Cloning and plasmids
Table of Contents
All PCRs were performed using the Fidelio polymerase (Ozyme) and cloning was performed using either Gibson assembly mix (NEB or Codex) or T4 DNA ligase (NEB). Exogenous expression of all Polθ constructs (GFP–Polθ) used in this study was achieved by cloning full-length POLQ fused with a C-terminal Flag-P2A-BLAST cassette in the PiggyBac CAG eGFP vector (Addgene, #40973) between BsrGI and SacI restriction sites. The P2A-BLAST cassette was amplified from the eFlut P2A-BLAST plasmid (gift from J. Stewart-Ornstein) with forward primer containing the Flag sequence. For degron experiments, the Flag-mAID cassette was amplified from pMK288 (mAID-Bsr, Addgene 72826), fused to P2A-BLAST and cloned in the PiggyBac CAG eGFP-POLQ. TIR1 expression was achieved using the retroviral vector: pBabe Blast osTIR1-9Myc (Addgene 80073). Polθ(10A) and Polθ(4A) were obtained by amplification of DNA fragments with the desired mutations (gBlocks from Integrated DNA Technologies) cloned into PiggyBac CAG eGFP-POLQ-Flag-P2A-BLAST between PflFI and NheI restriction sites. For knock-in experiments, the KI-POLQ-E30-NeonGreen cassette was obtained for POLQ knock-in, and the Flag-mAID-Zeocin and the Flag-mAID-Hygromycin cassettes were obtained for BRCA2 knock-in after assembly of PCR fragments amplified from mNeonGreen plasmid (gift from D. Fachinetti), pMK288 (mAID-Bsr, Addgene 72826) and pMK287 (mAID-Hygro, Addgene 72825) plasmids. PCRs were performed with 600 bp of homologous sequences flanking the gRNA site in the last exons of POLQ and BRCA2 and finally cloned in puc19 vector. pOZ-53BP1WT and pOZ-53BP1AA (containing the T1609A/S1618A mutations) plasmids were gifts from D. Chowdhury. Plasmids allowing bacterial expression of His–TOPBP1 BRCT0-2, GST–TOPBP1 BRCT4-5 and GST–TOPBP1 BRCT7-8 were gifts from J. N. Mark Glover.
Cell culture and generation of cell lines
We made use of hTERT-RPE-1 (human retinal epithelium), HEK-293T (human embryonic kidney) and HeLa (human cervical adenocarcinoma) cells. All cell lines were obtained from ATCC and were grown in Dulbecco’s modified Eagle’s medium (DMEM) with nutrient Mixture F12 (for RPE-1) or DMEM (for HEK and HeLa) (Gibco) and supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and 200 IU ml−1 penicillin. Cells were incubated at 37 °C in 5% CO2. Throughout, wild-type GFP–Polθ refers to the exogenous expression of GFP–Polθ in RPE-1 TP53−/−;POLQ−/− cells, BRCA2−/− GFP–Polθ refers to the exogenous expression of GFP–Polθ in RPE-1 TP53−/−;BRCA2−/−;POLQ−/− cells. When experiments were performed in HeLa or HEK-293T, the cell line origin is specified in the figure. Expression of GFP–Polθ was obtained after co transfection of GFP–Polθ with transposase (System Biosciences, PB210PA-1) according to the manufacturer’s instructions, with blasticidin selection.
All genome-editing experiments were performed using ribonuclear protein (RNP) complexes composed of recombinant S. pyogenes Cas9 nuclease, Alt-R CRISPR–Cas9 CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (Integrated DNA technologies 1081058 and 1072532). Knockout cell lines were obtained after transfection with RNP complexes using RNAiMax (ThermoFisher Scientific). RPE-1 TP53−/− wild-type and isogenic BRCA2−/− or POLQ−/− cell lines were described previously26.
Knockout cell lines were generated as follows: BRCA2−/− GFP–Polθ cells were generated by knocking out BRCA2 in POLQ−/− cells complemented with GFP–Polθ. To generate BRCA2−/− cells expressing GFP-Polθ-mAID, GFP-Polθ-mAID was first introduced in BRCA2−/− cells and then POLQ was knocked out. Knockout of the BRCA2 gene was obtained after transfection of RNP complexes targeting introns flanking exon2 (BRCA2-KO1: GGTAAAACTCAGAAGCGC; BRCA2-KO2: GCAACACTGTGACGTACT). Knockout of the POLQ gene was obtained after transfection of RNP complexes targeting introns flanking exon2 (POLQ-KO1: GGAAGGCTTTTAGGTCAGTA; POLQ-KO2: GGCAACGGGGGCAGCTCCGC). TIR1 (Addgene 80073) was expressed to allow Polθ degradation upon IAA treatment.
To generate knock-in cell lines, cells were co-transfected with cassettes (Flag-NeonGreen-mAID-BLAST for POLQ knock-in, Flag-mAID-Zeocin and Flag-mAID-Hygromycin for BRCA2 knock-in) and RNP complexes (POLQ-KI: GTGAAAATAGGCGCCAGC; BRCA2-KI: GGAGAGTTCCCAGGCCAGTA). Next, cells were selected for 2 weeks in appropriate antibiotic and the resistant population was subcloned in 96 well plate. Clones were screened by PCR.
Retroviral particles containing either TIR1, pOZ-53BP1WT or pOZ-53BP1AA plasmids were obtained by transfection of the retroviral vectors with an expression vector for the VSV-G envelope protein (Addgene, #8454) into HEK-293T cells expressing GAG-pol proteins (Phoenix cells) using LTX (ThermoFisher Scientific). Viral particles containing supernatants were collected and filtered before cell transduction.
Cell lines expressing mCherry–PCNA were generated after random integration by transfecting mCherry-PCNA-19-SV40NLS-4 plasmid (Addgene, #55117), antibiotic selection (neomycin) and cell sorting (mCherry) by flow cytometry. Blasticidin (HEK and HeLa: 5 μg ml−1, RPE-1: 21 μg ml−1), puromycin (HEK: 5 μg ml−1, HeLa: 1 μg ml−1, RPE-1: 21 μg ml−1), zeocin (HeLa: 600 μg ml−1), neomycin (RPE-1: 600 μg ml−1) and hygromycin (500 μg ml−1) were used at indicated concentrations for cell lines selection. Cell lines have been correctly identified by short tandem repeat (STR) analysis (Eurofins) and were tested every month by qPCR for mycoplasma (Eurofins).
Chemicals, siRNA and antibodies
Nocodazole (100 ng ml−1, Sigma), 3-Indoleacetic acid (IAA, 500 μM, Sigma), RO3306 (referred as CDKi, 5 μM, Sigma), Aphidicolin (0.4 μM to 1.6 μM, Abcam), SiR-DNA (250 nM for time-lapse video microscopy and 1 μM for laser micro-irradiation experiments, Spirochrome), ATRi (2.5 μM, Selleckchem, VE-821), ATMi (10 μM, Tocris, Ku-55933), Palbociclib (1 μM, Selleckchem, PD-0332991), Rucaparib (1 μM, Selleckchem), Volasertib (referred as PLK1i throughout, 200 nM, Selleckchem, Bi6727), Bi2536 (200 nM, Selleckchem), MG132 (10 μM, Sigma), Bleomycin (50 μg ml−1, Sigma, B7216), and Polθi (Novobiocin, 200 μM, Sigma) were used at the indicated concentrations.
For RNA interference, we used the following targeted sequences targeting MDC1 (GUCUCCCAGAAGACAGUGAdTdT), TOPBP1 (ACAAAUACAUGGCUGGUUAdTdT), BRCA2 (GAAGAAUGCAGGUUUAAUAdTdT), BRCA1 (CAGCAGUUUAUUACUCACUAAdTdT), FANCD2 (GGAGAUUGAUGGUCUACUATdT), 53BP1 (CAGGACAGUCUUUCCACGAdTdT) and LacZ (Control, CGUACGCGGAAUACUUCGAdTdT). Transfection of siRNA oligonucleotides (Eurofins) was performed using Lipofectamine RNAiMax (ThermoFisher Scientific) following the manufacturer’s instructions.
For western blotting, antibodies used in this study were purchased from Millipore (BRCA1 OP92; BRCA2 OP95), Sigma (Anti-phospho-Ser/Thr-Pro MPM2 05-36849; Anti-pan-phospho-Ser SAB5700550, β-actin A5316), Santa Cruz Biotechnology (PARP1 sc8007; BARD1 sc-74559), Abcam (DNA ligase III ab185815; MDC1 ab11171), Cell Signaling (His 2365), Abclonal (Pan-phospho-Ser/Thr antibody, AP0893) and Bethyl (EXO1 A302-639A, TOPBP1 A300-111, Biotin A150-109A). The Polθ antibody was a gift from J. S. Hoffmann. For immunofluorescence, antibodies were purchased from Institut Curie antibody platform (HA), Immunovision (CREST HCT-0100), Chromotek (mNeonGreen 32F6), Novus Biologicals (FANCD2 NB100-182), Bethyl Laboratories (H2A.X (Ser139) A300-081A; TOPBP1 A300-111), Abcam (MDC1 ab50003; 53BP1 ab21083), Millipore (H2A.X (Ser139) 05-636; phospho-Histone H3 (Ser10) 06-570; 53BP1 MAB3802), and Santa Cruz Biotechnology (cyclin A sc-271682; PCNA sc-56, PLK1 sc-5504). The rabbit phospho-specific antibodies serum against Polθ (pS1493), was generated by Eurogentec using the following phosphopeptide: LLFDpSFSDDYLV.
Laser micro-irradiation was performed with a two-photon Ti:Saphire laser (Mai Tai DeepSee, Spectra Physics) at 800 nm with 20% power (from 2.1 W) on an inverted laser scanning confocal microscope equipped with spectral detection and a multi-photon laser (LSM880NLO/Mai Tai Laser, Zeiss, Spectra Physics), using a 40× objective NA1.3 oil DICII PL APO (420852-9870). Images were analysed using the ‘plot Z-axis profile’ function of Fiji (version 2.1.0/1.53c). X-ray irradiations were performed using the CIXD Dual Irradiator (RadeXp platform, Institut Curie).
Cell extracts, immunoprecipitation and Western blotting
All immunoblotting and immunoprecipitation experiments were performed from nuclear extracts. Nuclei were obtained after cells lysis with nuclei extraction buffer (10 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 34 mM sucrose, 10% glycerol, 1 mM dithiothreitol (DTT), 0.1% Triton X-100). Cells lysates were incubated 5 min on ice and centrifuged for 5 min at 1,300g. Nuclei (pellet) were lysed in 300 mM RIPA lysis buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.2 mM EDTA, pH 8.0 and 1% NP-40) for 40 min on ice, benzonase (Sigma) was added during the final 10 min of incubation. Extracts were centrifuged for 20 min at 20,000g and protein concentration was determined using BCA kit (ThermoFisher Scientific). For immunoprecipitation experiments, 1 mg of protein extract was diluted to 1 mg ml−1 in 150 mM NaCl RIPA buffer, incubated with magnetic agarose beads (Chromotek, GFP Trap (GTMA-20), mNeonGreen-Trap (NTMA-20)) for 2 h at 4 °C. Prior to elution, beads were washed twice with 150 mM NaCl RIPA buffer and once with 300 mM NaCl RIPA buffer. All buffers were supplemented with phosphatase and protease inhibitors (PhosSTOP and cOmplete Protease Inhibitor Cocktail, Roche). Finally, lysates were resolved by NuPAGE Tris-acetate gel (4/8%, Invitrogen) and transferred onto nitrocellulose membrane (Bio-Rad) (wet transfer in 10% Tris-Glycine, 20% methanol, 0.05% SDS, 2 h at 40 V). Membranes were then blocked for 1 h in 5% milk or 10% BSA and hybridized with the desired antibody.
Polθ protein fragment expression and purification
The Polθ protein fragments (E1424–Q1503) and (E1540–S1660) were expressed using a pET-22b expression vector as a fusion protein with a C-terminal GB1 protein tag and 6×His tag. A TEV site was introduced between the Polθ protein fragment and GB1. The Polθ gene fragments were optimized and synthesized by Genscript for high-level expression in bacteria. For NMR studies, the 15N-labelled and 15N,13C-labelled Polθ samples were produced in E. coli BL21 (DE3) Star cells grown in M9 minimum medium containing 0.5 g l−1 of 15NH4Cl for the 15N-labelled sample or 0.5 g l−1 of 15NH4Cl and 2 g l−1 of 13C-glucose for the 15N/13C-labelled sample. Expression was induced with 0.5 mM IPTG at an optical density at 600 nm (OD600) of 0.6 and cells were incubated for 3 h at 37 °C. After centrifugation, cell pellets were resuspended in lysis buffer (TBS 1×, EDTA-free Protease Inhibitor Cocktail, Roche) and homogenized by sonication. The lysate was treated with benzonase nuclease and MgCl2 (5 mM) for 20 min at room temperature and then centrifuged (15,000g) for 20 min at 4 °C. The supernatant was filtered (0.44 µm) and loaded at 2 ml min−1 on a HisTrap HP 5 ml column (GE Healthcare) that was equilibrated with buffer A (TBS 1×, 20 mM imidazole). Proteins were eluted at 1 ml min−1 using a linear gradient of imidazole (30 min to reach 100% of buffer B: TBS 1×, 500 mM imidazole). Elution fractions of interest were pooled and diluted to 1 mg ml−1 with TBS 1× and the GB1-6×His tag was cleaved by the TEV protease (2% w/w) during an overnight dialysis at 4 °C against TBS 1×. Cleaved protein solution was loaded on a HisTrap HP 5 ml (GE Healthcare) column and the Polθ without GB1-6×His fragment was collected in the flow-through. The quality of the purified protein was analysed by SDS–PAGE and the protein concentration was determined using its absorbance at 280 nm.
For NMR studies, samples were dialysed against the NMR buffer (25 mM HEPES, 50 mM NaCl, pH 7) and concentrated at 100–150 μM in the case of the 15N-labelled samples used to monitor phosphorylation by PLK1, and at 200–300 μM in the case of the 15N,13C-labelled samples used for the 1H, 15N, 13C NMR chemical shift assignment experiments. The PLK1 sample was provided by the protein production facility of Institut Curie.
To monitor binding to TOPBP1 BRCT7-8, a sample of phosphorylated 15N-labelled Polθ (E1424–Q1503) was diluted down to 60–70 µM and dialysed against the NMR buffer. It was further diluted twofold with either the NMR buffer in order to obtain the reference NMR sample with the free peptide, or a solution of purified TOPBP1 BRCT7-8 (Polθ E1424–Q1503:TOPBP1 BRCT7-8 ratio 1:0.5) in this same buffer in order to obtain the NMR sample containing the bound peptide.
The NMR experiments were performed on the Polθ protein fragments (E1424–Q1503) and (E1540–S1660), either 15N- or 15N,13C-labelled, and dissolved in the NMR buffer (with in addition 7% v/v D2O and EDTA-free Protease Inhibitor Cocktail 1× (Roche)). Most experiments were recorded on a 700 MHz Bruker ADVANCE NEO spectrometer equipped with a triple resonance cryogenically cooled probe. 1HN, 13Cα, 13Cβ, 13CO and 15N resonance frequencies of the non-phosphorylated fragments were assigned at 283 K using 2D 1H–15N SOFAST HMQC, 3D HNCA, 3D HNCACB, 3D CBCA(CO)NH, 3D HNCO, 3D HN(CA)CO and 3D HN(CA)NNH experiments. All 3D experiments were recorded using non uniform sampling (NUS: 40–50%). To monitor phosphorylation by PLK1, either 15N- or 15N,13C-labelled Polθ protein fragments were mixed with PLK1, and a series of 16 2D NMR 1H-15N SOFAST HMQC experiments were recorded at 298 K during 8 h, each experiment being a 1,536 × 160 dataset acquired with 80 scans during 30 min50. The temperature was then set to 283 K and 2D 1H–15N SOFAST HMQC, 3D HNCA, 3D HNCACB, 3D CBCA(CO)NH and 3D HN(CA)NNH experiments were recorded, in order to assign the resonance frequencies of the phosphorylated fragments (here again with NUS at 40–50%). For Polθ (E1424–Q1503) fragment, resonance assignment was performed at two different stages of the phosphorylation kinetics, first after an incubation with a 1:200 ratio of PLK1, and second after an additional incubation with 1:200 and 1:150 ratios of PLK1. For Polθ (E1540–S1660) fragment, resonance assignment was performed after an incubation with a 1:200 ratio of PLK1.
To identify the TOPBP1 BRCT7-8 binding site in Polθ protein fragment F1, 2D 1H–15N HSQC experiments were recorded on a 15N-labelled Polθ (E1424–Q1503) fragment, either phosphorylated or not, in the absence and presence of TOPBP1 BRCT7-8 at a 1:0.5 ratio, each experiment being a 1,536 × 160 dataset acquired with 800 scans during 5 h. The data were processed using Topspin 3.6 (Bruker) and analysed with CCPNMR Analysis Software51. 1HN, 13Cα, 13Cβ, 13CΟ and 15N resonance assignments of Polθ protein fragments (E1424–Q1503) and (E1540–S1660) were deposited at the Biological Magnetic Resonance Data Bank (BMRB) under the accession numbers 51900 and 51939 for the non-phosphorylated forms and 51920 and 51940 for the phosphorylated forms, respectively.
Biomolecular interactions between the Polθ peptides and TOPBP1 BRCT7-8 were measured using the BLI technology on an Octet RED96 instrument (FortéBio) with streptavidin biosensors. The Polθ peptides (E1472–Y1498) were synthesized by Genecust with an N-terminal biotin. Peptides sequences are (pS being a phosphorylated serine): 4S-P: Biotin-GSG-EGENLPVPETpSLNMpSDpSLLFDpSFSDDY and 4S: Biotin-GSG-EGENLPVPETSLNMSDSLLFDSFSDDY. The biosensors were hydrated for 20 min in the TOPBP1 BRCT7-8 buffer (25 mM HEPES, 50 mM NaCl, 5 mM β-mercaptoethanol). Non-specific interactions between TOPBP1 BRCT7-8 and the biosensors were characterized by performing the binding assays without loading the biotinylated peptides onto the streptavidin biosensors. In these conditions, no interaction was observed. For the binding assays, a concentration of 5 μM of biotinylated peptides was used to attach them on the streptavidin sensors, and TOPBP1 BRCT7-8 was added at different concentrations (62.5–2,000 nM range for 4S and 7.8–500 nM range for 4S-P). 3 cycles of 10-s incubation in the regeneration buffer (1 M NaCl) followed by 10-s incubation in the assay buffer (25 mM HEPES, 50 mM NaCl, 5 mM β-mercaptoethanol, 0.05% Tween) were performed before every kinetics experiment. The Kd values were measured in the steady-state mode.
In vitro phosphorylation of full-length Polθ and Polθ E1424–Q1503
Immunoprecipitates of GFP–Polθ (2 h GFP trap at 4 °C, followed by 2 washes with 150 mM NaCl buffer and 1 wash with 300 mM NaCl) or 1.2 μg of RBER-CDC25tide (Reaction Biology, 0590-0000-1) were resuspended in 30 μl kinase buffer (Cell Signaling, 9802) containing 50, 100 or 500 ng of recombinant PLK1 (Reaction Biology, 0183-0000-1) and 5 μM ATP and 5 μCi [γ-32P]ATP. After incubation for 30 min at 30 °C, reactions were stopped on ice by the addition of 2× Laemmli buffer and boiling for 5 min at 95 °C. Samples were separated on a 4–8% Tris-acetate gel (or 10% SDS–PAGE for the CDC25 positive control), and phosphorylated 32P-Polθ and CDC25 were detected by autoradiography.
Additionally, in vitro phosphorylation assays were performed on the Polθ (E1424 to Q1503) fragment (Genscript) cloned in the pET-28(b+) vector. In brief, 3 μM of Polθ fragment (E1424 to Q1503) or RBER-CDC25tide (a PLK1 substrate used as a positive control, obtained from Reaction Biology, 0590-0000-1. The CDC25 sequence is: ISDELMDATFADQEAK; the threonine phosphorylated by PLK1 is indicated in bold) were incubated with 0.05 μM of PLK1 enzyme (Reaction Biology, 0183-0000-1) in 20 μl of kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2) (Abcam) and 0.2 mM ATP for 2 hours at 30 °C. Reactions were loaded on an acrylamide gel, in which phosphorylated proteins were detected using the Pro-Q Diamond Phosphoprotein Blot Stain Kit (ThermoFisher Scientific, P33356), and total proteins using SYPRO Ruby Protein Gel Stain (ThermoFisher Scientific, S12000) following the manufacturer’s instructions and revealed using ChemiDoc imaging system (Bio-Rad).
Expression and purification of TOPBP1 BRCT fragments
The TOPBP1 fragment (1–290), also known as TOPBP1 BRCT0-2, was cloned into a pET-22b plasmid (Genscript). The TOPBP1 fragments (559–746) and (1264–1493), also known as TOPBP1 BRCT4-5 and TOPBP1 BRCT7-8, were expressed using pGEX-6P-1 plasmids provided by J. N. Mark Glover52,53. The BRCT0-2 fragment contains an 8-his N-terminal tag, the BRCT4-5 and BRCT7-8 a GST tag. All BRCT domains contain a TEV cleaving site. Plasmids coding for TOPBP1 BRCT domains 0-2, 4-5, and 7-8 were transfected into E. coli BL21 (DE3) Star cells (Thermo Scientific, EC0114) grown at 37 °C up to an OD600 of 0.7, induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and incubated at 22 °C overnight (16 h). Cell cultures were harvested by centrifugation and resuspended in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT and 10% glycerol. The proteins were further purified as described52,53. Expression of TOPBP1 BRCT domains was confirmed by immunoblotting using His (BRCT0-2) or GST (BRCT4-5 and BRCT7-8) antibodies.
Biotinylated Polθ peptides containing desired phosphorylated serines were purchased from Genecust (4S and 4S-P; S1628/S1635 and S1628-P/S1635-P) and were resuspended in water at concentrations ranging from 0.5 to 2 mg ml−1 in accordance with the manufacturer’s instructions. Peptides were coupled to streptavidin beads in 150 mM NaCl on a rotating wheel at room temperature, for 1 h. Beads were washed twice in 150 mM NaCl prior to use in pull-down assays. Nuclear extracts from HeLa cells (350 μg) or 2 ml of bacteria protein extracts were incubated with 1 nmol of desired streptavidin beads-coupled peptide, on a rotating wheel, for 2 h, at room temperature. After incubation, beads were washed three times using 150 mM NaCl RIPA buffer, and twice using 300 mM NaCl before elution in 2× Laemmli buffer. All buffers used were supplemented with phosphatase and protease inhibitors. Eluted proteins were analysed by SDS–PAGE to identify peptide partners. The same amount of peptides used for pull-downs was loaded into a separate gel, and biotin signal was detected in the migration front. Peptide sequences are (pS being a phosphorylated serine): 4S-P (1482/86/88/93): biotin-GSG-EGENLPVPETpSLNMpSDpSLLFDpSFSDDY. 4S (1482/86/88/93): biotin-GSG-EGENLPVPETSLNMSDSLLFDSFSDDYS. 1628-P/S1635-P: biotin-GSGTRQNHpSFIWSGApSFDLSPGLQRILDKVSS. S1628/S1635: biotin-GSGTRQNHSFIWSGASFDLSPGLQRILDKVS.
Mass spectrometry analyses
For the identification of Polθ binding partners by mass spectrometry, Polθ immunoprecipitation was performed as described above in 5 mg of nuclear extract of HEK-293T NeonGreen-POLQ knock-in cells. Lysates were incubated with mNeonGreen-Trap Magnetic Agarose beads (Chromotek) for 2 h at 4 °C and beads were washed twice with 150 mM NaCl buffer, once with 300 mM NaCl and three times in 25 mM ammonium bicarbonate. Finally, beads were resuspended in 100 µl 25 mM ammonium bicarbonate and digested by adding 0.2 μg of trypsin/LysC (Promega) for 1 h at 37 °C. Samples were then loaded into custom-made C18 StageTips packed by stacking one AttractSPE disk (SPE-Disks-Bio-C18-100.47.20 Affinisep) and 2 mg beads (186004521 SepPak C18 Cartridge Waters) into a 200 µl micropipette tip for desalting. Peptides were eluted using a ratio of 40:60 MeCN:H2O + 0.1% formic acid and vacuum concentrated to dryness. Peptides were reconstituted in injection buffer (0.3% TFA) before liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis by using an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) as described previously54. Mass spectrometry analysis was performed on three independent experiments. NeonGreen-Trap Magnetic Agarose beads incubated in HEK native cell extracts were used as control. For Polθ phosphorylated sites identification by mass spectrometry, Hela cells expressing GFP–Polθ were blocked 16 h in nocodazole. DMSO or Volasertib was added during the last hour of nocodazole treatment. 5 mg of nuclear extract were incubated with GFP Trap Magnetic Agarose beads (Chromotek) for 2 h at 4 °C and beads were washed once with 150 mM NaCl buffer, twice with 300 mM NaCl and twice with 500 mM NaCl buffer. Finally, beads were resuspended in 100 µl 25 mM Ammonium bicarbonate and digested by adding 0.2 μg of trypsin/LysC (Promega) for 1 h at 37 °C. Samples were then desalted and peptides were reconstituted in injection buffer before LC–MS/MS analysis as described previously in the Polθ binding partners identification. Mass spectrometry analysis was performed on five independent experiments. GFP Trap Magnetic Agarose beads incubated in HeLa native cell extracts were used as controls. For Polθ peptide (4S and 4S-P) binding partners identification by mass spectrometry, peptide pull-downs were performed in 666 μg of nuclear extract of HeLa cells. Lysates were incubated with streptavidin beads-coupled peptide, on a rotating wheel for 2 h at room temperature. After incubation, beads were washed three times using 150 mM NaCl buffer and twice using 300 mM NaCl. Beads were resuspended, digested and desalted, and then peptides were reconstituted in injections buffer before LC–MS/MS analysis, as described for Polθ binding partners identification and by using an Orbitrap Eclipse mass spectrometer. Mass spectrometry analysis was performed on five independent experiments. Pull-downs of streptavidin beads incubated in nuclear extract of HeLa cells were used as control.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE55 partner repository with the dataset identifier PXD029585 (username: email@example.com, password: Tv3OwInu) for Polθ binding partners and project accession: PXD036486 (username: firstname.lastname@example.org, password: GqeYlT33) for Polθ phosphorylated sites quantification and project accession: PXD042167 (Username: email@example.com, password: JdyDITGR) for Polθ peptide 4S-P binding partners identification.
Mitotic DSB repair assays
For mitotic DSB induced by RNP–Cas9
Cells were plated in 4× 10-cm dishes per condition and synchronized by overnight treatment with nocodazole. Next, mitotic cells were collected by shake off, centrifuged and counted. 250,000 mitotic cells were plated in 1 ml of nocodazole containing medium and transfected with an RNP complex targeting one of two AAVS1 loci located on chromosome 19 (AAVS1 gRNA locus 1: TGTGGCCTGGGTCACCTCTA; AAVS1 gRNA locus 2: GGGGCCACTAGGGACAGGAT). A total of 7 pmol of Cas9 was incubated at room temperature with 8.4 pmol of annealed crRNA–tracrRNA in a total volume of 100 µl Opti-MEM for 10 min. RNAimax (4 µl) was then added to the mix, and cells were transfected after 5 min of incubation. Seven hours post transfection, cells were pelleted, and genomic DNA extraction was performed using Nucleospin tissue kit (Macherey-Nagel). A fraction of mitotic cells was kept for phospho-histone H3 (Ser10) immunostaining. PCR around the break was performed using Terra polymerase (Takara) (30 cycles: 95 °C 10 s, 59 °C 20 s, 68 °C 20 s) (locus 1: For2bp GAGGACATCACGTGGTGCAG, Rev2bp CTGCCGTCTCTCTCCTGAGT; locus 2: For8bp GGTGTGTCACCAGATAAGGAATCTGC, Rev8bp GAGCTGGGACCACCTTATATTCC). PCR products were digested for 2 h with HphI (25 U final)(NEB), purified, using Nucleospin PCR cleanup kit (Macherey-Nagel), ligated into pCR4 vector, (4 µl of PCR per ligation) (TOPO-TA cloning kit, ThermoFisher Scientific), and subsequently transformed into NEB5 bacteria (ThermoFisher Scientific). Colonies were subjected to Sanger sequencing using T7 or T3 primer (Eurofins). Repair products missing one or both primer sequences and/or retaining HphI cut site were excluded from analysis. Data were plotted in R using ggplot2.
Extrachromosomal substrate to monitor mitotic end-joining repair
Construction of a DNA repair substrate
To assess mitotic end-joining repair, a DNA repair substrate was designed to mimic resected DNA ends containing four microhomologies, as previously described29. The repair substrate was built by ligating a PCR product (GFP sequence) to DNA oligonucleotide duplexes on both sides. The PCR product (747 bp) was amplified from a GFP sequence (PCAG vector, Addgene) using the following oligonucleotide sequences (Fwd core GFP: CAAGTGGTCTCAGACTGTGAGCAAGGGCGAGGAGCTG, Rev core GFP: GCCGAGGTCTCCGTCAGCTTGTACAGCTCGTCCATGCCGAG), digested by the BsaI-HF v2 enzyme (NEB) to reveal 4 nucleotides (nts) complementary to the DNA oligonucleotide duplexes and finally, purified (Gel and PCR cleanup kit Macherey-Nagel). In parallel, two oligonucleotide duplexes (1 and 2) with ~70 nts overhangs were made after annealing (95 °C 10 min, 25 °C 30 min) of the following oligonucleotides. Oligonucleotides for duplex 1 (17 nt dsDNA, 69 nt ssDNA): oligo 1-A, CATCGCTTAGCTGTATA; oligo 1-B, TGACTATACAGCTAAGCGATGCTCTCACCGAGCGTATCTGCTGGG TTGTGGATGAATTACATATGCTGGGAGAACCAAGATTGGGCAGTT. Oligonucleotides for duplex 2 (15 nt dsDNA, 71 nt ssDNA): oligo 2-A, CTCACACCCATCTCA; oligo 2-B: AGTCTGAGATGGGTGTGAGAGTGAAGATCCTCACCTTCGGAGTACTCCTTCTTTTGACCATTGATACGATACTTCTCAGCCGAGCTGCTT.
Oligonucleotide duplex 1 and duplex 2 (100 pmol of each) were ligated to the core GFP PCR (10 pmol) using T7 ligase (NEB). The resulting duplex 1–GFP–duplex 2 DNA fragment was then phosphorylated (polynucleotide kinase, NEB) and purified (Nucleospin PCR Cleanup, Macherey-Nagel). In cells, two duplex 1–GFP–duplex 2 DNA fragments could anneal through sequence microhomology (four nucleotides in bold in oligonucleotide sequences) forming DNA end-joining repair product.
DNA repair substrate transfection and measurement of end-joining repair efficiency
Cells (interphase or mitotic) were transfected with 150 ng of duplex 1–GFP–duplex 2 substrate using RNAimax reagent and incubated for 1.5 h. Cells were then collected, centrifuged, and resuspended in 2 ml benzonase buffer (50 mM Tris-HCl, 1 mM MgCl2, 25 U benzonase), with the addition of nocodazole for mitotic cells, and incubated for 30 min at 37 °C to remove extracellular DNA. DNA was purified using the NucleoSpin tissue kit (Macherey-Nagel).
To measure DNA repair efficiency, a qPCR was performed both on the GFP sequence (to control for the DNA repair substrate transfection) as well as on potential end-joining repair products. Mitotic end-joining repair efficiency was calculated using ΔΔCt between GFP qPCR and end-joining qPCR using SYBR Green Master Mix (ThermoFisher Scientific).
Primers used for qPCR. GFP: For qPCR core, CTCACACCCATCTCAGACTGTGAGCAA; Rev qPCR GFP, CAGCTTGCCGTAGGTGGCATCG. End joining: For qPCR EJ, GGGTTGTGGATGAATTACATATGCTGG; Rev qPCR EJ, CGGAGTACTCCTTCTTTTGACCATTGATAC.
All oligonucleotides and primers were ordered from Eurofins.
Cells grown on coverslips were fixed for 15 min in 2% paraformaldehyde, permeabilized for 10 min in 0.5% Triton X-100, blocked for 30 min in BSA 5%, 0.1% Triton X-100, immunostained for 1 h with primary antibodies, 45 min with secondary antibodies (Highly Cross-Adsorbed Secondary Antibodies Alexa Fluor, ThermoFisher Scientific) and mounted Vectashield mounting medium containing DAPI (Vector Laboratories). The same protocol was applied to mitotic cells, which were collected by shake off and centrifuged onto glass slides using a cytospin centrifuge (400g, 5 min) before fixation. For staining with PCNA or TOPBP1 antibodies, cells were fixed with ice-cold methanol for 20 min on ice before blocking and immunostaining. Biotin azide-EdU click-it reaction was performed after fixation and permeabilization by incubating cells with a mix of Picolyl biotin, CuSO4, THPTA (Jena Bioscience) and sodium ascorbate in PBS for 30 min in the dark followed by an incubation with streptavidin conjugated Alex fluor 647 antibody (ThermoFisher Scientific) during 1 h at room temperature. Images were taken at 60× or 100× magnification using Upright Spinning disk Confocal Microscope (Roper/Zeiss) or at 40× magnification using DeltaVision (Deconvolution) Image Restoration Microscope. All images were taken with a Z-stack of 0.4 μM on 8 μM range around focus and projected using maximum intensity projection. Images were analysed using the Fiji software56. All scale bars represent 5 μm unless stated otherwise.
Automatic microscopy for screening Polθ foci formation
The Human DDR siRNA library comprising 1,746 single siRNAs representing 582 human genes (3 siRNAs per gene) was obtained from ThermoFisher Scientific (A30089). For controls, siRNAs targeting GL2, kinesin family member 11 (KIF11) and GM130 were used to assess transfection efficacy and induced cell toxicity. FANCD2 siRNA (GGAGAUUGAUGGUCUACUATdT, defined as FANCD2-ctrl in Supplementary Table 1) was used as a positive control inhibiting Polθ foci formation. RPE-1 cells expressing GFP–Polθ (250 cells per well in 384-well plates) were grown overnight. siRNAs were incubated 15 min with 0.05 μl Interferin reagent (Polyplus transfection) and Opti-MEM (ThermoFisher Scientific) prior to being transferred into each 384-well plate wells, to obtain a 10 nM final concentration. Cells were irradiated 72 h after transfection, at a dose of 5 Gy (226 s) with the CIXD irradiator (RadeXp platform, Institut Curie) and incubated at 37 °C, 5% CO2 for 2 h. Cells were fixed in 3% paraformaldehyde solution (Sigma-Aldrich, HT5014) for 15 min with MAP-C (Titertek), washed once in 1× PBS and quenched in 50 mM NH4Cl solution for 10 min. Cells were then blocked with 1% BSA solution for 15 min and permeabilized with 0.5% Triton X-100 for 5 min. Nuclei were stained with 0.2 μg ml−1 Hoechst 33342 (DNA marker,1:500, Sigma, 14533). Image acquisition was performed using the INCell analyser 6500 HS automated system (GE Healthcare) at a 40× magnification (Nikon 40×/0.95, Plan Apo), using the same exposure time for all plates in the experiment and across replicate experiments. Plates were loaded onto the INCell system with Kinedx robotic arm (PAA). 16-bit images of six fields in each well were acquired, each containing channels for Hoechst 33242 (DAPI, wave 1) and intrinsic GFP–Polθ (FITC, wave 2). Computational image processing operations were performed using INCell Analyser 3.7 Workstation software (GE Healthcare). Polθ was then identified as organelles in the nuclei area and quantified by the average intensity of pixels within the defined organelles region. A binary threshold was applied to discriminate cells containing at least of 5 Polθ foci resulting in “% cells multi-foci” (Supplementary Table 1). For each feature or phenotypic class depicted from the applied cell threshold described, plate positional effects were corrected using Tukey’s two-way median polishing57,58, which iteratively subtracts row, column and well median computed across all plates within a replicate. Corrected values were normalized as follows: sample median and median absolute deviation (MAD) were calculated from the Interferin-treated population of each internal plate data points (named as Ref pop) and used to compute robust Z-scores59 (RZ scores), according to the formula: RZ score = (compound value – median (reference population))/(1.14826 × MAD (reference population)). Where the compound value corresponds to the treated well and MAD is defined as the median of the absolute deviation from the median of the Interferin-treated population. A gene was identified as a hit if |RZ score| >2 points in the same direction for at least 2 siRNAs targeting the same gene in at least 2 replicate experiments. The screen was done in three biological replicates. For each siRNA, the median RZ score of each of the three replicate experiments is shown in Fig. 1a.
Time-lapse video microscopy
To determine Polθ foci formation during cell cycle progression, cells expressing GFP–Polθ and PCNA-mCherry were plated on 35 mm Ibidi μ-Dishes (Ibidi, 81156) and imaged every 10 min for 16 h on an Inverted Eclipse Ti-E (Nikon) microscope equipped with a stage-top incubator and CO2 delivery system, 40× (1.4 NA) oil objective, a Spinning disk Yokogawa CSU-X1 unit integrated in Metamorph software by Gataca Systems equipped with Camera EMCCD. Images were mounted in SoftWoRx and analysed using the Fiji software (1.53c). Aphidicolin (1.6 μM unless stated otherwise) was added to the cells for 24 h and removed 3 h before acquisition. For nuclear bodies resolution, palbociclib was added 30 min before starting acquisition.
To determine mitotic timing by phase-contrast video microscopy, 250,000 cells were seeded in 35 mm Ibidi μ-Dishes (Ibidi, 81156) 24 h before imaging. Cells were arrested in RO3306 for 4 h with or without IAA (Polθ depletion) and released in normal growth media before imaging. Cells were immediately imaged every 5 min for 16 h, at 20× using an inverted video microscope (Deltavision) equipped with CoolSNAP HQ2 camera controlled by Softworx software. Cell rounding (mitotic entry) and flattening (mitotic exit) were used as landmarks to calculate mitotic timing. In addition, mitotic timing was also measured from condensation to anaphase. In that case, DNA was stained 30 min prior acquisition by incubating cells with SiR-DNA (250 nM, Spirochrome) and acquisition was performed an Inverted Eclipse Ti-E (Nikon) microscope equipped with a stage-top incubator and CO2 delivery system, 40× (1.4 NA) oil objective, equipped with Intensilight Lamp Hg 130W (Nikon) and Camera CoolSNAP HQ (Photometrics). All images were mounted in SoftWoRx and analysed using the Fiji software (1.53c).
Clonogenic survival assays
For mitotic cells: cells were blocked in G2 using RO3306 (5 μM), released, then blocked in mitosis by nocodazole and subsequently seeded for clonogenic survival. Polθ-mAID cells were blocked 4 h in RO3306 (5 μM) with or without IAA (500 μM) and released in nocodazole with or without IAA for 1 h. In the case of PARPi treatment, cells were blocked 16 h in RO3306 (5 μM) and released in nocodazole with or without Rucaparib (1 μM) for 1 h. After shake off, mitotic cells were washed twice with warm media, counted and seeded for clonogenic survival. For interphase cells: growing cells were treated with IAA (500 μM), or Rucaparib (1 μM) for indicated time, washed twice with warm media and counted. For clonogenic assay upon siRNA transfection, 150,000 cells were plated per well of a 6-well plate and transfected the same day with indicated siRNA. The day after, cells were washed twice, counted and seeded for clonogenic survival. For each condition, two concentrations of cells (BRCA2−/− cells: 500 and 2000 cells; wild-type cells: 50 and 150 cells) were seeded per well of a 6-well plate. Finally, 10 to 14 days after seeding, colonies were stained with blue methylene (Sigma, 1808) for 2 h at room temperature, washed in water, air-dried overnight and scored manually.
Cells were blocked in G2 with RO3306 (5 μM) for 16 h, released, then blocked in mitosis by nocodazole and subsequently treated with Rucaparib for 1 h. After shake off, mitotic cells were centrifuged and subsequently resuspended in a pre-warmed hypotonic solution (0.075 M KCl) and maintained at 37 °C for 15 min. Cells were then centrifuged, and pellets were fixed in fresh methanol and stored at −20 °C. After metaphase spreading, air-dried slides were stained with a Gurr/Giemsa/Acetone solution (Gibco, 10582013; Giemsa (KaryoMAX Giemsa stain solution, Gibco, 10092013)) for 5 min and then rinsed twice with Gurr buffer. Slides were air-dried and neo-mounted with anhydrous mounting medium (Merck, 109016). Images were acquired at 100× magnification using DeltaVision (Deconvolution) Image Restoration Microscope.
A series of five models were calculated for the complex between TOPBP1 BRCT7-8 and the Polθ peptide E1472–Y1498 in its non-phosphorylated and phosphomimetic (phosphorylated serines being replaced by glutamic acids) states. All models gave a similar representation of the interface, characterized by an iPTM score of 0.36–0.51 and 0.48-0.61 in the case of the non-phosphorylated and phosphomimetic peptides, respectively. The weak effect of phosphomimetic mutations on the AlphaFold calculations is consistent with the poor sensitivity of AlphaFold to single mutations.
Data are shown as mean ± s.e.m. except in violin plots, which show median with quartiles, and box plots, which show median with minimum and maximum values. For all statistically significant comparisons P values are displayed on figures and statistical analyses are specidfied in the figure legends. Sample sizes and number of biological replicates are specified in the figure legends. Blinding and randomization were not employed in this study. All statistical analyses were performed with GraphPad Prism 9, except chi-square tests, which were performed using Chi-Square Test Calculator (https://www.socscistatistics.com/tests/chisquare2/default2.aspx).
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