Method Details

Vectors were transfected into cells using Lipofectamine 2000 (Invitrogen, Cat. No. 11668-019) and Opti-MEM Reduced Serum Medium (Invitrogen, Cat. No. 31985-070). For each transfection 1 μg of construct DNA was mixed with 400 μL Opti-Mem and 5 μL Lipofectamine-2000. To avoid aggregation of DNA and Lipofectamine-2000, the DNA was pre-mixed in 200 μL of Opti-Mem and separately, the 5 μL of Lipofectamine were mixed into 200 μL of Opti-mem. The two components were then mixed together and incubated for 20 min at RT. This transfection mixture was added to the tissue cultures in 2 mL of antibiotic-free media.

Cells were suspended in NuPAGE LDS sample buffer (ThermoFisher) with 10 mM DTT, incubated at 100°C for 5 min and sonicated briefly. Protein samples were resolved on 8% bis-tris gels (ThermoFisher) and transferred to Immobilon-P PVDF 0.45 μm membrane (Merck Millipore) by wet transfer. Membranes were probed with antibodies using standard techniques and detected by enhanced chemiluminescence. Antibodies used for western blotting were as follows: CAP-H (Bethyl A300-603A, 1:1000), CAP-D3 (Bethyl, A300-604A, 1:1000) and GAPDH (Cell Signaling 2118L, 1:5000).

Cells were synchronized at the G1/S boundary by addition of high dose aphidicolin (APH, Calbiochem). Media containing 5 μg/ml APH was added to cells for 2 h to block cell cycle and retain cells at the G1/S boundary. Cells were washed in PBS and released in normal growth media. FACS analysis and immunofluorescence of cell populations at 2 h – 10 h following release showed that cells progressed synchronously from S-phase into G2. Replication stress was induced by low dose treatment of APH (0.4 μM APH), for extended periods (12 – 24 h).

RPE1 cells were treated with 0.1 μg/ ml colcemid (Life Technologies, Cat No 15210-040) for 1 h prior to harvest, and HCT116 for 30 min to induce mitotic arrest and increase the number of mitotic cells. Cells were trypsinised and washed in PBS. Hypotonic solution, containing 75 mM KCl was added drop wise to a final 5 mL volume. Hypotonic treatment was performed at RT for 10 min, after which cells were pelleted by centrifugation at 1200 rpm for 5 min and fixed three times in 5 mL of freshly prepared solution of 3:1 ratio (v/v) methanol: acetic acid. The MAA fixative was added to the cell pellet dropwise with constant agitation. Chromosome preparations were stored at −20°C. To prepare slides with metaphase spreads, metaphase chromosome preparations were dropped onto glass slides. The glass slides were pre-treated in a dilute solution of HCl in ethanol for at least an hour prior to use. The chromosome preparations were pelleted by centrifugation at 1500 rpm for 5 min and resuspended in freshly prepared MAA solution until the suspension became cloudy. Two drops of the suspension were dropped onto a pre-treated glass slide from a height of 20 cm and dried at RT overnight before staining or hybridization.

To map the location of fragile sites two complementary approaches were used. First, a visual inference of the fragile locus position was made using reverse DAPI banding. Second, the position of the fragile site was determined by calculating the distance along the chromosome arm. In this ratio-based approach, the total length (a), in pixels, of the chromosome arm that the break occurred on and the pixel length of the distance between the centromere and the break (b) were measured. The ratio (b) / (a) was calculated and used on scaled models of banded chromosomes (from the International System for Human Cytogenetic Nomenclature) to infer genomic locations for the breaks. The ratios clustered along the chromosome arms, indicating recurrent breaks at CFS locations and the mid-point of each cluster was taken as a putative CFS location. However, as fixation and spreading of chromosomes is likely to cause some distortion, molecular fine-mapping of the most frequent CFS regions was also undertaken using FISH.

Probes used in this study are listed in Table S2. After mapping fragile sites, the following probes were used to interrogate genomic loci by FISH (probe ID: A14, A17, K5, C2, L12, A13, P21, C14). DNA was prepared from the BACs or Fosmids and labeled as previously described (Naughton et al., 2010). Probes were labeled using a nick translation reaction with the uridine analogs biotin-16-dUTP (Roche, CatNo 11093070910) or digoxigenin-11-dUTP (Roche, CatNo 11093088910). Nick translation was performed in a 20 μL reaction volume, containing 1-1.5 μg DNA with 5 μL each of 0.5 mM dATP, dCTP and dGTP and either 2.5 μL of 1 mM biotin-16-dUTP or 1 μL of 1 mM digoxigenin-11-dUTP. DNase I (Roche, Cat No 4716728001) was added to a final concentration of 1 U/ml and DNA polymerase I (Invitrogen, Cat No 18010025) was added to a final concentration 0.5 U/μl. The reaction was performed in 1 x nick translation salts (NTS) buffer, containing 50 mM Tris pH7.5, 10 mM MgSO4, 0.1 mM DTT and 50 μg/ml BSA for 90 min at 16°C. Unincorporated nucleotides were removed by gel filtration of the NTS reaction through a G50 Sephadex spin column (Roche, Cat No G50DNA-RO). Slides, containing either MAA-fixed chromosome spreads or PFA-fixed nuclei, were treated with 100 μg/ml RNaseA (Invitrogen, Cat No 12091039) in 2 x SSC for 1 h at 37°C, washed briefly in 2 x SSC and dehydrated through an ethanol series (2 min each in 70%, 90% and 100% ethanol). Slides were air-dried and baked at 70°C for five min before denaturing. Denaturation was performed in 70% formamide (v/v) in 2 x SSC (pH 7.5). Slides containing MAA-fixed chromosome spreads were denatured at 70°C for 1 min, while slides on which cells were cultured and then fixed in 4% PFA were denatured at 80°C for 20 min. Following denaturation, slides were submerged in ice-cold 70% ethanol for 2 min and then dehydrated through 90% and 100% ethanol for 2 min each at RT. For hybridization, 150 ng of labeled probe was combined with 5 μg of salmon sperm and 10 μg of human Cot1 DNA (Invitrogen, Cat No 15279011). Two volumes of ethanol were added and the probe mix was collected by centrifugation and dried. Dried probes were resuspended in 10 μL of hybridization buffer containing 50% formamide (v/v), 1% Tween-20 and 10% dextran sulfate (Sigma Aldrich, Cat No D8906-100G) in 2 x SSC. Probes were denatured at 70°C for 5 min and reannealed at 37°C for 15 min and chilled on ice. Probes were pipetted onto slides and hybridization was performed at 37°C overnight. Coverslips were then removed and slides were washed four times in 2 x SSC at 45°C for 3 min and four times in 0.1 x SSC at 60°C for 3 min. Slides were then blocked in 5% milk in 4 x SSC for 5 min at RT. Detection of biotin label was performed with sequential layers of fluorescein (FITC)-conjugated avidin, biotinylated anti-avidin and a further layer of FITC-avidin. Digoxigenin was detected with sequential layers of Rhodamine-conjugated anti-digoxigenin and Texas-Red (TR) –conjugated anti-sheep IgG. Slides were DAPI stained, mounted in Vectashield and imaged on a Zeiss epifluorescence microscope using a 100x objective. Data was collected using micromanager software and analyzed using custom scripts in iVision or ImageJ. FISH signals were scored based on symmetry and overlap with DAPI chromosome staining: FISH probes showing asymmetric signals or signals extending outside the chromosome scaffold were classified as irregular, while symmetric signals contained within the chromosome scaffold were classified as regular. Whenever possible, scoring was performed blindly to experimental conditions. In FISH experiments measuring inter-probe distance, ambiguous images in which the two alleles could not be distinguished, were not used in the analysis.

Premature chromosome condensation was induced using the protein phosphatase 1 inhibitor calyculin (Sigma, C5552-10UG). To determine the cell cycle stage of prematurely compacted chromosomes, asynchronously growing HCT116 cells were pulsed with EdU (5 μM) for 6 hours and then treated with 50 ng/ml calyculin for 1 hour. Cells were then harvested using trypsin/versene. As calyculin treatment induced significant cell detachment, media containing the detached cells was collected and centrifuged together with the trypsinised cells. Metaphase spreads were prepared and dropped onto glass slides using the methods described above. Incorporated EdU was labeled in a click reaction by incubating slides with a reaction mixture containing 500 μg/ml CuSO4, 40 μM Alexa Fluor 488 Azide (Thermo Fisher Cat. No. A10266) and 20 mg/ml ascorbic acid for 1 hour at RT. Slides were washed three times in PBS for 5 min, stained with DAPI and mounted in Vectashield. Slides were imaged on a Zeiss Epifluorescence microscope using 100x objective. Chromosomal morphology and EdU staining pattern were used to classify chromosomes into G1, S, G2 and M-stage chromosomes.

For immunofluorescence on metaphase chromosomes, cell suspensions fixed in 3:1 methanol: acetic acid were dropped onto glass slides, allowed to dry incompletely and immediately immersed in PBS for 5 minutes at room temperature. Slides were washed in TEEN buffer (10 mM Triethanolamine- HCl pH 8.5, 2 mM EDTA, 250 mM NaCl) and blocked in 10% fetal calf serum (FCS) at 37°C for 10 minutes. Primary antibodies were added at the required dilutions and incubated in a humidified chamber at 37°C for 30 minutes. Slides were then washed in KB buffer (100 mM Tris-HCl pH 7.7, 1.5 M NaCl, 1% BSA). Secondary antibodies, raised in donkey and conjugated to fluorophores (Jackson Immuno Research), were diluted 1:500 in TEEN buffer, added to the slides and incubated at 37°C for 30 minutes. Slides were washed in KB buffer and stained in 50 μg / ml DAPI for 3 min at RT to detect DNA and nuclei. Slides were mounted in Vectashield (Vector Laboratories, Cat No H-1000) and imaged on a Zeiss Epifluorescence microscope using 100x objective. Primary antibody anti-H3S10p (1:100 dilution, clone CMA313) was a gift from Hiroshi Kimura (Hayashi-Takanaka et al., 2009), and detected with a Texas Red anti-mouse secondary antibody (1:500 dilution, Jackson Immuno Research). Anti-SMC2 antibody was detected with an FITC labebelled anti-rabbit secondary antibody (1:500 dilution, Jackson Immuno Research).

EdU was added to exponentially growing cell cultures for 30 min at 5 μM for replication labeling or 20 μM for analyzing mitotic DNA synthesis. EdU was detected by incubating slides with a click reaction mixture containing 500 μg/ml CuSO4, 40 μM Alexa Fluor 488 Azide (Thermo Fisher Cat. No. A10266) and 20 mg/ml ascorbic acid for 1 hour at RT. Slides were washed three times in PBS for 5 min, stained with DAPI and mounted in Vectashield. Slides were imaged on a Zeiss Epifluorescence microscope using 100x objective.

For dual EdU and propidium iodide (PI) staining for cell cycle analysis, cells were trypsinised, pelleted and resuspended in PBS at a density of 1.5 × 10⁶ cells/ml. Ethanol was slowly added to the cell suspension to a concentration of 70% to fix and permeabilise the cells, which were incubated on ice for a minimum of 30 min or stored at 4°C for up to 2 weeks. EdU staining was performed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen, CatNo {"type":"entrez-nucleotide","attrs":{"text":"C10634","term_id":"1535705","term_text":"C10634"}}C10634) following the manufacturer’s instructions. Cells were stained in a solution containing 1 μg/ml PI and 4 μg/ml RNase A in PBS at 2 × 10⁶ cells/ml for a minimum of 30 min at RT. Cell cycle analysis was performed on a LSR Fortessa analyzer (BD Biosciences) and analyzed using FlowJo software.

SMC2-AID-mClover cells were a derivative of tet-OsTIR1 HCT116 cells established in the Kanemaki laboratory (Natsume et al., 2016). C terminus targeting constructs for the SMC2 gene contained a 5′ homology arm (410 bp), mAID tag, mClover, resistance cassette and a 3′ homology arm (482 bp) (mAID tag, mClover tag and Hygromycin or G418 resistance cassettes were taken from pMK289 and pMK290. The guide RNA target sequence was TCCACATGTGCTCCTTTGGG. Constructs and resultant cell lines were established using published approaches from the Kanemaki laboratory (Natsume et al., 2016). CAPH-AID-mCherry and CAPH2-AID-mCherry HCT116 cells were a kind gift from the Imamoto lab, RIKEN, Japan (Takagi et al., 2018). For SMC2 degradation SMC2-AID-clover cells were incubated with 1μg/mL doxycycline overnight to induce OsTir1 expression and treated with 500μM Indole-3-acetic acid (IAA) for 24 h. For CAP-H and CAP-H2 degradation, HCT116 cells expressing AID tagged proteins were incubated with 500 μM Indole-3-acetic acid (IAA) for 8 h.

All genomic coordinates are HG38. COSMIC mutations were assessed at CFSs by examining the COSMIC track on the UCSC browser.