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TopBP1 utilises a bipartite GINS binding mode to support genome replication

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Uncropped images of SDS PAGE gels are available as a Source data file. Information on antibodies and plasmids used are available in Supplementary Data 3.

Protein purification

Purification of recombinant TopBP1

Purification of TopBP1-331-766 (BRCT3-5) for structural studies was done using bacterial expression. BL21(DE3) cells carrying a pET17 plasmid encoding HIS-(3C)-TopBP1-331-766-Strep was were grown in TurboBroth at 37 °C to an OD600 of 2.0 and induced by with 0.5 mM IPTG prior to overnight incubation at 16 °C. Lysis by sonication was done in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM TCEP, 10 U DNASE Turbo and complete, EDTA-free Protease Inhibitor Cocktail (Merck). The resulting lysate was clarified by centrifugation at 40,000 × g for 60 min at 4 °C. The supernatant was applied to a 5 ml HiTrap TALON crude column (GE Healthcare, Little Chalfont, UK), washed with lysis buffer, followed by a wash with lysis buffer containing 10 mM imidazole. Elution followed by increasing imidazole to 250 mM. The eluted protein was separated on a 5 ml HiTrap STREP column (GE Healthcare, Little Chalfont, UK), and eluted with 2 mM desthiobiotin. The eluate was concentrated before separation on a Superdex200increase 10 300 column (GE Healthcare, Little Chalfont, UK) equilibrated in 10 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP.

Recombinant TopBP1-1-766-Strep-WT (BRCT0-5) and mutants for interaction studies and experiments in Xenopus egg extract were purified using SF9 insect cells. The cells were grown in suspension in insect cell media (Pan biotech, P04-850 500) at 27 °C. Baculoviruses were generated using pLIB-based constructs (Addgene, 80610). 500 ml Sf9 cells (1 × 106/ml) were infected with a 1:100 dilution of recombinant baculovirus carrying TopBP1-1-766-Strep-WT or mutants, and were incubated at 27 °C for 72 h. Cell pellets were lysed by douncing in 30 ml of lysis buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 0.1% (v/v) Tween-20, 0.5 mM TCEP, protease inhibitor cocktail (Roche Complete protease inhibitor cocktail, 05056489001)). The lysate was centrifuged at 44,800 × g for 45 min and the supernatant was loaded onto the StrepTrapHP-1ml column (Cytiva, 28907546). Elution was done using 2.5 mM desthiobiotin in elution buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 0.01% Tween-20 (v/v), 0.5 mM TCEP, 2% (v/v) glycerol, 2.5 mM desthiobiotin).

Purification of GINS

GINS for structural and fluorescence polarisation studies was purified in SF9 insect cells Psf2, Psf3 and HIS-(3C)-Sld5 were expressed together with Psf1 or Psf1 (1–151; B domain deleted)) from a single baculovirus, respectively, produced using pBig1a. Cell pellets were resuspended in lysis buffer containing 25 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM TCEP, 10 U DNASE Turbo and complete, EDTA-free Protease Inhibitor Cocktail (Merck), then disrupted by sonication. The resulting lysate was clarified by centrifugation at 40,000 × g for 60 min at 4 °C. The supernatant was separated on a 5 ml HiTrap TALON crude column (GE Healthcare, Little Chalfont, UK), using washing in lysis buffer supplemented with 10 mM imidazole. Elution was done using a gradient up to 250 mM imidazole. Peak fractions were pooled and concentrated. For subsequent size exclusion chromatography (analytical and for EM) a Superdex200increase 10 300 column (GE Healthcare, Little Chalfont, UK) equilibrated in 10 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP was used while for FP experiments a Superdex200 16 600 column (GE Healthcare, Little Chalfont, UK) equilibrated in 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.25 mM TCEP, 0.02% (v/v) Tween 20 was used.

GINS for pulldown experiments was purified using 6xHis-3xFlag-Sld5. Sf9 insect cells in suspension cultures were co-infected with 1:50 dilution of each four recombinant baculoviruses for expressing 6xHis-3xFlag-Sld5, Psf1, Psf2 and Psf3, respectively. The viruses were made using pLib-based plasmids. 72 h post infection, pellets from one-litre cells were lysed by douncing in 60 ml of lysis buffer (20 mM HEPES pH 8.0, 300 mM NaCl, 0.1% (v/v) Tween-20, 25 mM imidazole, 0.5 mM TCEP, protease inhibitor cocktail (Roche Complete protease inhibitor cocktail, 05056489001)). The lysate was centrifuged at 44,800 × g for 45 min and the supernatant was incubated with 2.5 ml Ni-NTA agarose beads (Qiagen, 30210) for 1 h at 4 °C. The bound protein was eluted with six bead volumes elution buffer (20 mM HEPES pH 8.0, 300 mM NaCl, 0.01% (v/v) Tween-20, 250 mM imidazole, 0.5 mM TCEP, 2% (v/v) glycerol). The eluate was dialysed into 20 mM HEPES pH 8.0, 150 mM NaCl, 0.01% (v/v) Tween-20, 0.5 mM TCEP, 2% (v/v) glycerol, and then applied onto an HiTrap HP Q column (Cytiva, 17115301). To elute the bound protein, a linear gradient of NaCl (from 150 mM to 1 M) in elution buffer (20 mM HEPES pH 8.0, 0.01% (v/v) Tween-20, 0.5 mM TCEP, 2% (v/v) glycerol) was applied. Peak fractions were pooled and separated by size exclusion chromatography (equilibration buffer: 20 mM HEPES pH 8.0, 300 mM NaCl, 0.01% (v/v) Tween-20, 0.5 mM TCEP, 2% (v/v) glycerol).

Purification of PolE2-N and geminin from E. coli

PolE2-N-1-75 (amino acids 1–75) with N-terminal MBP-TEV2 tag in pMal, and GST tagged Xenopus geminin (Xgeminin) in pGEX were expressed in Rosetta E. coli culture. Expressions were induced with 1 mM IPTG at OD600 = 0.6 at 20 °C (PolE2-N) or 25 °C (Xgeminin) overnight. Cells were harvested by centrifugation. For PolE2-N, cell pellets were resuspended in lysis buffer (20 mM Hepes pH 8.0, 150 mM NaCl, 0.5 mM TCEP, 2 (v/v) glycerol and protease inhibitor cocktail (Roche Complete protease inhibitor cocktail, 05056489001)) and lysed by sonication. The cell lysate was clarified by centrifugation. The supernatant was incubated with amylose resin (New England Biolabs, E8021S) for 1 h before elution with lysis buffer + 10 mM maltose. The peak fraction was dialysed into TopBP1 buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 0.5 mM TCEP, 0.01% (v/v) Tween-20 and 2% (v/v) glycerol). For Xgeminin, lysis by sonication was carried out in 5 mg/ml lysozyme, 20 mM Hepes pH 7.7, 200 mM NaCl, 5 mM β-mercaptoethanol, 5% glycerin. After a clarifying centrifugation step, the supernatant was incubated with glutathione-sepharose (0.5 ml per liter culture; GE Healthcare 17513201) for 3 h before elution with lysis buffer + 50 mM glutathione (Applichem A2084,0025). Peak fractions were pooled and dialysed into XBE2 buffer (100 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, 1.71% w:v sucrose, 5 mM K-EGTA, 10 mM HEPES–KOH, pH 7.7) and concentrated to 360 µM Xgeminin. Aliquots were shock frozen and stored at −80 °C.

Size exclusion chromatography for isolating the TopBP1-GINS complex

Recombinant TopBP1 and GINS proteins were mixed in an equimolar ratio to give a final concentration of 20 µM of each component and incubated on ice for at least 30 min prior to application to a Superdex200increase 10 300 column (GE Healthcare, Little Chalfont, UK) equilibrated in 10 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP.

Fluorescence polarisation experiments

Fluorescein-labelled peptides (WT: Flu-GYGAPSEKHEQADEDLLSQYENG or LLPP: Flu-GYGAPSEKHEQADEDPPSQYENG) (Peptide Protein Research Ltd, Bishops Waltham, UK) at a concentration of 100 nM, were incubated at room temperature with increasing concentrations of GINS in 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.25 mM TCEP, 0.02% (v/v) Tween 20 in a black 96-well polypropylene plate (VWR, Lutterworth, UK). Fluorescence polarisation was measured in a POLARstar Omega multimode microplate reader (BMG Labtech GmbH, Offenburg, Germany). Binding curves represent the mean of three independent experiments, with error bars representing SEM.

Immunoprecipitations from transiently transfected 293T cells

Transient transfections of 6xMyc-Tev2-TopBP1 into 293T cells (ATCC CRL-11268) were carried out using PEI (polyethyleneimine) according to a protocol kindly shared by David Cortez’ lab. 4 µg plasmid DNA in 100 µl DMEM (Thermofisher, 41965039) without penicillin-streptomycin were combined with 2.4 µl Polyethylenimin (Sigma, 408727; 10 mg/ml) and incubated for 20 min before addition to 4 × 106 cells on a 10-cm dish. Transfected cells were used 72 h post transfection. 72 h post transfection, 293T cells were lysed by douncing in ten times cell pellet volume of lysis buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 0.1% (v/v) Tween-20, 0.5 mM TCEP, 2% (v/v) glycerol, protease inhibitor cocktail (Roche, Complete protease inhibitor cocktail, 05056489001)), and centrifuged at 20,000 × g for 15 min at 4 °C. The soluble supernatant was added to magnetic anti-Myc beads (5 µl slurry per sample; Thermofischer, 88842) and incubated for 2 h at 4 °C rotating. Cells equivalent to 50% of a 15 cm dish were used per sample. Beads were washed three times in lysis buffer with 5 min incubation each, and finally boiled in 50 µl Laemmli loading buffer (6.5% glycerol, 715 mM β-mercaptoethanol, 3% SDS, 62.5 mM Tris-HCl pH 7.9, 0.005% bromphenol blue).

Pulldown experiments of recombinant PolE2-N or TopBP1 using immobilised GINS

Magnetic anti-Flag beads (1 µl slurry for western blot/silver staining-scale experiments, 4 µl for Coomassie-scale experiments) were coupled with 600 ng (western/silver-scale) or 2400 ng (coomassie-scale) GINS via 3xFlag-Sld5. 100 nM final concentration of TopBP1-BRCT0-5-Strep (WT or mutants) in 20 µl (western/silver-scale) or 80 µl (coomassie-scale) of interaction buffer (20 mM HEPES pH 8.0, 150 mM NaCl (or 100 mM NaCH3COO when indicated), 0.01% (v/v) Tween-20, 0.5 mM TCEP, 2% (v/v) glycerol) were added to GINS-coupled beads in 5 µl (western/silver-scale) or 20 µl (coomassie-scale) interaction buffer. For PolE2-N, 1 µM PolE2-N protein in 20 µl were used for GINS-coupled beads in 5 µl reaction buffer. After incubation for 45 min at 4 °C rotating, beads were washed three times for 5 min with interaction buffer and boiled in 50 µl Laemmli buffer before SDS PAGE.

Pulldown from cell lysates with recombinant TopBP1-1-766-strep (BRCT0-5)

For pulldown assays from soluble lysates of non-transfected 293T cells, 5 µl streptactin Sepharose HP (Cytiva, 28935599) beads were coupled with 10 µg recombinant TopBP1-1-766-strep-WT or mutants in coupling buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 0.1% (v/v) Tween-20, 0.5 mM TCEP, 2% (v/v) glycerol) for 45 min at 4 °C before washing two times with coupling buffer and then once in cell lysis buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 0.1% (v/v) Tween-20, 0.5 mM TCEP, 2% (v/v) glycerol, protease inhibitor cocktail (Roche Complete protease inhibitor cocktail, 05056489001). Control pulldowns were done with Flag-peptide-coupled beads or, when indicated, using GINS-coupled beads and the non-GINS binding TopBP1-Gcc-B4mut. 293T cells were lysed in ten times pellet volume of cell lysis buffer, and centrifuged at 20,000 × g for 15 min at 4 °C. Soluble lysate from cells equivalent to 50% of a 15 cm tissue culture plate was added to the TopBP1-coupled beads and incubated for 2 h at 4 °C rotating. Beads were washed three times with 5 min incubation in lysis buffer and boiled in 50 µl Laemmli buffer before SDS PAGE.

Generation of replicating Xenopus egg extracts

Our work with Xenopus laevis frogs complied with ethical regulations. The protocols used in this study, namely to handle the frogs, collect their eggs for cytosolic extract generation, and to prepare sperm, were approved by the Landesamt für Natur-, Umwelt- und Verbraucherschutz, Nordrhein-Westfalen (81-02.05.40.20.050).

Xenopus laevis egg extracts were prepared from metaphase II arrested eggs as described in ref. 79. After washing with MMR (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES–NaOH, pH 7.8) eggs were dejellied (2% cysteine w:v in H2O, pH 7.8 with NaOH). Eggs were rinsed in XBE2 (100 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, 1.71% w:v sucrose, 5 mM K-EGTA, 10 mM HEPES–KOH, pH 7.7). Then, the eggs were transferred into centrifuge tubes containing 1 ml XBE2 + 10 µg/ml protease inhibitor (aprotinin Sigma A6279, leupeptin Merck 108975, pepstatin Carl Roth 2936.1) + 100 µg/ml cytochalasin D (Sigma C8273). For packing, eggs were centrifuged for 1 min at 1400 × g at 16 °C in a swingout rotor. Excess buffer, activated and apoptotic eggs were removed before crushing the eggs at 16,000 × g for 10 min at 16 °C. The extract was collected with a 20 G needle and supplemented with 10 µg/ml cytochalasin D, 10 µg/ml protease inhibitors, 1:80 dilution of energy regenerator (1M phosphocreatine K salt, 600 µg/ml creatine phosphokinase in 10 mM HEPES–KOH pH 7.6) and LFB1/50 (10% w-v sucrose, 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, 2 mM DTT, 20 mM K2HPO4/KH2PO4 pH 8.0, 40 mM HEPES–KOH, pH 8.0) to 15% (v:v). The extract was cleared at 84,000 × g at 4 °C for 20 min in a swingout rotor. The cytoplasmatic layer was collected and supplemented with 2% glycerol v:v. The extract was frozen by dropping 20 µl aliquots into liquid nitrogen and stored at −80 °C.

Immunodepletion of TopBP1 from Xenopus egg extract

For XTopBP1 depletion from Xenopus egg extract, 0.5 µg antibody #1, #2 or IgG (rabbit; self-made) were coupled per µl magnetic Protein G dynabeads (Life Technologies 10004D) for 1 h at RT. Freshly thawed extracts were supplemented with 1/40 energy regenerator and 250 µg/ml cycloheximide and released into interphase by adding 0.3 mM CaCl2 for 15 min at 23 °C. Subsequently, extracts were depleted for 45 min on ice with 0.675 µg antibody per µl extract. After depletion the extract was aliquoted, snap frozen and stored at −80 °C for replication assays and chromatin isolations.

Xenopus egg extract replication assays

For TopBP1 addback experiments, 6 ng/µl recombinant TopBP1 protein or buffer were added to TopBP1 immunodepleted interphase Xenopus egg extract containing 50 nCi/µl α32P-dCTP (Perkin Elmer NEG513A250UC). To start the replication reaction the egg extracts were supplemented with 5 ng sperm DNA (kindly provided by the lab of O. Stemmann) per µl extract and incubated at 23 °C. 1 µl extract was spotted for each time point onto a glass fibre membrane. Unbound α32P-dCTP was removed by rinsing the membrane three times 15 min with ice cold 5% TCA in water and once with ice cold EtOH. Newly replicated DNA was detected by phospho-imaging. Standard error of the mean was calculated from three independent experiments.

Chromatin isolation from Xenopus egg extracts and analysis by mass spectrometry (CHROMASS) and western blotting

For chromatin isolation, XTopBP1-depleted interphase egg extract was supplemented with 50 µg/ml aphidicolin and aliquoted into 15 µl samples before addition of buffer or recombinant TopBP1 to a final concentration 6 ng/µl. 2.25 µM Xgeminin or buffer were added before incubation for 10 min on ice. To start the replication, 9 ng sperm DNA/µl extract was added and incubated for 75 min at 23 °C. Reactions were stopped with 300 µl ice cold ELB salt (10 mM HEPES–KOH pH 7.7, 50 mM KCl, 2.5 mM MgCl2) + 250 mM sucrose + 0.6% triton X-100. The diluted extract was loaded onto a sucrose cushion (150 µl ELB salt + 25% sucrose) and centrifuged for 10 min at 2500 × g at 4 °C in a swingout rotor. The supernatant above the sucrose cushion was removed and the cushion surface was washed twice with 200 µl ELB salt containing 250 mM sucrose. The cushion was removed leaving about 15 µl behind, followed by centrifugation for 2 min at 10,000 × g at 4 °C in a fixed angle rotor. For western blotting, the chromatin pellet was resuspended in 24 µl 1x Laemmli SDS sample buffer (6.5% glycerol, 715 mM β-mercaptoethanol, 3% SDS, 62.5 mM Tris-HCl pH 7.9, 0.005% bromphenol blue) and 5 µl were separated by denaturing SDS PAGE. For MS analysis, four biological replicates of chromatin pellets were analysed by CHROMASS45. In brief, chromatin pellets were resuspended in 50 µl denaturation buffer (8 M Urea; 100 mM Tris pH 8). Dithiothreitol (DTT) was added to a final concentration of 5 mM and samples were incubated at 22 °C for 30 min. To alkylate peptides, iodoacetamide (20 mM) was added and samples were incubated at 37 °C for 30 min. DTT (25 mM) was added, and samples were incubated at 22 °C for 5 min. 500 ng Lys-C (add Supplier) were added, and samples were incubated at 37 °C for 3 h. Samples were diluted with 100 mM ammonium bicarbonate to adjust the urea concentration to 1 M. Trypsin (Sigma, 500 ng per sample) was added and samples were incubated over night at 37 °C. Digested peptides were acidified with 1% trifluoroacetic acid (TFA) and desalted on Empore C18 material according to Rappsilber et al. https://www.nature.com/articles/nprot.2007.261). Eluted peptides were dried in a vacuum concentrator and reconstituted with 9 µl of buffer A (0.1% formic acid) containing 0.2% acetonitrile and 0.01% trifluoro acetic acid. For MS analysis, 4 µl of the reconstituted peptides were separated on an EASY-nLC™ 1200 chromatography system (Thermo Scientific) coupled to a Q Exactive HF-X Orbitrap LC-MS/MS System (Thermo Fisher Scientific) via a Nanoflex ion source. Peptide separation was carried out in analytical columns (50 cm, 75 µm inner diameter packed in-house with C18 resin ReproSilPur 120, 1.9 µm diameter Dr. Maisch) using a 3-h nonlinear gradient at a flow rate of 250 nl/min using buffer A (aqueous 0.1% formic acid) and buffer B (80% acetonitrile, 0.1% formic acid). MS data was acquired in data-dependent fashion using a Top15 method. The exact parameters for chromatography and MS instrument settings can be retrieved from the raw files available at ProteomeXchange (PXD040024).

MS data files from single-shot experiments were processed with MaxQuant (version 2.0.1.0) using a non-redundant Xenopus laevis data base available at ProteomExchange (PXD040024)45. Raw data were normalized using the label-free quantitation (LFQ) algorithm implemented in MaxQuant. MS Data with Perseus (version 1.6.15.0)80. Protein groups were filtered to eliminate contaminants, reverse hits, and proteins identified by site only. For the heat map (Fig. 6a) LFQ intensities were log2 transformed and for each protein z-scores were calculated across all replicates (N = 4) of all four conditions. Subsequently, the average of the z-scores was calculated for each condition and selected proteins were plotted (see supplementary data 2 for the z-score of all proteins). Proteins were manually annotated and sorted according to their function in DNA replication and DNA repair. To identify proteins with significant abundance changes between the four conditions, LFQ intensities were log2 transformed and missing values were imputed with random values drawn from a normal distribution centred around the detection limit of the MS instrument (Perseus imputation; width = 0.3, down shift = 1.8). Two sample Student’s t-tests were carried out in Perseus. Student’s t-tests were carried out in Perseus. For these tests three valid values in at least one quadruplicate of either of the tested conditions was required. FDR was adjusted for multiple testing by the Benjamini–Hochberg procedure using a significance threshold of FDR<0.05 (see supplementary data 2). Data visualisation was carried out in R. All scripts are available upon request.

Determination of TopBP1-proximal proteins by APEX2 biotinylation

Sample preparation

Proximity labelling was performed in isogenic stable 293 Flip-In (Thermo Fisher; R75007) cells stably expressing TopBP1 N-terminal tagged with APEX2 or in 293T cells transiently expressing either N-terminal or C-terminally APEX2-tagged TopBP1. 24 h before transfection, 0.8 × 106 cells were seeded onto a 6-cm plate. For each condition, four dishes were separately transfected (biological replicates). One day after transfection, cells were incubated for 30 min with 500 µM biotin phenol at 37 °C, before incubation for 1 min at RT with or without (controls) 1 mM H2O2. The medium was discarded, and cells were washed three times with quenching buffer (10 mM sodium azide, 10 mM sodium ascorbate and 5 mM Trolox in Dulbecco’s PBS (Life Technologies 14190169). Cells were rinsed off the dishes with DPBS, transferred into reaction tubes before washing again with DPBS. Cells were lysed at 95 °C for 10 min in TSD buffer (50 mM Tris pH 7.5, 5 mM DTT and 1% SDS). Samples were diluted ten times with TNN buffer (20 mM Tris pH 7.9, 200 mM NaCl and 0.5% NP-40 alternative) and benzonase (75 U per sample) (Sigma E1014). Samples were incubated for 15 min at 4 °C and centrifuged at 21,000 × g for 2 min at 4 °C. Supernatants were incubated over night with 15 µl streptavidin sepharose beads (Sigma GE17-5113-01) at 4 °C. Beads were washed once with TNN buffer + 0.1% SDS and twice with 25 mM ammonium bicarbonate before processing for mass spectrometry. For this, captured proteins were washed two times with H2O prior to on-bead digestion to remove MS incompatible buffer components. The beads were taken up in 100 µl 0.8 M urea, 50 mM ammonium bicarbonate buffer (ABC) and supplemented with 5 mM DTT. After incubation at 37 °C for 30 min, 10 mM iodoacetamide (IAM) was added and incubated for 30 min at room temperature in the dark while shaking at 1500 rpm (Thermomixer C, Eppendorf). The IAM was quenched with 11 mM DTT. Trypsin was added to a total of 300 ng Trypsin per sample. The samples were incubated over night at 37 °C, shaking at 1150 rpm before stopping by 1% (vol/vol) formic acid (FA). After bead collection by centrifugation (650 × g, 5 min) 100 µl supernatant were transferred to a Eppendorf Lo-bind 1.5 ml tube. The remaining beads were incubated with 50 µl 1% formic acid for (1150 rpm, 5 min rpm, RT) and after collecting the beads once more by centrifugation (650 × g, 5 min) the supernatant was combined with the first one. The sepharose beads were discarded. To remove residual sepharose beads, the combined peptide containing solutions were passed over pre-equilibrated (50 µl 0.5% formic acid) home-made 2-disc Glass microfiber StageTip (disc material: GE Healthcare; pore size: 1.2 µM; thickness: 0.26 mm; 50 × g, 2 min). The cleared tryptic digests were then desalted on home-made C18 StageTips as described81. Briefly, peptides were immobilized and washed on a 2 disc C18 (Empore) StageTip. After elution from the StageTips, samples were dried using a vacuum concentrator (Eppendorf) and the peptides were taken up in 0.1% formic acid solution (10–15 μl) and directly used for LC-MS/MS experiments.

LC-MS/MS settings

MS Experiments were performed on an Orbitrap Fusion LUMOS instrument (Thermo) coupled to an EASY-nLC 1200 ultra-performance liquid chromatography (UPLC) system (Thermo). The UPLC was operated in the one-column mode. The analytical column was a fused silica capillary (75 µm × 46 cm) with an integrated fritted emitter (CoAnn Technologies) packed in-house with Kinetex 1.7 µm C18-XB core shell beads (Phenomenex). The analytical column was encased by a column oven (Sonation PRSO-V2) and attached to a nanospray flex ion source (Thermo). The column oven temperature was set to 50 °C during sample loading and data acquisition. The LC was equipped with two mobile phases: solvent A (0.2% FA, 2% Acetonitrile, ACN, 97.8% H2O) and solvent B (0.2% FA, 80% ACN, 19.8% H2O). All solvents were of UPLC grade (Honeywell). Peptides were directly loaded onto the analytical column with a maximum flow rate that would not exceed the set pressure limit of 980 bar (usually around 0.4–0.6 µl/min). Peptides were subsequently separated on the analytical column by running a 105 min gradient of solvent A and solvent B (start with 3% B; gradient 3% to 9% B for 6:30 min; gradient 9% to 30% B for 62:30 min; gradient 30% to 50% B for 24:00 min; 50% to 100% B for 2:30 min; 100% for 9:30 min) at a flow rate of 300 nl/min. The mass spectrometer was controlled by the Orbitrap Fusion Lumos Tune Application (version 3.3.2782.28) and operated using the Xcalibur software (version 4.3.73.11). The mass spectrometer was set in the positive ion mode. The ionization potential (spray voltage) was set to 2.5 kV. Source fragmentation was turned off. Precursor ion scanning was performed in the Orbitrap analyser (FT; Fourier transform mass spectrometer) in the scan range of m/z 370–1500 and at a resolution of 240,000 with the internal lock mass option turned on (lock mass was 445.120025 m/z, polysiloxane)82. AGC (automatic gain control) was set to standard and acquisition time to auto. Product ion spectra were recorded in a data-dependent fashion in the IT (IT; ion trap mass spectrometer) in a variable scan range (auto setting) and at rapid scan rate. Peptides were analysed using the setting top speed (repeating cycle of full precursor ion scan (AGC target set to 300%; acquisition time set to auto) followed by dependent MS2 scans for 3 s (minimum intensity threshold 4 × 103)). The MS2 precursor ions were isolated using the quadrupole (isolation window 1.6 m/z) and fragmentation was achieved by Higher-energy C-trap dissociation (HCD) (normalized collision mode (stepped setting) and normalized collision energy set to 27, 32, 40%). During MS2 data acquisition dynamic ion exclusion was set to 20 s. Only charge states between 2-7 were considered for fragmentation.

Data processing

RAW spectra were submitted to an Andromeda83 search in MaxQuant (v2.0.3.0) using the default settings (Cox and Mann, 2008)84. Label-free quantification and match-between-runs was activated85. The MS/MS spectra data were searched against the Uniprot H. sapiens reference database (one protein per gene; UP000005640_9606_OPPG.fasta, 20585 entries, downloaded 1/10/2022) and a dedicated database containing the APEX2-myc sequence (ACE_0741_SOI_v01.fasta; 1 entry). All searches included a contaminants database search (as implemented in MaxQuant, 246 entries). The contaminants database contains known MS contaminants and was included to estimate the level of contamination. Andromeda searches allowed oxidation of methionine residues (16 Da) and acetylation of the protein N-terminus (42 Da) as dynamic modifications and the static modification of cysteine (57 Da, alkylation with iodoacetamide). Enzyme specificity was set to Trypsin/P with two missed cleavages allowed. The instrument type in Andromeda searches was set to Orbitrap and the precursor mass tolerance was set to ± 20 ppm (first search) and ± 4.5 ppm (main search). The MS/MS match tolerance was set to ±0.5 Da. The peptide spectrum match FDR and the protein FDR were set to 0.01 (based on target-decoy approach). Minimum peptide length was 7 aa. For protein quantification unique and razor peptides were allowed. Modified peptides were allowed for quantification. The minimum score for modified peptides was 40. Label-free protein quantification was switched on, and unique and razor peptides were considered for quantification with a minimum ratio count of 2. Retention times were recalibrated based on the built-in nonlinear time-rescaling algorithm. MS/MS identifications were transferred between LC-MS/MS runs with the match-between-runs option in which the maximal match time window was set to 0.7 min and the alignment time window set to 20 min. The quantification is based on the value at maximum of the extracted ion current. At least two quantitation events were required for a quantifiable protein. Further analysis and filtering of the results was done in Perseus v1.6.10.0.80. For quantification, we combined related biological replicates to categorical groups and investigated only those proteins that were found in at least one categorical group in a minimum of 3 out of 4 biological replicates. Comparison of protein group quantities (relative quantification) between different MS runs is based solely on the LFQ’s as calculated by MaxQuant, MaxLFQ algorithm85. Statistical evaluation was done by two-sided Student’s t-testing (FDR = 0.05, S0 = 0.1)

Analysis of the TopBP1-GINS by cross-linking mass spectrometry

Sample preparation

50 μg (1.15 µg/µl) of each GINS and TopBP1-BRCT0-5-strep (amino acids 1–766) were incubated for 45 min on ice in cross-linking buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 0.01% Tween-20, 2% Glycerol, 0.5 mM TCEP). 600 µM or 2500 µM of BS3 cross-linker were added and incubated for 30 min at 35 °C shaking. The reaction was stopped by adding ammonium bicarbonate to a final concentration of 100 mM. 90% of the cross-linked sample was used for mass spectrometry analysis of cross-links. 10% of the reaction were separated by SDS-PAGE. The coomassie-stained gel was cut in slices above the molecular weight of monomeric TopBP1-BRCT0-5-strep for subsequent mass spectrometry.

Sample processing for mass spectrometry

Sample preparation of cross-linked samples for LC/MS/MS is based on the SP3 protocol86. 30 µg of protein from each cross-linking sample was taken up in 100 µl 1× SP3 lysis buffer (final concentrations: 5% (wt/vol) SDS; 10 mM TCEP; 200 μl 40 mM chloracetamide; 200 mM HEPES pH 8) and heated for 5 min at 90 °C. After cooling the samples to room temperature (on ice) a mix of 150 µg hydrophobic (#65152105050250) and 150 µg hydrophilic (#45152105050250) SeraMag Speed Beads (Cytiva) was added (bead to protein ratio 10 to 1) and gently mixed. Then 100 µL 100% vol/vol Ethanol (EtOH) was added before incubation for 20 min at 24 °C shaking vigorously. The beads were collected on a magnet and the supernatant aspirated. The beads were then washed 4 times with 180 µL 80% EtOH (collection time on the magnet minimum of 4 min). The beads were then finally taken up in 100 µl 25 mM ammoniumbicarbonate (ABC) containing 1 µg Trypsin (Protein:Trypsin ratio 30:1). To help bead dissociation, samples were incubated for 5 min in a sonification bath (preheated to 37 °C). Samples were incubated over night shaking at vigorously. Samples were acidified with formic acid (FA, final 1% vol/vol) before collection on a magnet. The supernatants were transferred to a fresh Eppendorf tube, before removing trace beads using a magnet for 5 min. The tryptic digests were then desalted on home-made C18 StageTips as described81. Briefly, peptides were immobilized and washed on a 2 disc C18 StageTip. Samples were then dried using a vacuum concentrator (Eppendorf) and the peptides were taken up in 0.1% formic acid solution (10 μl) and directly used for LC-MS/MS experiments.

LC-MS/MS settings cross-linking mass spectrometry

MS Experiments were performed on an Orbitrap Fusion LUMOS instrument (Thermo) coupled to an EASY-nLC 1200 ultra-performance liquid chromatography (UPLC) system (Thermo). The UPLC was operated in the one-column mode. The analytical column was a fused silica capillary (75 µm × 41 cm) with an integrated fritted emitter (CoAnn Technologies) packed in-house with Kinetex 1.7 µm C18-XB core shell beads (Phenomenex). The analytical column was encased by a column oven (Sonation PRSO-V2) and attached to a nanospray flex ion source (Thermo). The column oven temperature was set to 50 °C during sample loading and data acquisition. The LC was equipped with two mobile phases: solvent A (0.2% FA, 2% Acetonitrile, ACN, 97.8% H2O) and solvent B (0.2% FA, 80% ACN, 19.8% H2O). All solvents were of UPLC grade (Honeywell). Peptides were directly loaded onto the analytical column with a maximum flow rate that would not exceed the set pressure limit of 980 bar (usually around 0.4–0.6 µL/min). Peptides were subsequently separated on the analytical column by running a 70 min gradient of solvent A and solvent B (start with 2% B; gradient 2% to 6% B for 5:00 min; gradient 6% to 25% B for 42:00 min; gradient 25% to 40% B for 15:00 min; 40% to 98% B for 1:00 min; 98% for 7:00 min) at a flow rate of 350 nl/min. The mass spectrometer was controlled by the Orbitrap Fusion Lumos Tune Application (version 3.3.2782.28) and operated using the Xcalibur software (version 4.3.73.11). The mass spectrometer was set in the positive ion mode. The ionization potential (spray voltage) was set to 2.5 kV. Source fragmentation was turned off. Precursor ion scanning was performed in the Orbitrap analyser (FT; Fourier transform mass spectrometer) in the scan range of m/z 370–1600 and at a resolution of 120,000 with the internal lock mass option turned on (lock mass was 445.120025 m/z, polysiloxane)82. AGC (automatic gain control) was set to standard and acquisition time to auto. Product ion spectra were recorded in a data-dependent fashion in the FT in a variable scan range (auto setting) and at 15,000 resolution. Peptides were analysed using a top speed regime (repeating cycle of full precursor ion scan (AGC target set to standard; acquisition time 200 ms) followed by dependent MS2 scans for 5 s (minimum intensity threshold 2 × 104). The MS2 precursor ions were isolated using the quadrupole (isolation window 2.0 m/z) and fragmentation was achieved by Higher-energy C-trap dissociation (HCD) (normalized collision mode set to stepped and normalized collision energy set to 27, 30, 33%). During MS2 data acquisition dynamic ion exclusion was set to 60 s. Only charge states between 3–7 were considered for fragmentation.

Data processing protocol

The searches for cross-linked peptides were performed with two different search engines: MetaMorpheus (MM) version 0.0.32087 and pLink2 version v2.3.988. Searches were performed on the Thermo RAW files using the database ACE_0704_SOI_plus_con_v01.fasta (244 entries). The database contains the sequences for TopBP1 (1–766), PsfI, PsFII, PsfIII, Sld5 and 239 known contaminating proteins found in MS samples. The peptide spectrum match FDR for MM was 0.01 and for pLink2 0.05 (based on target-decoy approach, decoys are generated by the software). The settings for MetaMorpheus were: cross-linker name = DSS _KSTY (note: DSS and BS3 have identical cross-linker size); cross-linker type = non-cleavable; cross-linker mass = 138.06808; cross-linker modification site 1 = K; cross-linker modification site 2 = KSTY; protease = trypsin; maximum missed cleavages = 3; minimum peptide length = 5; maximum peptide length = 60; initiator methionine behaviour = Variable; max modification isoforms = 1024; fixed modifications = Carbamidomethyl on C, variable modifications = Oxidation on M; parent mass tolerance(s) = ±10 ppm; product mass tolerance = ±20 ppm. The settings for pLink2 were: cross-linker name = DSS-BS3-KSTY; cross-linker type = non-cleavable; cross-linker mass = 138.06808; cross-linker modification site 1 = K; cross-linker modification site 2 = KSTY; protease = trypsin; maximum missed cleavages = 3; minimum peptide length = 6; maximum peptide length = 60; fixed modifications = Carbamidomethyl on C, variable modifications = Oxidation on M; parent mass tolerance(s) = ±20 ppm; product mass tolerance = ±20 ppm. The results from both searches were converted to the ProXL XML format using the respective converters (metaMorph2ProxlXML.jar, plink2toProxlXML.jar, follow links on the ProXL website; https://proxl-ms.org/) and uploaded to the ProXL server89. Analysis and evaluation of cross-links was performed on our local ProXL Server. The results from both searches was analysed together.

Computational modelling

Locally implemented AlphaFold2-Multimer version 2.2.090 was used to predict the binding interfaces of GINS and TopBP1 using the full-length sequences of each GINS subunit and residues 301–766 of human TopBP1. 5 models were obtained, using 5 seeds per model, by default and ranked with the combined score of ipTM+pTM implemented in AlphaFold2-Multimer90,91. The top ranked model used for figure production and statistical plots were produced using Alphapickle 1.5.4 for the top seed from each model.

Blind molecular docking for insight into the GINI binding region used the GINI sequence DEDLLSQY of TopBP1 (residues 487–494) in the servers Cluspro92 (accessed on 13/06/2022), HPEPDOCK93 (accessed on 13/06/2022), and MDockPeP94,95,96 (accessed on 13/06/2022) web servers with 200 solutions per server. We further extended, refined, and re-scored the sampling, by generating 10 additional conformations per web server solution (for a total of 6000 solutions) with FlexPepDock97. The top-10 best-scored solutions were selected for analysis and visualization.

The binding free energies of GINS with BRCT4 and GINI were estimated based on the best scored AlphaFold2 model using the web servers PRODIGY98 and PPI-Affinity99. The region of GINI employed for the estimation comprised residues 481 to 496. Both PRODIGY and PPI-Affinity analyse the residues in the binding interface for free energy estimation. PRODIGY uses a linear amino acid chain model, whilst PPI-Affinity uses a structure-based machine learning algorithm.

Cryo-electron microscopy

Grid preparation

Quantifoil 1.2/1.3, 300 mesh copper grids (Quantifoil) were glow discharged using a Tergeo Plasma Cleaner (Pie Scientific) with an indirect plasma treatment for 30 s. Grids were loaded into a Leica EM GP2 (Leica microsystems) and 3 μl of peak fractions from size exclusion chromatography were diluted to 0.1 mg/ml and applied to the front of the grid, with an additional 0.5 μl buffer applied to the grids rear, before back blotting for 3 s and plunging into an ethane propane mix.

Electron microscopy and data processing

For dataset 1, grids were stored in liquid nitrogen prior to imaging at 300 kV on a FEI Titan Krios (Thermo Fisher Scientific) equipped with K3 detector (Gatan). 18,945 movies were collected, using data acquisition software EPU (Thermo Fisher Scientific), at a magnification of 105,000 and a pixel size of 0.85 Å using a total dose of 50 e-/Å2. These were motion corrected in 5 × 5 patches using MOTIONCOR2100 implemented in RELION4.044 before importing into cryoSPARC43. Micrographs were CTF corrected prior to blob picking and extraction of an initial set of particles. Subsequent filtering by 2D classification removed most of these leaving a cleaned set of particles that were used to produce 4 initial models. The 2D classes and initial models appeared to have at least two distinct sub populations, one containing just the GINS complex alone and one that also had some additional density. In order to distinguish between these two sets of particles heterorefinement was performed to split the two groups. The first population of  particles containing only the GINS complex were then imported into RELION4.0 before several cycles of 3D classification, to yield a final set of 111,455 particles that gave a volume at 5.9 Å resolution used to produce the GINS alone volume used in (Supplementary Fig. 16a). The second group of TOPBP1-GINS particles were used to train a TOPAZ model before implementing further picking using TOPAZ101. The resulting particles were again filtered by rounds of 2D classification and heterorefinement before exporting the particles, that appeared to consist of the full TOPBP1-GINS complex, into RELION4.0. Several cycles of 3D classification were then performed with classes appearing to lack the additional density eliminated, to yield a final set of 208,115 particles. 3D refinement and post-processing of these gave a volume at 4.71 Å resolution into which previously solved crystal structures of the GINS complex (29EX) and the central BRCT4/5 module of TopBP1 (3UEN) could be docked.

For dataset 2, grids were stored in liquid nitrogen prior to imaging at 300 kV on a FEI Titan Krios (Thermo Fisher Scientific) equipped with Fakcon 4i detector (Thermo Fisher Scientific). 8808 movies were collected, using data acquisition software EPU (Thermo Fisher Scientific), at a magnification of 120,000 and a pixel size of 0.74 Å using a total dose of 39.69 e-/Å2. The same processing schedule was followed as for dataset one to yield a final set of 154,278 particles. 3D refinement and post-processing of these gave a volume at 4.1 Å resolution into which the previously solved model could be docked alongside the predicted GINI helix from the AlphaFold2-Multimer model. Minor adjustments to the model were made in Coot before refinement of the final model using PHENIX102.

Data presentation

Figure panels for all structural data were produced using ChimeraX-1.3.

Reporting summary

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

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