MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks

Cell lines, cell culture conditions, and treatments

All cell lines were grown in a sterile cell culture environment and were routinely tested for mycoplasma contamination. U2OS (U2OS, human bone osteosarcoma cell line; ATCC) cells were cultured in DMEM. All cell culture medium was supplemented with 10% FBS, 2 mM L-glutamine and penicillin-streptomycin antibiotics. Cell lines were grown under standard cell culture conditions in CO₂ incubators (37°C; 5% CO₂). Genetically modified U2OS cell lines U2OS ΔMDC1, U2OS ΔMDC1 + GFP-MDC1 WT and U2OS ΔMDC1 + GFP-MDC1-AQXF were described (Leimbacher et al, 2019). Stably transfected U2OS cell lines were cultured in the presence of 400 μg/ml Geneticin (G418). Irradiation of cells was performed in an YXLON, Y.SMART583 custom-made X-ray device and an IR dose of 3 Gy followed by 3 h of incubation at 37°C was applied. Laser microirradiation of cells was carried out using a MMI CELLCUT system containing an ultraviolet (UV) A laser of 355 nm where energy output was set to 50%, and each cell was exposed to laser beam for <300 ms, following incubation for 30 min at 37°C (described in Eid et al [2010]).

Cloning

To generate the GFP-PELI1 expression construct, the full-length PELI1 cDNA sequence was synthesized by BioCat GmbH and pcDNA4/TO-Strep-HA-GFP-Treacle plasmid, which was described (Larsen et al, 2014), was digested by KpnI and ApaI restriction enzymes to delete Treacle’s coding sequence. PCR with Phusion High-Fidelity DNA Polymerase was performed to amplify PELI1 cDNA sequence with primers partially overlapping the cohesive ends of the above-mentioned digested pcDNA4/TO vector. Sequences of these PCR primers were, forward: 5′-ATGAGCTGTACAAGGGTACCATGTTTTCTCCTGATCAAG-3′, and reverse: 5′-CAGCGGGTTTAAACGTCTAGATTAGTCTAGAGGTCCTTGA-3′. The amplified PELI1 cDNA sequence was cloned into the KpnI-ApaI-digested pcDNA4/TO-Strep-HA-GFP vector by In-Fusion HD Cloning (Takara) to produce plasmid pcDNA4/TO-Strep-HA-GFP-PELI1. The construct was transformed into Stellar Competent E. coli cells (Takara) for further colony sequence verification by Sanger sequencing and plasmid DNA extraction. To generate the RFP-PELI1 expression construct, the RFP coding sequence was amplified by PCR from vector pTurboRFP-B (Evrogen), with Phusion High-Fidelity DNA Polymerase (NEB) using the following primers, forward: 5′-AGCACAGTGGCGGCCATGAGCGAGCTGATCAAGGA-3′, and reverse: 5′-GAGAAAACATGGTACTCTGTGCCCCAGTTTGCTAGG-3′. The previously produced pcDNA4/TO-Strep-HA-GFP-PELI1 plasmid was digested with NotI-HF (NEB) and KpnI-HF (NEB) restriction enzymes to delete the GFP coding sequence. The amplified RFP sequence was cloned into the NotI-KpnI–digested pcDNA4/TO-Strep-HA-PELI1 vector by In-Fusion Snap Assembly (Takara) to produce plasmid pcDNA4/TO-Strep-HA-RFP-PELI1. The construct was transformed into Stellar Competent cells for sequence verification by Sanger sequencing and further plasmid DNA extraction. To generate the GST-PELI2-FHA expression construct, the full-length PELI2 cDNA sequence was synthesized by BioCat GmbH. The FHA domain of the PELI2 protein (comprising aminoacids 1–275) was amplified by PCR from the full-length cDNA sequence with Phusion High-Fidelity DNA Polymerase using the primers forward: 5′-CGCGGATCCATGTTTTCCCCTGGCCAGGAGGAAC-3′, and reverse: 5′-CCGGAATTCCTGCCGGAGGGCTTCTATGTGCTTC-3′, which contained BamHI and EcoRI restriction sites, respectively, as overhangs. The PCR product was digested with BamHI-HF (NEB) and EcoRI (NEB) restriction enzymes as well as the bacterial expression vector pGEX-4T-3 (containing the GST-tag), followed by ligation with T4 DNA Ligase (NEB). This resulted in the GST-PELI2-FHA construct, which was transformed into Stellar Competent cells for Sanger sequencing and plasmid DNA extraction. To generate the HA-Flag-MDC1(800)-AQXF expression construct, PCR with Q5 High-Fidelity 2X Master Mix (NEB) was performed using 15-nucleotide overlapping primers to amplify the AQXF mutant sequence (forward: 5′-GAAGACAACTATGGTGATTCTGAAGATCTGGACCTACAAG-3′, and reverse: 5′-AGGTGGAGACAGGCAAGGTCCATAGGCCTCAAGGTG-3′) and the plasmid backbone sequence (forward: 5′-TGCCTGTCTCCACCTAGGG-3′, and reverse: 5′-ACCATAGTTGTCTTCAGAGTCC-3′), deleting the WT TQXF cluster in the process. These original expression plasmids were pcDNA3.1-GFP-MDC1-AQXF which was a gift from Stephen Jackson’s laboratory and was described elsewhere (Kolas et al, 2007), and pcDNA3.1-HA-Flag-MDC1(800)-WT which was described in Jungmichel and Stucki (2010). The two resulting DNA fragments were then fused by In-Fusion Snap Assembly to produce plasmid pcDNA3.1-HA-Flag-MDC1(800)-AQXF. The construct was transformed into Stellar Competent cells for sequence verification by Sanger sequencing and further plasmid DNA extraction.

Cell transient transfection with DNA

Plasmid DNA was transiently transfected using jetOPTIMUS DNA Transfection Reagent (Polyplus) in six-well plates following the standard protocol provided by the manufacturer. Cells were then incubated for 24 h before proceeding with further analysis.

Expression and purification of recombinant proteins for biochemical analysis

For recombinant human PELI2-FHA expression in bacteria and further purification, the GST-tagged PELI2-FHA construct was transformed into E. coli BL21(DE3)pLysS Competent Cells (Promega). 100 μl of the transformation mixture were used to inoculate a 500 ml flask containing 100 ml of Luria–Bertani broth (LB) supplemented with carbenicillin (100 μg/ml) and chloramphenicol (34 μg/ml), and inoculated culture was incubated overnight at 27°C without shaking. On the next day, the overnight culture was grown at 37°C in an orbital shaking incubator until the optical density at 600 nm reached 0.6–0.8, moment when culture was removed from the incubator and rapidly cooled on ice to 25°C. Recombinant protein expression was induced by the addition of 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubation of the culture for a further 4 h at 25°C shaking at 220 rpm was performed. Cells were then harvested by centrifugation, and the resulting pellet was stored at −80°C until required. Bacterial pellet was lysed through repeated freezing (down to −80°C) and thawing and by resuspension in 5 ml of ice-cold lysis/wash buffer (25 mM HEPES pH 7.5, 1 M NaCl, 5% glycerol) supplemented with complete protease inhibitor cocktail (Roche), followed by vigorous sonication on ice delivered by a Jencons Ultrasonic Processor (15 s bursts at 70% amplitude). Triton X-100 was added to the lysate to a final concentration of 1% before incubation for 45 min rolling at 4°C and clearance by centrifugation for 30 min at 15,500 rcf and 4°C. After this step, the cleared extract was loaded onto a 5 ml Bio-Scale Mini Profinity GST Cartridge (Bio-Rad) for GST affinity purification on an NGC Quest 10 chromatography system (Bio-Rad). For this run, lysis/wash buffer and elution buffer pH 7.5 (25 mM HEPES pH 7.5, 1 M NaCl, 5% glycerol, 30 mM Glutathion) were used. Expression and purification levels of the purified protein fraction were tested on SDS–PAGE followed by gel staining with QuickBlue Protein Stain (LubioScience) for protein visualization, and fractions with reasonable protein content and purity were pooled. Dialysis of pooled fractions was performed overnight at 4°C with slow stirring against Buffer A (50 mM Tris–HCl pH 7.5, 50 mM NaCl, 10% glycerol, 1 mM DTBA). Next, pooled and dialysed fractions were loaded onto a 1 ml ENrich Q 5 × 50 Column (Bio-Rad) for ion-exchange purification on the NGC system (Bio-Rad). For this second run, Buffer A and Buffer B (50 mM Tris–HCl pH 7.5, 1 M NaCl, 10% glycerol, 1 mM DTBA) were used. Finally, the purified protein fraction was collected. Protein concentration was measured after every purification step.

Expression and purification of recombinant proteins for biophysical and X-ray structure analysis

The regions corresponding to the PELI1 and 2 FHA domains (residues 13–273 and 15–275, respectively) were cloned into the pGEX-6P1 vector using BamHI/XhoI restriction sites and expressed as a 3C protease-cleavable N-terminal GST fusion protein in BL21 (DE3) cells (NEB). A 20 ml LB overnight culture was inoculated into 1L of LB medium supplemented with 100 μg/ml ampicillin to an OD600 of 0.05 and incubated at 37°C, 250 rpm until an OD600 of 0.6 was reached, whereupon expression was induced with 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) and overnight incubation at 18°C. Overnight cultures were harvested for 20 min at 4,000g, 4°C and resuspended in 20 ml lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.2 mg/ml lysozyme, 20 μg/ml DNase, and 1x complete, EDTA-free protease inhibitor cocktail tablet) before sonication-mediated cell lysis. Insoluble material was removed by centrifugation (21,000g, 20 min) and the GST-FHA fusion proteins were purified from the clarified lysate containers using 1 ml bed volume of pre-equilibrated Glutathione Sepharose 4B beads. After washing with 20 column volumes of Pellino FHA buffer (50 mM HEPES pH 7.5, 150 mM NaCl), the protein was cleaved overnight at 4°C from GST with 1% (vol/vol) GST-3C protease and the flow-through was further purified by gel filtration using a HiLoad 16/60 Superdex 200 pg (Cytiva) column. Purified Pellino FHA domains were concentrated to 50 mg/ml with a 10 kD molecular weight cut-off concentrator (Sartorius).

ITC

For ITC analysis protein and peptides were buffer exchanged into 25 mM HEPES pH 7.0, 200 mM NaCl, 1 mM TCEP. Titrations were carried out using a Microcal PEAQ-ITC (Malvern) using 13 injections of peptide at a typical syringe concentration of 160 μM (1 × 0.4 μl, 12 × 3.0 μl) and FHA concentration of 16 μM in the cell. Data were analyzed with a single-site binding model using Microcal PEAQ-ITC software. Peptide sequences are: MDC1 pT699–AYDLQApTQCFA, MDC1-pT719–ATEDEPpTQAFA, MDC1-pT752–AYEVLApTQPFC, MDC1-pT765–AYEDSEpTQPFD, IRAK-pT141(+3Y)–AYSSASpTFLYP, γH2AX–GGKATQApSQEY. All peptides were synthesises with N-terminal acetylation and a C-terminal amide. MDC1 pT699 and pT719 were made with alanine substitutions at the pT+4 position (Leu and Met, respectively) to aid solubility and monodispersity.

Crystallographic analysis

For crystallisation, the MDC1-pT765 phosphopeptide with the Pellino 1FHA fragment in a 1,170 μM:754 μM stoichiometric excess in Pellino FHA buffer. Crystallisation using the sitting drop vapour diffusion method was conducted at 18°C and was achieved by mixing the 200 nl of the Pellino FHA:MDC1 phosphopeptide complex in a 1:1 ratio with 200 nl of mother liquor. Initial trials were performed using the Proplex crystal screen (Molecular Dimensions) and crystals grew after 1 wk in 1.5 M ammonium sulphate, 0.1 M Tris pH 8. Additional trials were performed using the Ångstrom Additive Screen (Molecular Dimension) in a 1:9 ratio with 1.67 M ammonium sulphate, 0.11 M Tris pH 8.0, whereby single crystals grew from drops consisting of 1.5 M ammonium sulphate, 0.1 M Tris pH 8, 20% (vol/vol) PEG400. Crystals were cryoprotected by soaking in mother liquor supplemented with 25% (vol/vol) PEG400. Diffraction data were collected at the Diamond Light Source beamline I04 at a wavelength of 0.9795 Å and processed using autoPROC (Vonrhein et al, 2011), and the structure was phased by molecular replacement using PHASER from the CCP4i suite with a search model comprised of coordinates from the human Pellino 2FHA structure (PDB accession code 3EGA; Lin et al, 2008). The model was manually rebuilt and adjusted in Coot and refined using PHENIX (Table 2). Coordinates for the Pellino-phosphopeptide complex have been deposited with the Protein Data Bank: Accession code 9IF9.

Peptide pull-downs

Streptavidin-coupled Dynabeads MyOne Streptavidin T1 (Invitrogen) were washed four times with PBS pH 7.4. To conjugate the Dynabeads with biotinylated peptides (21st Century Biochemicals, Inc.), 30 μl of beads were incubated with 0.2 μmol of each peptide with end-over-end rotation for 90 min at RT. Conjugated Dynabeads were then washed once with PBS and 3 × 2 min with 0.1% BSA in PBS. HeLa nuclear extracts (Ipracell) were diluted 1:1 in 2X Dilution buffer (100 mM NaCl, 10 mM NaF, 0.2 mM EDTA, 0.4% Igepal CA-630, 20 mM HEPES-KOH pH 7.4) supplemented with complete protease inhibitor cocktail (Roche), and cleared by centrifugation at 16,000g for 15 min at 4°C. Purified GST-PELI2-FHA recombinant protein was also diluted in 2X Dilution buffer containing protease inhibitors. MDC1-[p]TQXF peptide-conjugated Dynabeads were incubated either with clarified HeLa nuclear extracts for 4 h or with purified GST-PELI2-FHA for 5 h. H2AX-[p]S139 peptide-conjugated Dynabeads were incubated with clarified HeLa nuclear extracts for 4 h. In all the cases, incubation was performed with end-over-end rotation at 4°C. Beads were washed three times with Peptide pull-down buffer (50 mM NaCl, 50 mM KCl, 5 mM NaF, 0.1 mM EDTA, 0.2% Igepal CA-630, 10% glycerol, 20 mM HEPES-KOH pH 7.4) supplemented with complete protease inhibitor cocktail (Roche). Then, they were either resuspended in Peptide pull-down buffer and brought to the Functional Genomics Center of the University of Zurich (FGCZ) for further mass spectrometry, or elution was carried out in 2X SDS sample buffer (100 mM DTT, PLD001; Geneaid) for SDS–PAGE. Peptide sequences were as follows: Biotin-SGS-DSEDLDLQA[p]TQCFLE-NH₂ (MDC1-[p]T699), Biotin-SGS-AVQSMEDEP[p]TQAFML-NH₂ (MDC1-[p]T719), Biotin-SGS-LDEPWEVLA[p]TQPFCL-NH₂ (MDC1-[p]T752), Biotin-SGS-CLRESEDSE[p]TQPFDT-NH₂ (MDC1-[p]T765), Biotin-SGS-TVGPKAPSGGKKATQA[p]SQEY-OH (H2AX-[p]S139).

Immunoprecipitations

293FT cells were grown in six-well plates overnight and then transfected with pcDNA3.1-HA-Flag-MDC1(800) WT or AQXF mutant plasmids. After 24 h, cells were treated with 3 Gy of IR as described above. To prepare cell extracts for Flag immunoprecipitation, cells were washed in cold PBS buffer pH 7.45, harvested with a cell scraper and centrifuged at 500g for 3 min. PBS was then discarded and cells were resuspended with an appropriate volume of IP lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with complete EDTA-free protease inhibitor cocktail (Roche) and 25 U/ml Benzonase nuclease (Sigma-Aldrich). Cells were incubated on ice for 30 min and vortexed every 10 min to dissolve cell clumps. After incubation, lysates were cleared by centrifugation at 13,200 RPM for 15 min at 4°C and transferred into fresh tubes for protein quantification. Anti-FLAG M2 affinity gel (A2220; Sigma-Aldrich) was added to fresh tubes, centrifuged at 5,000g for 30 s and washed 3 × 2 min in cold TBS (50 mM Tris–HCl pH7.4, 150 mM NaCl) with end-over-end rotation at 4°C. Then, 20 μl of packed gel were added to 2 mg of the soluble cell extract and samples were incubated for 2 h with end-over-end rotation at 4°C. After incubation, the unbound fraction was discarded, and immunoglobulin-antigen complexes were washed 3 × 15 min in cold TBS with end-over-end rotation at 4°C. After the last wash, protein complexes were eluted using 50 μl of 2X SDS sample buffer (100 mM DTT; Geneaid), denatured at 95°C for 5 min, and vortexed before loading on the gel.

Mass spectrometry

MDC1-pTQXF and H2AX-pS139 peptide pull-downs from HeLa nuclear extracts were performed as described above, followed by mass spectrometry analysis. After the last wash, precipitated material was resuspended in peptide pull-down buffer and subjected to on-bead trypsin digestion according to the following protocol. Dynabeads were washed twice with 50 μl of digestion buffer (10 mM Tris, 2 mM CaCl₂, pH 8.2). Then, the buffer was discarded and beads were resuspended in 45 μl of digestion buffer supplemented with 5 μl of trypsin (100 ng/μl in 10 mM HCl). pH was adjusted to 8.2 by adding 1 M Tris pH 8.2. Digestion was carried out at 37°C for 5 h shaking at 800 rpm. Supernatants were collected, and peptides were extracted from beads using 150 μl of 0.1% trifluoroacetic acid (TFA) and 50% acetonitrile (ACN). Digested samples were dried and reconstituted in 20 μl ddH2O + 0.1% formic acid before performing liquid chromatography-mass spectrometry analysis (LC-MS/MS). For the analysis, 1 μl was injected on a nanoACQUITY UPLC system coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). MS data were processed for identification using the Mascot search engine (Matrixscience), and the spectra were searched against the Swissprot protein database. Protein and peptide identification results were visualized in Scaffold Proteome Software (Proteome Software, Inc., https://www.proteomesoftware.com/products).

SDS–PAGE and Western blotting

SDS–PAGE of the peptide pull-down samples and the immunoprecipitated samples was performed using 4–20% Mini-PROTEAN TGX Stain-free Protein Gels (Bio-Rad). As a reference to molecular weights, Prestained Protein Ladder 245 kD (Geneaid) was also loaded on the gel. SDS–PAGE of the MDC1-[p]TQXF peptide pull-down from purified GST-PELI2-FHA recombinant protein was followed by gel staining with QuickBlue Protein Stain (LubioScience) for protein visualization. SDS–PAGE of the MDC1-[p]TQXF and H2AX-[p]S139 peptide pull-downs from HeLa nuclear extracts and the Flag immunoprecipitation were followed by Western blotting, which was performed using the Trans-Blot Turbo Transfer System (Bio-Rad) on 0.2 μm nitrocellulose membranes (Bio-Rad). Membranes were blocked for at least 2 h with 5% milk in TBS-Tween. Primary antibodies were diluted in 2.5% milk in TBS-Tween and incubation was performed at 4°C overnight. Blots were then washed 3 × 5 min with TBS-Tween. Secondary antibodies were diluted in 2.5% milk in TBS-Tween and incubation was performed for 1 h at RT. Blots were washed again 3 × 5 min with TBS-Tween and then developed with Amersham ECL Prime Western blotting Detection Reagent (Cytiva), and image acquisition was performed on a ChemiDoc MP Imaging System (Bio-Rad). The following primary antibodies were used at the indicated dilutions: anti-RNF8 (1:5,000; Rabbit), anti-PELI1/2 (Rabbitpolyclonal, 1:1,000, ab13812; Abcam), anti-MDC1 (Rabbit polyclonal, 1:5,000, ab11171; Abcam), anti-flag M2 (Mouse monoclonal, 1/1,000, F1804; Sigma-Aldrich). The following secondary antibodies were used: ECL HRP-conjugated donkey anti-rabbit (1:10,000, NA934; Cytiva), ECL HRP-conjugated sheep anti-mouse (1:10,000, NA931; Cytiva).

Immunofluorescence

U2OS WT and ΔMDC1 cells were grown on glass coverslips (Thermo Fisher Scientific Menzel) overnight and then transfected with pcDNA4/TO-Strep-HA-GFP-PELI1 plasmid. After 24 h, coverslips were transferred into two-well chamber imaging slides (Ibidi) and treated with 10 μM 5-bromo-2′-deoxyuridine (BrdU) for 24 h before being subjected to laser microirradiation as described above. U2OS ΔMDC1 cells stably expressing either GFP-MDC1 WT or GFP-MDC1-AQXF were grown on glass coverslips overnight and then transfected with pcDNA4/TO-Strep-HA-RFP-PELI1 plasmid. After 24 h, cells were either treated with BrdU and laser microirradiation on Ibidi slides or with ionizing radiation as described above. Microlaser-treated cells were then washed twice with PBS and irradiated cells were washed once with PBS, before fixation with 4% buffered formaldehyde for 12–15 min at RT. Formaldehyde was discarded and cells were washed three times with PBS at RT, subsequently permeabilized for 5 min with 0.3% Triton X-100 in PBS, washed again three times with PBS and incubated with blocking buffer (10% FBS in PBS) for at least 1 h at RT. Primary antibodies were diluted in 10% FBS in PBS and incubation was performed at 4°C overnight. Coverslips were then washed 3 × 10 min with PBS. Secondary antibodies were diluted in 5% FBS in PBS and incubation was performed for 1 h at RT in the dark. After washing twice with PBS, coverslips were mounted on glass microscopy slides (Thermo Fisher Scientific) with VECTASHIELD PLUS Antifade Mounting Medium containing 0.5 μg/ml DAPI (Vector Laboratories). For the GFP-PELI1-transfected U2OS WT and ΔMDC1 cells, the following primary antibodies were used at the indicated dilutions: anti-γH2AX (Mouse monoclonal, 1:500, 05-636-I, RRID: AB_2755003; Sigma-Aldrich), anti-MDC1 (Rabbit polyclonal, 1:200, ab11171, RRID: AB_297810; Abcam). Also, these secondary antibodies were used: Alexa Fluor 568 Goat Anti-Mouse IgG H&L (1:1,000, A11031, RRID: AB_144696; Thermo Fisher Scientific), Alexa Fluor 700 Goat Anti-Rabbit IgG H&L (1:500, A21038; RRID: AB_2535709; Thermo Fisher Scientific). For the RFP-PELI1-transfected U2OS GFP-MDC1 WT and U2OS GFP-MDC1-AQXF cells, the following primary antibody was used at the indicated dilution: anti-γH2AX (Rabbit monoclonal, 1:400, 9,718; Cell Signaling Technology), and this secondary antibody: Alexa Fluor 700 Goat Anti-Rabbit IgG H&L.

In situ proximiy-ligation assay (PLA)

U2OS WT and ΔMDC1 cells were grown on glass coverslips in six-well plates at a density of 2 × 10⁵ cells per well (and two coverslips per well) for transfection with pcDNA4/TO-Strep-HA-GFP-PELI1 plasmid on the next day. After 24 h, coverslips were transferred into 24-well plates and subjected to ionizing radiation as described above. Cells were then washed once with PBS for 5 min on ice before fixation with ice-cold methanol for 12 min also on ice. Methanol was discarded and cells were washed 3 × 5 min with PBS at RT. All the following steps were performed using the Duolink PLA Reagents (DUO92001, DUO92005, DUO92008, DUO82049; Sigma-Aldrich) and according to the manufacturer’s protocol. The blocking solution was applied for 1 h in a humidified incubator at 37°C, followed by antibody incubation overnight at 4°C under humid conditions. The following antibodies were used at the indicated dilutions: anti-MDC1 (Mouse monoclonal, 1:300, ab50003, RRID: AB_881103; Abcam), anti-HA (Rabbit polyclonal, 1:250, ab9110, RRID: AB_307019; Abcam). Next, coverslips were washed 2 × 5 min with wash buffer A at RT and the PLUS and MINUS PLA probes were applied for 1 h at 37°C in a humidified incubator. Cells were washed again 2 × 5 min with wash buffer A at RT and incubated with the ligation solution for 30 min in a humidified incubator at 37°C. After washing the coverslips 2 × 5 min with wash buffer A at RT, the amplification solution was applied for 100 min at 37°C under humid conditions. Final washes were carried out at RT, 2 × 10 min with wash buffer B and for 1 min with 0.01X wash buffer B. Coverslips were then mounted on glass microscopy slides using VECTASHIELD PLUS Antifade Mounting Medium with DAPI.

Widefield and confocal microscopy

Widefield images of the PLA experiment were acquired with a Leica DMI6000B inverted fluorescence microscope, equipped with Leica K5 sCMOS fluorescence camera (16-bit, 2,048 × 2,048 pixel, 4.2 MP) and Las X software version 3.7.2.22383. An HC Plan Apochromat 40X/0.95 phase contrast air objective and Plan Apochromat 100X/1.40 phase contrast oil-immersion objective were used for image acquisition. For triple-wavelength emission detection, we combined DAPI with EGFP and Texas Red.

Confocal images of the microlaser and IRIF experiments were acquired with a Leica SP8 confocal laser scanning microscope coupled to a Leica DMI6000B inverted stand, with a 63X, 1.4-NA Plan Apochromat oil-immersion objective. For quadruple-wavelength emission detection, we combined DAPI with EGFP, Alexa Fluor 568 or TurboRFP and Alexa Fluor 700. The sequential scanning mode was applied, and the number of overexposed pixels was kept at a minimum. 5 z-sections were recorded with optimal distances based on Nyquist criterion, a resolution of 512 × 512 pixels and 8-bit depth.

For optimal representation in figures, maximum intensity projections were calculated using Fiji (Schindelin et al, 2012). Unprocessed grayscale tagged image files (TIFs) and maximum intensity projections of confocal z-stacks were exported from Fiji, followed by pseudo-colouring and adjustment of exposure or brightness/contrast in Affinity Photo V2 (Affinity Package V2, Serif Europe Ltd, affinity.serif.com/en-us/photo/). For maximum data transparency and preservation, unprocessed grayscale images or maximum intensity projections were imported as smart objects and adjustments were performed using adjustment layers. Processed images were saved as multilayer PSD files.

Image quantification

Adjusted volume (intensity) of Western blot bands was calculated with Image Lab (Bio-Rad, Image Lab Software Version 6.1, Bio-Rad Laboratories, Inc.) for every biotinylated p-peptide from the MDC1-[p]TQXF peptide pull-down from HeLa nuclear extracts, as a way of analyzing the binding affinity of either RNF8 or PELI1/2 for each pTQXF cluster. For quantitative assessment of protein colocalization in the IRIF experiment, maximum intensity projections of two channel confocal micrographs of single cells (EGFP and TurboRFP) were used for the SQUASSH analysis with the SQUASSH plugin (part of the MosaicSuite) for ImageJ and Fiji (Rizk et al, 2014; Leimbacher et al, 2019), imagej.net/Squassh. Widefield images of the PLA experiment were analyzed for quantification of the PLA signal using CellProfiler 4.0 (McQuin et al, 2018). Nuclei and GFP-positive regions were segmented with the intensity-based primary object detection module using the DAPI and GFP signals, respectively. GFP-positive nuclei were masked, and intensity of the PLA signal was measured on the respective channel and on the masked objects. The CellProfiler pipeline used is available upon request.

Statistics and reproducibility

The appropriate statistical test was chosen as follows: to compare two populations, unpaired normal distributed data were tested with a two-tailed t test (in case of similar variances) or with a two-tailed t test with Welch’s correction (in case of different variances). Unpaired non-continuous (count) data or non-normal distributed data were tested with two-tailed Mann-Whitney test (in case of similar variances) or with a two-tailed Kolmogorov–Smirnov test (in case of different variances). Three or more groups were analyzed by one-way ANOVA with Dunnett’s correction for multiple comparison. In case of non-continuous (count) data or non-normal distribution, Kruskal-Wallis with Dunn’s correction for multiple comparison was used.

All statistical analyses were performed with GraphPad Prism v9.00 (GraphPad Prism version 9.00 GraphPad Software, La Jolla California USA, graphpad.com). Sample sizes, α levels, and the statistical tests used are specified in the figure legends.