. The absorbance at 450 nm was measured using a Spark 10M microplate reader (Tecan).

siRNA and plasmid transfections

Transfections of siRNAs (at a final concentration of 40 nM) were performed with the cells at 60% confluency using INTERFERin (Polyplus, catalogue no. 409-01) or Lipofectamine RNAiMAX Transfection Reagent (Thermo Scientific, catalogue no. 13778075) according to the manufacturer’s instructions. TIMELESS depletion was achieved by transfection of 10 nM siRNA. Medium change was performed 24 h after siRNA transfection. The following siRNAs were used: negative control (AllStars Negative Control siRNA, Qiagen, catalogue no. 1027281), siTIM (TIMELESS; Qiagen, catalogue no. SI04142194), siTIMb (Dharmacon, 5′-GUAGCUUAGUCCUUUCAAATT-3′), siTIP (TIPIN; Invitrogen, catalogue no. S29864), siPARP1 (Qiagen, catalogue nos. SI02662989 and SI02662996) and (Invitrogen, catalogue no. s1097), siPARP1b (Dharmacon, 5′-GGAAAGAUGUUAAGCAUUUTT-3′ and 5′-CAUGGGAGCUCUUGAAAUATT-3′ and 5′-AGAAAAGGCUGGAGAGAGATT-3′), siPARP2 (Invitrogen, catalogue no. S19504), siBRCA2 (Qiagen, catalogue no. SI02653434). Efficiency of siRNA-mediated depletion was performed 72 h after transfection by western blotting. Empty vector, full length GFP-TIMELESS WT, GFP-TIMELESS R1081G, full length FLAG-PARP1 WT and FLAG-PARP1 D993G plasmids³⁷ were transfected using the FuGENE HD transfection reagent (Promega, catalogue no. E2311) according to manufacturer’s instructions. Efficiency of plasmid transfection was performed by western blot for detection of TIMELESS and PARP1 proteins.

Immunoblotting

Protein cell extracts were resolved by SDS–PAGE in precast protein gels (4–15% Mini-PROTEAN TGX, Bio-Rad, catalogue no. 4561083, or 3–8% Criterion XT Tris-Acetate Protein Gel, Bio-Rad, catalogue no. 3450129) and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% milk powder diluted in TBS-Tween 20 (0.01%) for 1 h at room temperature. Incubation with primary antibodies in blocking solution was applied for 1 h at room temperature. The following primary antibodies were used for western blot analysis: PCNA mouse monoclonal (1:1,000, clone PC10, Millipore, catalogue no. MABE288); alpha-Tubulin mouse monoclonal (1:1,000, Calbiochem, catalogue no. CP06); GAPDH mouse monoclonal (1:10,000, Abcam, catalogue no. ab8245); TIMELESS rabbit polyclonal (1:1,000, Abcam, catalogue no. ab109512); TIPIN rabbit polyclonal (1:250, Bethyl Laboratories, catalogue no. A301-474A); PARP1 rabbit polyclonal (1:1,000, Abcam, catalogue no. ab32138); PARP2 rabbit polyclonal (1:500, Active Motif, catalogue no. 39743); BRCA2 mouse monoclonal (1:1,000, Calbiochem, catalogue no. OP95); RNase H1 rabbit polyclonal (1:500, ProteinTech, catalogue no. 15606-1-AP); FLAG mouse monoclonal (1:1,000, Sigma-Aldrich, catalogue no. M2 F1804) and GFP rabbit polyclonal (1:500, Abcam, catalogue no. ab290). Following incubation with primary antibodies, three washes with TBS-Tween 20 (0.01%) were performed. Membranes were incubated with secondary horseradish antimouse or antirabbit peroxidase-coupled antibodies IgG (1:2,500, Promega, catalogue nos. W401B and W402B, respectively) for 1 h at room temperature, before detection by ECL-based chemiluminescence. Uncropped western blot images are provided in Supplementary Fig. 1.

Flow cytometry

Following siRNA transfection or drug treatment and, optionally, as indicated, following pulse-labelling with 10 μΜ EdU for 30 min, cells were collected by trypsinization and fixed in 90% methanol overnight at −20 °C. EdU detection was performed using the Click-it EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen catalogue no. C-10424) according to the manufacturer’s instructions. Detection of γH2AX phosphorylation was performed using the Guava Histone H2AX Phosphorylation Assay Kit (Luminex, catalogue no. FCCS100182) according to the manufacturer’s instructions. The genomic DNA was stained by incubating the cells in PBS containing RNase (Roche, catalogue no. 11119915001) and propidium iodide (Sigma-Aldrich catalogue no. 81845). EdU-DNA-γH2AX profiles were acquired by flow cytometry (Gallios, Beckman Coulter); more than 20,000 cells were analysed per sample using Kaluza software (Beckman Coulter). The gating strategy is provided in Supplementary Fig. 2.

EdUseq

The EdUseq protocol was performed as previously described³⁵. Briefly, HeLa cells were transfected with siRNA; 30 h later thymidine (Sigma-Aldrich) at 2 mM final concentration was added for 18 h, at which time the cells had reached 70–80% confluency. The cells were washed four times with warm PBS and released in fresh medium for 90 or 120 min. EdU (25 μM) was added 30 min before the cells were collected, and the cells were then fixed with 90% ice-cold methanol overnight. Cells were stored until processed for isolation of EdU-labelled DNA. Following fixation, the cells were permeabilized with 0.2% triton X in PBS; then, the EdU incorporated into genomic DNA was coupled to a cleavable biotin-azide linker (Azide-PEG(3+3)-S–S-biotin; Jena Biosciences, catalogue no. CLK-A2112-10), using the reagents of the Click-it Kit (Invitrogen, catalogue no. C-10424). Extraction of genomic DNA was performed with phenol-chloroform ethanol precipitation, followed by isolation of EdU-labelled DNA. Briefly, genomic DNA was sonicated to 100–500 bp nucleotide-long fragments using a bioruptor sonicator (Diagenode). EdU-labelled DNA fragments were captured on Dynabeads MyOne streptavidin C1 (Invitrogen, catalogue no. 65001). The beads were washed three times with Binding and Washing Buffer 1× (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCL, 0.5% Tween 20) and then were resuspended to twice the original volume with Binding and Washing Buffer 2×, mixed with an equal volume of sonicated EdU-labelled DNA incubated for 15 min on a rotating wheel at room temperature. Following three washes of the beads with Binding and Washing Buffer 1× and once with TE (10 mM Tris-HCl pH 8, 1 mM EDTA), the EdU-labelled DNA was eluted by incubating the streptavidin beads with 2% β-mercaptoethanol (Sigma, catalogue no. M6250) for 1 h at room temperature. The eluted DNA was used for library preparation using the TruSeq ChIP Sample Prep Kit (Illumina, catalogue no. IP-202-1012). High-throughput 100-base-pair single-end sequencing was performed on an Illumina Hi-Seq 4000 sequencer.

EdUseq data processing

Sequencing reads were aligned on the non-masked human genome assembly (GRCh37/hg19) using the Burrows–Wheeler Aligner software as described previously^35,56. Only the reads with the highest quality score were retained. Previously described custom Perl scripts were used to assign the aligned reads to 10 kb genomic bins. Sigma (σ) values were calculated as the normalized number of reads per bin divided by its standard deviation. The data were visualized using previously described scripts³⁵. Assignment of replication timing was performed with REPLI-seq data generated previously³⁵.

Cell viability and clonogenic assays

Viability assays were performed with DLD1 BRCA2^+/+ and DLD1 BRCA2^−/− cells treated with various inhibitors and DNA damaging agents, and following siRNA transfections, as indicated. In brief, 2,000 cells per well were seeded in Advanced TC 96-well microplates; 24 h later, PARP inhibitors were dispensed using a D300e digital dispenser (Tecan) at final concentrations ranging from 0.6 nM to 10 µM using fourfold dilution steps. The cells were incubated with the compounds for 5 days before adding CellTiter-Glo 2.0 reagent (Promega, catalogue no. G9242) to each well, according to the manufacturer’s instructions. Luminescence was measured using a Spark 10M microplate reader (Tecan).

Clonogenic assays were performed with a variety of cell lines following siRNA transfection or drug treatment. Briefly, following the indicated treatments, the cells were replated in triplicate in six- or 12-well plates (500–3,000 cells per well, depending on cell line) and cultured for an additional 14 days (or more, depending on cell line) in fresh medium. The cell culture medium was changed every two days. At the end of the experiment, medium was removed, and cells were rinsed with PBS and stained with 0.5% (w/v) crystal violet (Sigma-Aldrich) in 20% (v/v) methanol for 30 min in the dark. The staining agent was removed and the plates were rinsed three times in ddH₂0, air-dried and the cell colonies were counted.

Immunofluorescence assays

Cells were seeded onto autoclaved 12 mm glass coverslips or multiwell plates (μ-Plate 96 Wells, catalogue no. 89626) at 70–90% confluency. Following any indicated treatment, the cells were pre-extracted for 2 min with ice-cold 1× PBS containing 0.2% (v/v) Triton X-100 and then fixed with 4% formaldehyde for 10 min at room temperature. After three washes with 1× PBS, the cells were permeabilized in 1× PBS containing 0.2% (v/v) Triton X-100 for 15 min at room temperature. For detection of trapped PARP1 and PARP2, the cells were pre-extracted with cold cytoskeleton buffer (0.5% Triton X-100, 10 mM PIPES pH 6.8, 3 mM MgCl₂, 200 mM NaCl, 300 mM sucrose) for 10 min at 4 °C, followed by fixation with ice-cold methanol for 15 min at −20 °C. Following three washes with PBS, the cells were blocked with 5% BSA/1× PBS solution for 1 h at room temperature. Then, coverslips or multiwell plates were incubated for 2 h at room temperature with primary antibodies diluted in 5% BSA/1× PBS. Following incubation with primary antibodies, coverslips or multiwell plates were washed three times with 1× PBS and incubated for 1 h at room temperature with secondary antibodies diluted in 5% BSA/1× PBS. After three washes with 1× PBS, incubation with 1 μg ml⁻¹ 4,6-diamidino-2-phenylindole (DAPI)/1× PBS for 15 min in dark at room temperature was performed. Then, three washes with 1× PBS were performed and coverslips were mounted on slides using the Fluoromount-G (Thermo Fisher Scientific, catalogue no. 00-4958-02). For multiwell plates, following incubation with DAPI, 1× PBS was added in the wells. The primary antibodies used for the immunofluorescence were: γH2AX (S139) mouse monoclonal (1:1,000, clone JBW301, Millipore, catalogue no. 05-636); RAD51 rabbit polyclonal (1:1,000, Bioacademia, catalogue no. 70-002); 53BP1 rabbit polyclonal (1:1,000, Novus Biologicals, catalogue no. NB100-304); poly (ADP-ribose) mouse monoclonal (1:500, Trevigen, catalogue no. 4335-MC-100 and 1-500, Enzo Life Sciences, catalogue no. ALX-804-220-R100); PARP1 rabbit polyclonal (1:1,000, ProteinTech, catalogue no. 13371–1-AP); PARP2 rabbit polyclonal (1:1,000, Active Motif, catalogue no. 39743). Secondary antibodies used: Alexa Fluor 488 goat-antirabbit IgG (1:500, Invitrogen, catalogue no. A110334); Alexa Fluor 488 goat-antimouse IgG (1:500, Invitrogen, catalogue no. A11001); Alexa Fluor 594 goat-antirabbit IgG (1:500, Invitrogen, catalogue no. A11037); Alexa Fluor 594 goat-antimouse IgG (1:500, Invitrogen, catalogue no. A11005); Alexa Fluor 647 goat-antirabbit IgG (1:500, Invitrogen, catalogue no. A21244); Alexa Fluor 647 goat-antimouse IgG (1:500, Invitrogen, catalogue no. A21235). For micronuclei detection, following any indicated treatment, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature. Nuclei were countestained with 1 μg ml⁻¹ DAPI for 1 min at room temperature in dark, washed three times with PBS and mounted with Fluoromount-G.

In situ PLA

Following the indicated treatments, cells grown on coverslips were washed twice with 1× PBS and pre-extracted for 10 min with ice-cold 1× PBS containing 0.5% (v/v) Triton X-100 and protease inhibitor cocktail (Complete, EDTA-free; Roche); they were then washed twice with 1× PBS and fixed with 4% (v/v) formaldehyde for 10 min at room temperature, followed by two washes with 1× PBS. The cells were then incubated with 1× PBS containing 0.2% (v/v) Triton X-100 for 10 min at room temperature, washed again twice with 1× PBS and blocked with 5% BSA/1× PBS solution for 1 h. The coverslips were then incubated O/N at 4 °C with primary antibodies diluted in 5% BSA/1× PBS. Following incubation with primary antibodies, the coverslips were washed twice with 1× PBS and PLA was performed using Duolink PLA technology (Sigma-Aldrich, catalogue no. DUO92008) according to the manufacturer’s instructions. Briefly, coverslips were incubated with antirabbit PLUS and antimouse MINUS PLA probes (Sigma-Aldrich, catalogue no. DUO92002 and DUO92004 respectively) for 1 h at 37 °C, followed by two washes in Wash Buffer A (0.01 M Tris, 0.15 M NaCl and 0.05% Tween 20, pH 7.4) for 5 min. Then, PLA probes were ligated for 30 min at 37 °C, followed by two washes for 5 min in Wash Buffer A and amplification using the Duolink In Situ Detection Reagents Red (Sigma-Aldrich, catalogue no. DUO92008), performed at 37 °C for 100 min. After amplification, the coverslips were washed twice in Wash Buffer B (0.2 M Tris and 0.1 M NaCl, pH 7.5) for 10 min and then incubated with 1 mg ml⁻¹ DAPI/1× PBS for 15 min in the dark at room temperature. Finally, the coverslips were washed three times with 1× PBS and mounted on slides using Fluoromount-G. Primary antibodies used: RNAPII, H5 (1:500, BioLegend, catalogue no. 920204), RNAPII, CTD4H8 (1:500, Millipore, catalogue no. 05-623) and PCNA (1:500, Abcam, catalogue no. ab18197).

Quantification of nascent RNA production by EU labelling

Cells grown on multiwell plates were pulse-labelled with 1 mM EU for 30 min, washed twice with 1× PBS and fixed in 4% formaldehyde for 10 min at room temperature. After three washes with 1× PBS, the cells were permeabilized with 1× PBS containing 0.2% (v/v) Triton X-100 for 10 min at room temperature. EU incorporation was detected with Click-iT EU Alexa fluor 488 Imaging Kit (Thermo Fisher Scientific, catalogue no. C10329). Multiwell plates were incubated with Hoechst 33342 for 15 min in the dark at room temperature and subsequently washed three times with 1× PBS.

Detection of EdU incorporation in mitosis

For detection of mitotic EdU foci, cells, cultured on coverslips, were synchronized at the G1/S transition with 2 mM thymidine (Sigma-Aldrich, catalogue no. T1895) for 18 h, washed three times with 1× PBS and released in fresh medium containing 6 μM RO-3306 (Sigma-Aldrich, catalogue no. SML0569) for 11 h. The cells were then washed three times with warm medium and released in medium containing 100 ng ml⁻¹ nocodazole (Tocris, catalogue no. 1228) and 20 μM EdU (Invitrogen, catalogue no. A10044) for 1 h. The cells were then fixed and permeabilized with 4% paraformaldehyde, 20 mM HEPES, 10 mM EGTA, 0.2% Triton X-100, 1 mM MgCl₂ for 20 min at room temperature and then washed three times with 1× PBS. EdU incorporation was performed using the Click-it EdU Alexa Fluor 647 Kit (Invitrogen, catalogue no. C10340), after which the cells were washed twice with 1× PBS, incubated with DAPI (0.5 mg ml⁻¹, Thermo Fisher, catalogue no. D1306) in 1× PBS for 15 min at room temperature, washed three times with PBS and rinsed in distilled water. The coverslips were then mounted on slides using ProLong Gold Antifade Mountant (Thermo Fisher, catalogue no. P10144). Quantification of EdU foci on metaphase chromosome spreads was performed manually.

Image acquisition and analysis

Images from coverslips were acquired with a Zeiss Imager M2 AX10 microscope equipped with ApoTome2 and a Plan-APOCHROMAT ×100/1.4 oil immersion objective, using the ZEN3.4 (blue edition) software. Images were analysed with ZEN3.4 (blue edition) or ImageJ/FIJI software (National Healthcare Institute, USA). The threshold to determine whether a cell was positive for γΗ2ΑΧ or 53BP1 foci was set at 20 foci per nucleus and for RAD51 at ten foci per nucleus. Micronuclei, PLA foci and GFP-RNaseH1^D210N foci were quantified manually. Automated, multi-channel image acquisition of multiwell plates was performed in an unbiased fashion with an ImageXpress spinning disc confocal microscope (Molecular Devices), equipped with a sCMOS camera (Andor), and with a Nikon ×20 water 1.20 numerical aperture or ×60 water immersion 1.20 numerical aperture objective. The spinning disc confocal images were analysed using MetaXpress Custom Module Editor. The analysis pipeline started with the detection of the nuclei using the DAPI channel. This mask was then applied to quantify pixel intensities for γΗ2ΑΧ, PARP1/2 and EU incorporation for each individual cell. A light deconvolution was applied on the RAD51 and 53BP1 channel to ease the detection of the small foci. The segmentation of foci was then performed using shape, size and intensity above local background parameters. The masked foci were then attributed to their corresponding nuclei before the quantification of relevant parameters. Quantified values for each cell, were exported and were subsequently used to generate graphs using GraphPad Prism 9 software.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 software (v.9.4.1). Detailed description of means or medians, error bars and the number replicates and/or cells analysed is reported in the figure legends. Statistical differences for grouped analyses were performed using repeated-measures one-way analysis of variance (ANOVA) followed by a Tukey’s multiple comparisons test. Statistical test results are provided as P values in the figures. Dose–response curves were plotted using GraphPad Prism using as model the concentration of the inhibitor versus response, variable slope (four parameters). No statistical methods were used to determine the size sample size before conducting experiments. Experiments were not randomized and the investigators were not blinded to allocation. Data were assembled into figures using Adobe Illustrator CS6.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.