Cell culture

HCT116 cells and derivative cell lines (Supplementary Table 1) were grown in DMEM (Life Technologies, 41966-029) supplemented with 10% fetal bovine serum (FBS, GIBCO), 100 U ml⁻¹ penicillin and 100 mg ml⁻¹ streptomycin in an atmosphere containing 6% CO₂ at 37 °C. HCT116 OsTIR (F74G), mAID-mClover-RAD21 HCT116 OsTIR (F74G)⁶, WAPL-mClover-mAID HCT116 OsTIR (F74G)⁴ and CTCF-mClover-mAID HCT116 OsTIR (F74G)⁶ were provided by M. Kanemaki. None of the cell lines were authenticated in house. All cell lines used in this study were subjected to regular mycoplasma testing in house, and all of them consistently reported negative results. To degrade mAID-tagged proteins, cells were treated with 2 µM 5-Ph-IAA (aux; BioAcademia). To degrade Halo-SMARCAL1, cells were treated with Halo-PROTAC3 at 1:1,000 for the indicated times.

Cell line generation

Cell lines were generated following published protocols^56,57. HCT116 OsTIR (F74G) cells were transfected with CRISPR-Cas9 and donor plasmids using FuGENE HD Transfection Reagent (Promega) in a 12-well plate following the instructions of the manufacturer. Two days after transfection, cells were plated in 10 cm² dishes and selected with antibiotics. Selected clones were isolated and confirmed for protein expression of the modified alleles. Oligonucleotides to generate sgRNA or amplify homology arms to build donor plasmids are provided in Supplementary Table 1.

RNA interference

For RNA interference, cells were transfected with the indicated siRNAs (Supplementary Table 1) for the indicated times and concentrations. Transfections were carried out using RNAiMax (Thermo Fisher) according to the instructions of the manufacturer.

Lentiviral transduction

For cell lines expressing SMC1^WT, SMC1^3D or SMC1^4E, SMC1-3×Flag variant genes were cloned into LT3GEPIR (generated by GeneScript) and transfected into to HEK293T cells together with the lentiviral packaging vectors. After 48 h, lentiviral culture medium was harvested, filtered and added to SMC1-mClover-mAID HCT116 OsTIR (F74G) cells. After infection for 24 h, puromycin (2 μg ml⁻¹, InvivoGen) resistant single cells were isolated. To induce the expression of the SMC1 variants, cells were treated with 1 µg ml⁻¹ dox (Sigma-Aldrich).

iPOND–MS

iPOND was performed as described^33,58 with minor modifications. HCT116 cells were labelled with 10 µM EdU for 10 min and treated with the different drugs as indicated in Extended Data Fig. 1a. Cells were crosslinked with 1% formaldehyde for 20 min at room temperature (RT), quenched with 0.125 M glycine for 5 min, and washed three times with cold PBS. EdU was linked to biotin, after permeabilization with 0.25% Triton X-100/PBS for 30 min, by incubating in click reaction buffer (10 mM sodium-l-ascorbate, 20 µM biotin azide (Vanderbilt University) and 2 mM CuSO₄) at RT for 1 h on a rotator. Cells were washed twice with PBS, resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0 and 1% SDS) supplemented with protease inhibitors, and chromatin was solubilized by sonication in a Bioruptor Pico (Diagenode) at 4 °C 10 min (30 s on and 30 s off cycles). After centrifugation for 10 min at 16,000g, supernatants were diluted with 1:1 PBS (vol/vol) containing protease inhibitors and incubated overnight with myOne streptavidin C1 dynabeads (Thermo Fisher). Beads were washed once with lysis buffer, once with 1 M NaCl, twice with lysis buffer and once with PBS. Captured proteins were digested on beads using 500 ng of sequencing grade modified trypsin (Promega, V5111), including reduction and alkylation of cysteines with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 2-chloroacetamide (ClAA) addition, respectively. Resulting peptides were cleaned using the Phoenix kit (Preomics) according to instructions in the kit. Liquid chromatography-tandem mass spectrometry analysis of peptide mixture was conducted on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher) directly coupled to an ACQUITY UPLC M-Class System (Waters) configured for 75-μm scale single-pump trapping. Peptides were separated on a nanoEase HSS C18 T3, 100 A, 75 μm × 250 mm analytical column (Waters, PN: 186008818) at a constant flow rate of 300 nl min⁻¹ applying a peace-wise linear gradient from 5% to 33% solvent B in 45 min (solvent A: water, including 0.1% formic acid; solvent B: acetonitrile, including 0.1% formic acid). MS data acquisition was conducted in data-independent mode. DIA scans were acquired in the Orbitrap mass analyser at 15,000 Resolution (normalized AGC target: 3,000%, maxIT: 23 ms) covering the m/z range from 350 to 1,050 in 70 non-overlapping isolation windows. Precursors were quadrupole isolated at 10m/z and HCD fragmented at an NCE of 28 (calculated for the middle of the isolation window and charge 2).

Data analysis was performed using the PROSTAR package (v.1.34.6) in R (v.4.3.3). Protein intensity values were log₂-transformed, and missing values for partially observed values were imputed using the SLSA (Structured Least Squares Algorithm). Data normalization was carried out first within groups and subsequently globally (excluding no biotin and thymidine chase controls) using the LOESS method. The normalized dataset was further processed in Perseus (v.1.6.10.50), in which remaining missing values were imputed from a normal distribution. For each comparison, proteins were retained only if at least two non-imputed valid values were present in at least one group. The resulting dataset was re-imported into PROSTAR, and differential abundance analysis was performed using the LIMMA test with a log₂ fold-change threshold of 0.5. Benjamini–Hochberg correction was used to calculate P-values and FDR. Volcano plots were generated using GraphPad Prism (v.10).

PLA and SIRF

Cells were asynchronously grown on poly-lysine-coated coverslips. Cells were labelled with 25 µM EdU (Thermo Fisher) for 10 min. After the indicated treatments, cells were washed with PBS and pre-extracted for 5 min using CSK buffer (10 mM HEPES, 50 mM NaCl, 0.3 M sucrose, 3 mM MgCl₂, 1 mM EDTA or 1 mM EGTA and 0.5% Triton X-100) at 4 °C, followed by fixation in 4% formaldehyde at RT for 15 min. Following fixation, cells were washed three times with PBS. In SIRF experiments, EdU was linked to biotin by incubating in click reaction (0.1 M Tris, pH 8.5, 0.1 M sodium-l-ascorbate, 2 mM CuSO₄ and 0.1 mM biotin azide) for 1 h at 37 °C in a humidified chamber. After three washes with PBS, the cells were incubated in blocking buffer (5% BSA in PBS) at 37 °C for 1 h and incubated overnight at 4 °C with the indicated antibodies (Supplementary Table 1). After washing twice for 5 min with TBST (0.05% Tween-20 in TBS), cells were incubated with PLA probes (Merck) for 1 h at 37 °C, ligated for 30 min at 37 °C and subjected to a polymerase reaction for 100 min at 37 °C according to the instructions of the manufacturer. After washing twice for 5 min with TBS, cells were incubated at 37 °C for 30 min with fluorescent-labelled secondary antibodies (Supplementary Table 1), against the individual targets of the PLA, in TBS containing DAPI (0.5 µg ml⁻¹). Following two 5-min washes in TBS and letting coverslips air-dry in the dark, coverslips were mounted with Prolong Gold antifade reagent. Microscopy imaging was performed using a Leica DM6 B microscope (HCX PL APO 63× objective). SIRF/PLA and individual channel quantification was performed using Cell Profiler, and plotted and statistically assessed using GraphPad Prism 10. At least 100 cells were measured per condition.

Biochemical fractionation, co-immunoprecipitation assays and Western blotting

Biochemical fractionation of cells was performed as previously described^33,58. Co-immunoprecipitation experiments were performed by lysing cells in a buffer consisting of 20 mM Tris (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, 2 mM NaF, 10% glycerol, 0.2% NP40, 20 mM β-glycerophosphate, 0.5 mM DTT, protease inhibitor cocktail and Benzonase (Millipore), and rotated at 4 °C for 4 h. Lysates were centrifuged at 16,000g for 30 min at 4 °C. Supernatants were incubated with anti-Flag M2 affinity beads (Merck) overnight at 4 °C. Beads were washed three times with the lysis buffer. Biochemical fractions, co-immunoprecipitation beads and total cell extracts were prepared in Laemmli sample buffer (4% SDS, 20% glycerol and 120 mM Tris-HCl, pH 6.8) and loaded onto 4–20% Mini-PROTEAN TGX Precast Protein Gels (BioRad). For BRCA2 detection, extracts were run in 3–8% NuPAG Tris-Acetate Midi Protein Gels (Thermo Fisher). Proteins from all gels were separated by electrophoresis at 180 V followed by protein transfer to a nitrocellulose membrane in transfer buffer (25 mM Tris and 192 mM glycine) containing 20% methanol. Before addition of primary antibodies, membranes were blocked in 5% milk in 0.1% TBST (1× TBS supplemented with 0.1% Tween-20) for 1 h and incubated in 3% BSA with primary antibodies overnight at 4 °C (Supplementary Table 1). Secondary antibodies were added for 1 h at RT (in blocking solution; Supplementary Table 1). Membranes were washed three times with 0.1% TBST, 10 min each, after primary and secondary antibody incubations and detected with ECL detection reagent (GE Healthcare).

Flow cytometry

Cell lines were labelled with 25 µM EdU for 30 min, harvested by standard trypsinization, washed with PBS once and fixed for 12 min with 4% formaldehyde in PBS. Cells were permeabilized with 0.25% Triton X in PBS, washed twice with PBS, and EdU was labelled by incubating in click reaction (0.1 M Tris pH 8.5, 0.1 M sodium-l-ascorbate, 2 mM CuSO₄ and 0.1 mM Alexa Fluor-linked azide (Thermo Fisher)) for 1 h at RT. Cells were washed twice with PBS and DNA was stained with 1 μg ml⁻¹ DAPI in PBS for 1 h. Samples were run on Attune NxT Flow Cytometer (Thermo Fisher) and analysed using FlowJo software v.10.0.8 (FlowJo). At least 10,000 cells were measured per condition. For gating strategy, see Supplementary Fig. 2.

Metaphase spreads

Asynchronous mAID-mClover-Sororin cells were treated with 2 μM aux and/or nocodazole for a total of 4 h. Mitotic cells were collected by shake-off, incubated in hypotonic buffer (0.03 M sodium citrate), fixed in Carnoy’s solution, dropped on acid-washed slides and left overnight at RT. After staining with 1 μg ml⁻¹ DAPI, slides were mounted with ProLong antifade and left to dry overnight before imaging in a Leica DM6000 microscope with LAS AF software.

DNA fibre assays

All cell lines subjected to this analysis were grown asynchronously and labelled with 30 μM of the thymidine analogue 5-chloro-2′-deoxyuridine (CldU; Sigma-Aldrich) for 30 min, washed three times with warm PBS and exposed to 250 μM of 5-iodo-2′-deoxyuridine (IdU) for 30 min. For fork progression and S1 DNA fibre assessments, IdU was added alone or in combination with the indicated genotoxic treatments and concentrations. For fork degradation assays, IdU was added alone for 30 min, cells were washed three times with PBS and media containing 4 mM HU was added for 3 h. All cells were collected by trypsinization. For S1 assays, cells were incubated with S1 and prepared for spreading as described in ref. ⁴⁹. All cells or nuclei were resuspended in cold PBS at 5 × 10⁵ cells ml⁻¹ or 2 × 10⁶ nuclei ml⁻¹. A total of 2 μl of this cell suspension was mixed with 7 μl of lysis buffer (200 mM Tris-HCl, pH 7.5, 50 mM EDTA and 0.5% SDS) on a glass slide. After incubating for 6 min at RT, the slides were tilted to a 45° angle to stretch the DNA fibres onto the slide. The resulting DNA spreads were air-dried and fixed in 3:1 methanol:acetic acid. The DNA fibres were denatured by incubating them in 2.5 M HCl for 1 h at RT, washed three times with PBS and blocked with 1% BSA in PBS-T (0.05% Tween-20 in PBS) for 1 h at RT. CldU and IdU tracks were stained for 2 h at RT using two different anti-BrdU antibodies recognizing CldU and IdU, respectively (Supplementary Table 1). After washing three times with PBS, slides were stained with secondary antibodies (Supplementary Table 1) for 1 h at RT in the dark. The slides were mounted in 30 μl Prolong Gold antifade reagent (Invitrogen). Microscopy was done using a Leica DM6 B microscope (HCX PL APO 63× objective). At least 100 fibres per sample were measured using ImageJ. Values were plotted and statistically assessed using GraphPad Prism 10.

Electron microscopy

Cells were asynchronously grown, treated with 1 µg ml⁻¹ dox for 48 h, 2 µM aux for 5 h and 50 µM HU where indicated. Cells were collected, resuspended in ice-cold PBS and crosslinked with 10 μg ml⁻¹ 4,5′,8-trimethylpsoralen and pulses of 365 nm ultraviolet (UV) irradiation with monochromatic light (UV Stratalinker 1800; Agilent Technologies). DNA was extracted as previously described⁴⁸. Briefly, cells were lysed (1.28 M sucrose, 40 mM Tris-HCl [pH 7.5], 20 mM MgCl₂, and 4% Triton X-100; Qiagen) and digested (800 mM guanidine-HCl, 30 mM Tris-HCl pH 8.0, 30 mM EDTA pH 8.0, 5% Tween-20 and 0.5% Triton X-100) at 50 °C for 2 h in presence of 1 mg ml⁻¹ proteinase K. The DNA was purified using chloroform:isoamylalcohol (24:1) and precipitated in one volume of isopropanol. DNA was washed with 70% EtOH and resuspended in 200 μl TE (Tris-EDTA) buffer. A total of 6 μg of was incubated with 120 U of PvuII HF (New England Biolabs) for 5 h at 37 °C. RNase A (Sigma-Aldrich, R5503) was added to a final concentration of 250 µg ml⁻¹ for the last 2 h of this incubation. Digested DNA was purified using a Silica Bead Gel Extraction kit (Thermo Fisher Scientific) according to the instructions of the manufacturer. DNA was spread on carbon-coated 400-mesh nickel grids (G2400N, Plano, using benzyl-dimethyl-alkyl-ammonium chloride). DNA was then coated with platinum using a High Vacuum Evaporator (EM BAF060, Leica) as previously described⁴⁸. The grids were imaged automatically at 28,000× using a Talos 120 transmission electron microscope (FEI; LaB6 filament, high tension ≤120 kV) with a bottom-mounted CMOS camera BM-Ceta (4,096 × 4,096 pixels) and the MAPS 3 software (Thermo Fisher Scientific). For analysis, the samples were annotated for replication intermediates using the MAPS offline viewer (v.3.28, Thermo Fisher Scientific), and the corresponding images were extracted. The replication intermediates were scored blind to the experimental condition using Fiji⁵⁹. For each experimental condition, at least 65 replication fork molecules were analysed in two distinct biological replicates. The values were plotted and statistically assessed using GraphPad Prism 10.

Proliferation and clonogenic assay

Cells were treated with 1 µg ml⁻¹ of dox for 48 h (for WT and 4E-SMC1 substituted cells) and 2 µM aux (1 day before treatment in NIPBL-cells and 3 days before treatment in SMC1-substituted cells). In each well of a 6-well plate, 1,000 cells were seeded. The cells were then optionally treated with 90 nM MMC (Sigma-Aldrich) for 24 h, washed, and incubated in normal media, or treated with 150 µM HU (Sigma-Aldrich) or 50 nM Aph (Sigma-Aldrich) for 9 days to allow formation of colonies. Subsequently, colonies were stained with 0.5% (wt/vol) crystal violet in 20% ethanol. Plates were imaged using a plate reader, and the number of colonies was quantified using ImageJ. Values were normalized to the UNT control, and then plotted and statistically assessed using GraphPad Prism 10.

Micro-C experiments

The Micro-C library was prepared using the Dovetail Micro-C Kit according to the protocol of the manufacturer. Briefly, cells were treated with 1 µg ml⁻¹ dox for 48 h and 2 µM aux for 5 h where indicated. The chromatin was fixed with disuccinimidyl glutarate (DSG) and formaldehyde in the nucleus. The crosslinked chromatin was then digested in situ with micrococcal nuclease (MNase). After digestion, the cells were lysed with SDS to extract chromatin fragments, which were then bound to Chromatin Capture Beads. Next, the chromatin ends were repaired and ligated to a biotinylated bridge adapter, followed by proximity ligation of adapter-containing ends. After proximity ligation, the crosslinks were reversed, the associated proteins were degraded, and the DNA was purified and then converted into a sequencing library using Illumina-compatible adaptors. Biotin-containing fragments were isolated using streptavidin beads before PCR amplification.

Repli-C experiments

Cells were treated with 1 µg ml⁻¹ dox for 48 h, 2 µM aux for 5 h where indicated. A total of 10 µM EdU was added for 15 min, either before collection or before washing and treating cells with 2 mM HU for 2 h. Repli-C was performed using the Dovetail HiChIP MNase Kit with modifications. The cells were collected by trypsinization, the chromatin was fixed with DSG and formaldehyde and permeabilized with 0.25% Triton X in PBS. EdU was clicked to digoxigenin by incubating in a click reaction (0.1 M Tris, pH 8.5, 0.1 M sodium-l-ascorbate, 2 mM CuSO₄ and 0.3 mM digoxigenin-azide (ATTBio)). Then, crosslinked chromatin was digested in situ with MNase and extracted after cell lysis using a combination of RIPA lysis and gentle sonication using Bioruptor Pico in RIPA buffer. The chromatin fragments were incubated with anti-digoxigenin antibody pre-coupled to A/G-coated beads, overnight rotating at 4 °C. Next, the chromatin ends were repaired and ligated to a biotinylated bridge adapter followed by proximity ligation of adapter-containing ends. After proximity ligation, the crosslinks were reversed, the associated proteins were degraded, and the DNA was purified and converted into a sequencing library using Illumina-compatible adaptors. Biotin-containing fragments were isolated using streptavidin beads before PCR amplification.

Micro-C and Repli-C analyses

Micro-C and Repli-C data were processed using a standardized Hi-C analysis pipeline. Raw paired-end sequencing reads were quality-trimmed using Trim Galore (quality threshold = 20, minimum length = 30 bp) and aligned to the hg38 human reference genome using BWA-MEM⁶⁰) with Hi-C-specific parameters (−5SP −T0). Aligned reads were processed with the pairtools suite: contacts were parsed with a minimum mapping quality of 40 (–min-mapq 40), allowing walks of up to five unique alignments (–walks-policy 5unique) with a maximum inter-alignment gap of 30 bp. Parsed pairs were sorted, and PCR duplicates were removed while retaining duplicate marking statistics. Valid contact pairs were converted to ‘.hic’ format using Juicer Tools (v.1.22.01) and to multi-resolution cool format (.mcool) using HiCExplorer with Knight–Ruiz (KR) matrix balancing normalization. For comparative analyses between conditions, biological replicates were merged using pairtools merge, and datasets were downsampled to equalize sequencing depth across conditions using pairtools sample. Downsampling fractions were calculated to match the condition with the lowest sequencing depth, ensuring unbiased comparisons (Supplementary Tables 2–6). Contact probability as a function of genomic distance was calculated as an intra-chromosomal contact frequency distribution, using logarithmically increasing genomic distance bins. The insulation score was computed using a custom Python script following the methodology described in ref. ⁶¹, extracting 10-kb resolution raw matrices and using a sliding window of 100 kb × 100 kb. Chromatin loops were detected using Chromosight⁶² on balanced contact matrices at 25 kb resolution, with a scanning distance range of 50 kb to 1 Mb. Moreover, architectural stripes (left and right patterns) were identified at 25 kb resolution.

Replication fountains, characterized by enriched long-range chromatin interactions radiating from replication origins, were identified using the FUN (Fountains Using Neighborhoods) algorithm⁵ on balanced contact matrices at 10 kb and 25 kb resolutions. Signal-over-noise (SoN) scores were calculated using a sliding window approach with an extension length of 500 kb, offset of 50 kb and padding widths of three bins (10 kb) or 1 bin (25 kb). Fountain structures were detected by extending outwards from SoN summits (extension pixels: 6–100 bins, step size 2 at 10 kb; 3–35 bins, step size 1 at 25 kb) and evaluated for statistical significance (P-value < 0.05, signal-to-background ratio >1.3).

Metaplots were created using coolpuppy⁶³ on Knight–Ruiz balanced matrices. In the inter-replication fork metaplots, the –scale option was added to scale all the regions to the same size. Intra-replicon was quantified from metaplot diagonal decay profiles by calculating the log₂ fold-change ratio between short-range (≤50 kb) and mid-range (50–100 kb) normalized contact frequencies. For each condition, diagonal decay was computed by extracting contact values at increasing distances from the diagonal using a sliding window approach (three bins each side of the centre point), with background normalization calculated from an extended window (10 bins). Values were log₂-transformed and smoothed using a three-point moving average kernel. The intra-replicon contact index was defined as log₂(mean contacts ≤50 kb/mean contacts 50–100 kb), in which higher values indicate increased short-range contacts. Values were plotted and statistically assessed using GraphPad Prism 10. Inter-replication fork interaction strength was quantified by extracting the sum of log₂ contact frequencies from a 6 × 6 pixel region centred in the origin–origin interaction, representing the enrichment of contacts between paired replication origins.

Identification of replication origins

Replication fork directionality (RFD) profiles were obtained from published Pu-seq data⁴³, which quantifies the differential usage of leading and lagging strand DNA polymerases during replication. To identify replication origins, we analysed the RFD signal across the genome at 10-kb resolution. For each chromosome (excluding chrM and chrY), binned mean values were extracted and smoothed using a moving average filter (window size = 5) followed by Savitzky–Golay filtering (window = 15, polynomial order = 3) to preserve signal features while reducing noise. Replication origins and termination zones were identified by detecting inflection points in the smoothed RFD profile through second-derivative analysis. The second derivative captures the curvature of the signal, identifying the precise genomic positions at which replication fork dynamics fundamentally change. Replication origins were called as downward inflection points (second-derivative changes from positive to negative), corresponding to bidirectional fork movement away from the origin. For each putative origin, replication strength was calculated as the difference between the maximum RFD value in the downstream region (between the origin and the next termination zone) and the minimum RFD value in the upstream region (between the previous termination zone and the origin). Origins with replication strength below a threshold of 0.05 were considered weak and excluded from further analysis. When weak origins were removed, adjacent termination zones were merged by averaging their genomic positions. The remaining strong origins were reassigned to their flanking termination zones to ensure consistency between origin and termination zone annotations. Origins lacking flanking termination zones on either side were discarded^64,65,66,67.

Reporting summary

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