ORIGINAL RESEARCH published: 28 September 2021 doi: 10.3389/fcell.2021.745195 Breakage of CRISPR/Cas9-Induced Chromosome Bridges in Mitotic Cells Marina Rodriguez-Muñoz, Martina Serrat, David Soler, Anna Genescà* † and Teresa Anglada* † Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain Chromosomal instability, the most frequent form of plasticity in cancer cells, often proceeds through the formation of chromosome bridges. Despite the importance of these bridges in tumor initiation and progression, debate remains over how and when they are resolved. In this study, we investigated the behavior and properties of chromosome bridges to gain insight into the potential mechanisms underlying bridge- induced genome instability. We report that bridges may break during mitosis or may remain unbroken until the next interphase. During mitosis, we frequently observed Edited by: discontinuities in the bridging chromatin, and our results strongly suggest that a Yu-Sheng Cong, substantial fraction of chromosome bridges are broken during this stage of the cell Hangzhou Normal University, China cycle. This notion is supported by the observation that the chromatin flanking mitotic Reviewed by: Marin Barisic, bridge discontinuities is often decorated with the phosphorylated form of the histone Danish Cancer Society Research H2AX, a marker of DNA breaks, and by MDC1, an early mediator of the cell response Center (DCRC), Denmark Jin He, to DNA breaks. Also, free 30 OH DNA ends were detected in more than half of the Michigan State University, bridges during the final stages of cell division. However, even if detected, the DNA United States ends of broken bridges are not repaired in mitosis. To investigate whether mitotic *Correspondence: bridge breakage depends on mechanical stress, we used experimental models in Anna Genescà anna.genesca@uab.cat which chromosome bridges with defined geometry are formed. Although there was Teresa Anglada no association between spindle pole separation or the distance among non-bridge teresa.anglada@uab.cat kinetochores and bridge breakage, we found a direct correlation between the distance † These authors have contributed equally to this work and share last between bridge kinetochores and bridge breakage. Altogether, we conclude that the authorship discontinuities observed in bridges during mitosis frequently reflect a real breakage of the chromatin and that the mechanisms responsible for chromosome bridge breakage Specialty section: This article was submitted to during mitosis may depend on the separation between the bridge kinetochores. Cell Growth and Division, Considering that previous studies identified mechanical stress or biochemical digestion a section of the journal as possible causes of bridge breakage in interphase cells, a multifactorial model Frontiers in Cell and Developmental Biology emerges for the breakage of chromosome bridges that, according to our results, can Received: 21 July 2021 occur at different stages of the cell cycle and can obey different mechanisms. Accepted: 07 September 2021 Published: 28 September 2021 Keywords: DNA bridges, genomic instability, chromosome damage, mitosis, DNA repair Citation: Rodriguez-Muñoz M, Serrat M, Soler D, Genescà A and Anglada T INTRODUCTION (2021) Breakage of CRISPR/Cas9-Induced An important characteristic of tumors is their ability to adapt. In the face of changes, be they Chromosome Bridges in Mitotic Cells. endogenous or exogenous, the neoplastic cell undergoes alterations to generate a variety of Front. Cell Dev. Biol. 9:745195. phenotypes, some of which give the cell the ability to survive. An important difficulty in combating doi: 10.3389/fcell.2021.745195 the adaptive capacity of neoplastic cells is that there is no single mechanism responsible for Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis genome instability, but there are at least four different that determine the breakage of bridges may be different in the mechanisms: excessive erosion of telomeric sequences, different stages of the cell cycle. accelerated DNA replication, impaired DNA repair, and defects By using different methods for the detection of DNA in chromosome segregation during mitosis. However, although breakage, we found that the discontinuities frequently observed intrinsically different, the four mechanisms share, to a greater in bridging chromatin at the end of mitosis mostly correspond or lesser extent, a common intermediary: the chromosome to real breaks. Whereas some bridges remain unbroken and bridge. This abnormal structure is frequently observed in tumor form nucleoplasmatic bridges, by employing experimental samples; it occurs 10 times more frequently in neoplasms and models to produce bridges with defined characteristics, we premalignant lesions than in healthy tissue (Rudolph et al., 2001; demonstrate that others resolve during mitosis, and their Gisselsson, 2003). Chromosome bridges associate with genomic breakage is associated with the distance between the kinetochores instability as they can cause the regression of the cleavage involved in the bridge. furrow and lead to the formation of tetraploid cells in which supernumerary centrosomes hinder the correct segregation of chromosomes (Ganem et al., 2009; Pampalona et al., 2012). MATERIALS AND METHODS Alternatively, bridges can break and lead to DNA damage restricted to the chromosome involved in the bridge, a process Cell Culture known as chromothripsis (Maciejowski et al., 2015, 2020). HEK 293T and 293T Phoenix cells were cultured in MEM, and Despite its close association with genome instability and its U2OS cells were propagated in 1:1 DMEM:Ham’s F10 medium. importance in tumor initiation and progression, the mechanisms All media were supplemented with 10% Fetal Bovine Serum that determine how and when bridges are resolved are unclear. (FBS). MCF10A were grown in DMEM:F12 with 5% horse According to the Breakage-Fusion-Bridge (BFB) model serum, 100 ng/mL cholera toxin, 10 µg/mL insulin, 20 ng/ml (McClintock, 1939), chromosome bridges can break and initiate epidermal growth factor, and 0.5 µg/mL hydrocortisone. RPE1 a cycle capable of self-feeding when a broken DNA end fuses Cas9 Tet-ON cells were obtained from Iain Cheeseman’s with another broken end and forms a new unstable chromosome Laboratory and cultured in DMEM:F12 with 10% tetracycline- structure. In this way, the BFB cycle can instigate a profound free FBS (FBS-TET-12A, Labclinics, Barcelona, Spain). U2OS reorganization of the genome. Based on time-lapse observations LacO/LacI-GFP-CENPT were a kind gift from Iain Cheeseman’s of living cells, classical studies placed the breakage of chromatin Laboratory (Gascoigne et al., 2011) and were cultured in 10 mM bridges during the last stages of cell division (Hoffelder IPTG (Isopropyl β-D-1-thiogalactopyranoside, I6758-1G, Merck, et al., 2004; Shimizu et al., 2005). However, studies aimed at Kenilworth, NJ, United States) and 200 µg/mL Hygromycin B determining the causes of bridge rupture during mitosis reached (H3274, Merck). U2OS GFP-MDC1 were obtained from the controversial conclusions. A rapid shrinking of the chromatin Laboratory of Manuel Stucki and were grown in 250 µg/mL of fiber compatible with tangling of the severed chromatin prior geneticin (11811023, Thermo Fisher Scientific, Waltham, MA, to nuclear membrane reformation and completion of cytokinesis United States). Penicillin/streptomycin (100 U/mL) was added was reported by Shimizu et al. (2005). Whereas these observations to the media, and cell cultures were maintained in a humidified indicate that mechanical tension would be responsible for incubator at 37◦ C and 5% CO2 . severing anaphase bridges, other studies suggest that constricting forces generated by the cleavage furrow break chromosomal DNA Plasmid Construction (Janssen et al., 2011). A retroviral plasmid was obtained by cloning the GFP-Lap2β More recent studies argue that the often-visible discontinuities sequence (a gift from Ulrike Kutay’s Laboratory) into the in bridging chromatin during the last stages of mitosis do not pLNCX2-mCherry-CHMP4B plasmid (a gift from Sanford correspond to actual breaks (Maciejowski et al., 2015; Umbreit Simon; Addgene plasmid #116923) between the AgeI and et al., 2020). Unlike the classical models, Maciejowski et al. (2015) NotI restriction sites, thus removing the mCherry-CHMP4B and Umbreit et al. (2020) state that chromosome bridges cannot gene. Guide RNA for the subtelomeric region of chromosome be resolved in anaphase and, therefore, inevitably lead to the 2 (50 -ATATTAAGGGCTCCCCGTCG-30 ) was designed using presence of chromatin in the cleavage plane. According to these the CCTop - CRISPR/Cas9 target online predictor platform authors, stabilized nucleoplasmic bridges persist in daughter cells (Stemmer et al., 2015) and was cloned into lenti-sgRNA for a considerable period of time as the presence of chromatin blast (gift from Brett Stringer, Addgene #104993) between in the cleavage plane inhibits abscission, the final stage of the BsmBI restriction sites. The cloning protocol for lenti- cytokinesis (Steigemann et al., 2009). From there, destabilization sgRNA blast is described in addgene website1 . All plasmids were of the bridge due to mechanical stress (Umbreit et al., 2020) verified by Sanger sequencing. After sequencing, plasmids were or enzymatic digestion after loss of nuclear envelope function amplified by transformation into Stbl3 (C737303, Invitrogen, (Maciejowski et al., 2015, 2020) will eventually lead to bridge Waltham, MA, United States) or DH5α competent cells resolution in the interphase. In summary, different models have (18258012, Thermo Fisher Scientific) and purified using the been proposed to explain how and when chromosome bridges NucleoBond PC kit (740573.100, Macherey-Nagel, Bethlehem, are resolved, and it is unclear at present whether they can break PA, United States) following the manufacturer’s instructions. during mitosis or, alternatively, remain invariably unbroken until the end of mitosis to break in the next interphase. It is important 1 https://media.addgene.org/data/plasmids/104/104993/104993-attachment_ to distinguish between these two possibilities, as the mechanisms fbZFcCQmsWQB.docx Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis FIGURE 1 | Frequencies of chromosome bridges in RPE1 Cas9 sgRNA Chr4 cells. (A) Representative images of bridges during the last stages of mitosis (EA, early anaphase; LA, late anaphase; ET, early telophase; LT, late telophase) and interphase. Chromosome bridges are visualized with DAPI (blue) during mitosis and with GFP-BAF (green) during interphase. To restrict the analysis to G1 cells, immunofluorescence of cyclin D1 (red) was performed, and its labeling is shown in red. Scale bar = 10 µm. (B) Percentage of bridges during the last stages of mitosis and in interphase cells at the G1 stage. Chromosome bridges were induced with the CRISPR/Cas9 Chr4 methodology, and cells were synchronized with RO3306 and released for 43–120 min to enrich the mitotic or interphase cell populations, respectively (n = 227 cells in mitosis and 1032 cells in G1). The DNA concentration was measured with a NanoDrop 2000 irradiator (dose rate of 5.10 Gy/min) and incubated at 37◦ C and spectrometer (Thermo Fisher Scientific). 5% CO2 for 24 h prior to fixation. (2) To generate bridges using an inducible ectopic kinetochore function, IPTG was removed Viral Particle Production and from the media to allow the interaction between LacO and LacI- Transduction GFP-CENPT (after seven washes with PBS), and the cells were Lentivirus or retrovirus particles were produced in HEK 293T fixed 24 h later. (3) To generate bridges with the CRISPR/Cas9 or 293T Phoenix, respectively. Cells were transfected at 70% based model in cells expressing sgRNA targeting subtelomere confluency with 1 µg/mL of total DNA plasmid following regions, Cas9 was expressed by adding 1 µg/ml doxycycline the protocol from Trono’s Laboratory website2 . For lentivirus (631311, Clontech, Mountain View, CA, United States) to the particle production, the DNA concentration of all plasmids medium for 15 h, and a washout of 24 h was performed before was adjusted to a ratio 4:3:1 (transfer plasmid:psPAX2:pMD2.G; the cells were fixed. When indicated, cells were synchronized Supplementary Table 1). The supernatant was harvested 48 h in G2 phase with 9 µM RO3306 (SML0569, Sigma-Aldrich, later, concentrated through centrifugal filters (UFC910008, Saint Louis, MO, United States) for 18 h and were released Merck), and stored at −80◦ C. for 43–120 min. Cells were transduced with viral particles (Supplementary Table 1) overnight using medium with 4 µg/mL polybrene. For BrdU Incorporation retroviral transduction, the procedure was repeated three times. For the BrdU pulse, cells were seeded in 35-mm-diameter petri Cells with the antibiotic-resistance cassette were selected, with dishes and after 24 h, 10 µM 5-bromo-20 -deoxyuridine (BrdU) 2.5 µg/mL blasticidin (15205, Merck) or 250 µg/mL geneticin was added to the culture for 8 h and then substituted with 10 µM (11811023, Thermo Fisher Scientific) for 2 weeks or 2 µg/mL BrdU for an additional 16 h. puromycin (P8833, Merck) for 1 week. Transduced fluorescent cells were enriched by fluorescent activated cell sorting (FACS) Immunofluorescence by using a BD FACSJazz (BD Biosciences, Franklin Lakes, NJ, Cells were fixed with 4% paraformaldehyde (PFA) for 15 min, United States) equipped with a 488 nm laser and two detectors permeabilized with 1x PBS/0.5% Triton-X-100 solution for (530/40 and 692/40 nm). 20 min, and blocked with 1x PBS/0.5% BSA/0.15% glycine for 15 min. For BrdU labeling, after permeabilization, cells were Methods to Generate Chromosome treated with 2 M HCl for 20 min and incubated in a borate Bridges buffer (pH = 9) for 5 min at room temperature. For the CtIP Chromosome bridges were generated using different labeling, after PFA, cells were additionally incubated in ice- methodologies: (1) To generate bridges with irradiation, cold methanol and acetone for 30 min and 1 min, respectively. cells were exposed to 2.5 Gy using an IBL-437C R-137 Cs Then, a blocking step was performed in 1x PBS/1% FBS/5% BSA. After the blocking step, primary antibodies were incubated 2 https://www.epfl.ch/labs/tronolab/wp-content/uploads/2019/06/LV_production. overnight at 4◦ C. Alternatively, BrdU antibody was incubated pdf for 3 h at room temperature. All antibodies are listed in Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis FIGURE 2 | Chromosome bridge breakage is marked with γH2AX. (A) Representative images of chromosome bridges (DAPI, blue) that exhibit γH2AX labeling (red). Representative bridges with (i) a single γH2AX focus in the middle of the bridge, (ii, iii) two γH2AX foci flanking the discontinuity of a chromosome bridge, and (iv) γH2AX labeling spanning the chromosome bridge. Scale bar = 5 µm. (B,C) Frequency of chromosome bridges γH2AX-positive and -negative classified according the continuity or discontinuity of the DAPI staining for (B) all mitotic stages together and for (C) cells segregated by phase (EA, early anaphase; LA, late anaphase; ET, early telophase; LT, late telophase). Asterisks indicate statistical differences between continuous and discontinuous bridges regarding the γH2AX labeling (Fisher’s exact test, ****p < 0.0001; n = 609 from 5 replicates). Chromosome bridges were induced with the CRISPR/Cas9 Chr4 methodology, and cells were synchronized with RO3306 and released for 43 or 50 min to enrich the number of cells in the anaphase and telophase stages of mitosis, respectively. Supplementary Table 2. After three washes with 1x PBS/0.1% on glass microscope slides with Vectashield mounting medium Tween 20 or after a 15 min wash in PBS/1% Triton-X-100 (for (Vector Laboratories, Inc., Burlingame, CA, United States) the BrdU immunofluorescence), the secondary antibodies listed containing 0.25 µg/mL 40 ,6-diamidino-2-phenylindole (DAPI). in Supplementary Table 2 were incubated for 1 h at room Sample visualization and image acquisition were performed temperature. Finally, all samples were washed, briefly rinsed with using an Olympus BX61 epifluorescent microscope (Olympus, distilled water, progressively dehydrated in alcohol, and mounted Hamburg, Germany) equipped with a CV-M4 + CL camera (JAI, Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis at 73◦ C in formamide 70%/2x SSC buffer and underwent progressive dehydration. The Chr2 whole-chromosome probe (Vysis probes, Des Plaines, IL, United States) was also denatured at 73◦ C for 5 min prior to hybridization for 12 h at 37◦ C. Instead, the LacO-TxRed probe (50 - CATGTGGAATTGTGAGCGGATAACAATTTGTGG-30 ) was hybridized for 1 h at room temperature. Following hybridization, slides were rinsed with 0.4x SSC/0.3% NP-40 for 2 min at 55◦ C and 2x SSC/0.1% NP-40 for 2 min at room temperature. Finally, samples were dehydrated and counterstained with DAPI (0.25 µg/mL). Samples were visualized using an Olympus BX61 microscope, as described in the immunofluorescence section. SensiTive Recognition of Individual DNA Ends For the STRIDE assay, cells were seeded into 24 × 24-mm coverslips, and chromosome bridges were induced with the CRISPR/Cas9 system. Samples were fixed in PFA 4%, and coverslips were sent embedded in 1x PBS to intoDNA (Krakow, Poland). The dSTRIDE methodology was applied according to the protocol previously described by Kordon et al. (2019). Live-Cell Imaging For time-lapse experiments, cells were seeded onto MatTek dishes (P35G-1.5-14-C, MatTek, Ashland, Massachusetts, MA, United States) and allowed to attach for 48 h. The day of the experiment, metaphase or early anaphase (EA) cells were identified using the RFP-H2B signal. Images from five z-stacks (with a 2-µm separation between planes) were acquired every 3 min for 30–60 min with a Zeiss Axio Observer Z1 inverted fluorescent microscope (Zeiss, Oberkochen, Germany) equipped with Zen blue software (Zeiss) and an AxioCam MRm camera (Zeiss). Cells were maintained under controlled conditions (5% CO2 and 37◦ C) during the experimental time course. FIGURE 3 | Broken chromosome bridges exhibit 30 OH DNA ends. (A) Representative image of STRIDE (red) detecting the DNA ends flanking the Statistics discontinuity of the chromosome bridges (DAPI, blue). Kinetochores are labeled with CREST antibody (green). Arrowheads indicate the 30 OH DNA Fisher’s exact test was used to determine differences between ends of a broken bridge. Scale bar = 5 µm. (B) Frequency of STRIDE-positive categorical variables. For continuous variables, data normality and -negative chromosome bridges classified according to the continuity or was analyzed using the D’Agostino-Pearson omnibus K2 discontinuity of DAPI staining. Chromosome bridges were induced with the normality test. If the data were normally distributed, a t-test CRISPR/Cas9 methodology, and cells were synchronized with RO3306 and was used for unpaired statistical analysis; the Mann–Whitney released for 43 min to enrich the anaphase and telophase populations (Fisher’s exact test, ***p < 0.001; n = 82 chromosome bridges). test was applied for unpaired non-parametric analysis. Statistical analysis and graph plotting were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, United States) and Großwallstadt, Germany) and CytoVision software (Applied p-values < 0.05 were considered statistically significant. Imaging, Newcastle, United Kingdom). Colocalization was determined visually as the coincidence in space of the protein RESULTS of interest with each γH2AX focus. When indicated, image analysis and measurements were performed using ImageJ We used three different methods to generate chromatin bridges: (Schindelin et al., 2012). moderate-dose radiation exposure, inducible function of an ectopic kinetochore by expression of the LacI-GFP-CENPT Fluorescence in situ Hybridization chimeric protein in cellular clones stably transfected with Cells were fixed in methanol:acetic acid (3:1) for 10 min, rinsed LacO (Gascoigne et al., 2011; Supplementary Figure 1A), twice with 2x SSC, and dehydrated in ethanol. Additionally, and CRISPR/Cas9-mediated DNA double-strand breaks on for oligo-FISH, samples were incubated with formaldehyde- chromosome 2 (Supplementary Figure 1B) or chromosome 4 MgCl2 buffer for 10 min. Samples were denatured for 3 min (Umbreit et al., 2020; Supplementary Figure 1C). In addition to Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis FIGURE 4 | MDC1 recruitment to chromatin bridges in mitotic cells. (A) Frequency of MDC1-positive and -negative staining chromosome bridges during the last stages of mitosis (EA, early anaphase; LA, late anaphase; ET, early telophase; LT, late telophase). Frequencies of MDC1 recruitment to continuous and discontinuous chromosome bridges (continuity was assessed with DAPI; Fisher’s exact test, ****p < 0.0001; n = 208 chromosome bridges from two replicates). Chromosome bridges were induced with the CRISPR/Cas9 Chr4 methodology, and cells were synchronized with RO3306 and released for 43 or 50 min. (B) Percentages of MDC1/γH2AX colocalization in interphase cells vs. in chromosome bridges during mitosis. Error bars indicate SD (Fisher’s exact test, ns p > 0.05; n = 213 γH2AX foci in interphase cells, n = 146 γH2AX foci in mitotic bridges; two replicates). (C) Representative images for the colocalization of MDC1 (red) and γH2AX (green) at chromosome bridges (DNA in blue). Scale bar = 10 µm. (D) Representative images of a time-lapse of MDC1 (green) recruitment to chromosome bridges (red) in U2OS GFP-MDC1/RFP-H2B cells. Images from anaphase entrance to 24 min later are shown. Bridges were spontaneously induced. Arrowheads highlight an MDC1 focus at the end of a broken bridge. Scale bar = 10 µm. chromosome bridges generated by the three methods described and mostly involved the chromosomes against which guide RNAs above, we examined those spontaneously formed in genomically were designed (Supplementary Figures 1B,C; Umbreit et al., unstable U2OS cells. While spontaneously formed bridges and 2020). As for the inducible kinetochore experimental model, those induced by radiation were randomly generated throughout although they theoretically should also be specific, we frequently the cell genome, those obtained by the CRISPR/Cas9 system in observed the formation of non-specific bridges. In this model, RPE1 cells were formed under exquisite spatio-temporal control the inhibition of the interaction between the LacI-GFP-CENPT Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis frequency of chromosome bridges as cells advanced from cell division to interphase (Figure 1), a reduction that was consistent with a progressive resolution of chromosome bridges during the last stages of cell division. While we observed bridges in 83.33% of cells in EA and 81.13% in late anaphase (LA), the frequency of cells with bridges decreased to 40.91 and 37.09% in early and late telophase (ET and LT), respectively, and we only detected bridges with GFP-BAF (a sensitive reporter of chromosome bridges in interphase as its signal is not compromised by bridge stretching) in 9.79% of interphase cells at the G1 stage of the cell cycle (positive for cyclin D1). The differences in the frequencies of bridges between mitotic and interphase cells cannot be attributed to a low proliferation index as most of the interphase cells were cycling (92.12% of interphase cells incorporated BrdU after a 24- h pulse) (Supplementary Figure 2). A similar stage-associated reduction in the frequencies of bridges was also observed using other experimental systems (83.33, 58.49, 32.89, and 24.39% of irradiated MCF10A cells at EA, LA, ET, and LT, respectively, and 6.90% in interphase visualized by GFP-Lap2β; Supplementary Figure 3). These findings suggested that fundamental aspects of bridge-breakage mechanisms remained to be clarified, as the observed decrease in the frequency of bridges is not compatible with the claim that all or most bridges persist through mitosis and cytokinesis. Breakage of Chromosome Bridges During Mitosis Indirect evidence for mitotic bridge breakage came from the observation that bridges in mitotic cells were frequently labeled with markers of DNA rupture. To determine the extent to which mitotic bridges were labeled in a manner compatible with their severing, we immunodetected the phosphorylated form of FIGURE 5 | Replication protein A recruitment to chromosome bridges in the histone variant H2AX (then termed γH2AX), which is a mitotic cells. (A) Representative image for the RPA32 labeling on chromatin marker that flags regions in the genome that contain chromosome bridges. RPA32 (red), flanked by γH2AX (green), forms a filament-like structure that connects the two groups of segregated DNA breaks (Rogakou et al., 1998, 1999). In the CRISPR/Cas9 chromosomes (blue). Arrowhead highlights the RPA filament. Scale experimental cell model, a visible discontinuity in the chromatin bar = 10 µm. (B) Distribution of RPA-positive and -negative staining in fiber was observed in 43.84% of mitotic bridges (considering continuous and discontinuous chromosome bridges during the last stages of all stages of mitosis together), and 73.03% of them presented mitosis (EA, early anaphase; LA, late anaphase; ET, early telophase; LT, late γH2AX signaling (Figures 2A,B). Conversely, 91.52% of the telophase). Chromosome bridges were induced with the CRISPR/Cas9 Chr4 methodology, and cells were synchronized with RO3306 and released for 43 continuous bridges did not show γH2AX signaling (Fisher’s or 50 min (n = 127 chromosome bridges). exact test, p < 0.0001; Figure 2B). Regarding the location of the γH2AX signal on the DNA bridge (Figure 2A), three main categories were defined: (a) a single γH2AX focus usually in chimeric protein and the ectopic LacO sequences is not complete the middle of the bridge; (b) two γH2AX foci flanking the and consequently, DNA bridges accumulate with cell passage. discontinuity of the intervening chromatin fiber; or (c) multiple γH2AX foci spanning the chromatin bridge. Interestingly, most of the continuous bridges positive for γH2AX staining displayed The Frequency of Chromosome Bridges a single or two very close γH2AX foci in the middle of the bridge Decreases as Cells Exit Mitosis and (82.14%), probably indicating a recent breakage. In contrast, Progress to Interphase the predominant labeling pattern in the discontinuous subset It has been proposed that mammalian dicentric chromosomes of chromatin bridges was a γH2AX focus located at each end can withstand mitotic spindle and cytokinetic ring forces to of the discontinuity (76.92%). Only a small fraction (9.74%) of invariably persist through cell division and form long chromatin the discontinuous bridges positive for γH2AX displayed multiple bridges between daughter cells at the interphase (Maciejowski γH2AX foci over the chromatin. et al., 2015; Umbreit et al., 2020). Using the CRISPR/Cas9 The observed association between the morphology of the experimental system, we detected a progressive reduction in the bridges and their γH2AX labeling pattern was especially Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis FIGURE 6 | 53BP1, BRCA1, and CtIP are not recruited to broken chromosome bridges in mitotic cells. Representative images for the colocalization of (A) 53BP1 (green) with γH2AX (red), (B) BRCA1 (red) with γH2AX (green), and (C) CtIP (red) with γH2AX (green). Upper panels correspond to interphase cells, and lower panels show mitotic cells with chromosome bridges. DNA is counterstained with DAPI (blue). All images correspond to RPE1 cells with CRISPR/Cas9-induced chromosome bridges using sgRNA Chr4. Cells were synchronized with RO3306 and fixed after a 45-min release. Scale bar = 10 µm. Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis the continuous bridges also presented STRIDE labeling. Taken together, these results indicate that most of the discontinuities observed in chromosome bridges reflect an actual breakdown of the chromatin fiber. DNA Ends in Mitotic Chromosome Bridges Are Sensed as Broken Ends Upon appearance of a DSB, cells activate the DNA damage response (DDR), which comprises two main stages: the initial detection of DNA breaks followed by downstream events leading to cell cycle arrest and DNA repair (Jackson and Bartek, 2009). Numerous factors involved in DNA break signaling, processing, and repair accumulate at damaged sites and form focal structures. However, although the recruitment of DDR factors to DSBs in interphase cells is efficient, the condensed structure of chromatin in mitosis is known to make it difficult to recruit or retain DDR factors to the chromatin flanking the broken ends (Giunta et al., 2010; Heijink et al., 2013; Alcaraz Silva et al., 2014). Given the important role of MDC1 (mediator of DNA damage checkpoint 1) in the early steps of the DDR (Coster and FIGURE 7 | Distance between pericentrins is not associated with bridge Goldberg, 2010; Jungmichel and Stucki, 2010), we investigated breakage in RPE1 Cas9 sgRNA Chr4 cells. (A) Representative images of its recruitment to broken bridges in the CRISPR-cell model by mitotic chromosome bridges (DAPI, blue) immunolabeled with γH2AX (red) immunodetecting MDC1 in fixed cells. We found that, similar and pericentrin (green). Scale bar = 5 µm. (B) Schematic illustration of the to γH2AX, MDC1 labeled the chromatin flanking chromosome measurement of the distance between pericentrins. Pericentrins are depicted in green, d = distance between pericentrins. (C) Distance between bridge discontinuities (Fisher’s exact test for MDC1 labeling pericentrins classified by γH2AX labeling of the bridge. The mean and SD are continuous and discontinuous bridges, p < 0.0001; Figure 4A). indicated (t-test, ns p > 0.05; n = 183). After CRISPR/Cas9 chromosome Furthermore, given that γH2AX provides a docking site for the bridge induction, cells were synchronized with RO3306 and fixed after a DDR-mediator protein MDC1, we wanted to assess whether 43-min release. γH2AX and MDC1 coincided at the broken ends of chromosome bridges. We found that MDC1 colocalized with γH2AX in the bridges of mitotic cells as it does in interphase cells (94.52 and informative when we classified the bridges according to the 91.08% colocalization in mitotic bridges and interphase cells, stage of mitosis (Figure 2C). Whereas the fraction of continuous respectively; Fisher’s exact test, p = 0.31; Figures 4B,C). In a bridges decreased from EA (63.04%) to LT (42.25%), and most complementary manner, the MDC1 signal was absent from those of them were devoid of γH2AX foci, bridges with visible bridges without γH2AX labeling. discontinuity increased complementarily, and most of them Next, we evaluated in living cells the dynamics of MDC1 showed a γH2AX signal. Therefore, the γH2AX-based method recruitment to chromatin bridges in mitosis. To this end, we for the indirect detection of DNA breaks indicates that a used a human osteosarcoma cell line U2OS stably expressing substantial fraction of chromosome bridges are broken during the GFP-tagged MDC1 and RFP-H2B in which the endogenous final stages of cell division. MDC1 was knocked out using CRISPR/Cas9 (Leimbacher et al., In addition to methods based on monitoring of histone 2019). Time-lapse recording of living cells showed recruitment modification at the damage sites, we used a method for the direct of the fluorescent protein on bridges in mitotic cells followed by detection of DNA DSBs (double-strand breaks) in chromosome retraction of the chromatin fiber (Figure 4D and Supplementary bridges during mitosis. The method we used is abbreviated Video 1). Therefore, we conclude that DNA ends in broken STRIDE (section “Sensitive Recognition of Individual DNA bridges of mitotic cells are detected and signaled by the DDR Ends), and it enables the direct in situ detection of 30 OH cellular machinery. DNA ends even when they are individual DNA double-strand When instead of DSBs the lesion produces single-stranded cuts (Kordon et al., 2019). It consists of the conjugation DNA fragments, replication protein A (RPA) is known to detect of deoxynucleotide analogues to free DNA ends and their them and act as a platform to recruit other factors involved in detection by hybridization with fluorescent nucleotides after DNA repair. Using an anti-RPA32 antibody in the CRISPR/Cas9 rolling circle amplification. The STRIDE assay was applied to Chr4 experimental model, we found that although most mitotic the detection of free DNA ends in compacted mitotic chromatin bridges showed no RPA32 signal, in 22.05% of the bridges in in RPE1 cells. In the RPE1 CRISPR/Cas9 experimental model, mitotic cells, we observed a long RPA32 signal extending like a total of 82 chromosome bridges were identified in mitotic a filament physically connecting the two groups of recently cells, and 65.15% presented STRIDE labeling at the chromatin segregated chromosomes (Figure 5A). As shown in Figure 5B, flanking the discontinuity (Figure 3). In addition, 12.5% of RPA32 signaling was mostly detected at telophase, indicating that Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis FIGURE 8 | Chromosome bridge breakage is associated with the separation between bridge kinetochores in RPE1 Cas9 sgRNA Chr4 cells. (A) Representative images for the immunofluorescent labeling of kinetochores (CREST, green) and γH2AX (red) on chromosome bridges (DAPI, blue). (i) Arrowheads indicate the CREST signals of a continuous bridge (γH2AX-negative) that are displaced away from the non-bridge kinetochores of the cell. (ii) Representative image of a broken bridge (γH2AX-positive) with no displacement of the bridge kinetochores. (iii) Ring chromosome with one broken and one unbroken bridge sharing kinetochores (arrowheads). Scale bar = 5 µm. (B) Distance between the non-bridge kinetochores of cells with bridges classified according to their γH2AX labeling in unbroken (γH2AX-negative) and broken (γH2AX-positive) bridges. The mean and SD are indicated (Mann–Whitney test, ns p > 0.05; n = 216 cells from two replicates). (C) Schematic illustration for measurements taken of the distances between kinetochores (green): distance between bridge kinetochores (dark gray line), regions including the non-bridge kinetochores (yellow highlighted), centroid of the region (black cross), distance between centroids (pale purple line), distance between the bridge kinetochores and the centroid referred to as displacement (orange line). (D,E) Displacement of bridge kinetochores from the cluster of non-bridge kinetochores classified according to (Continued) Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis FIGURE 8 | (Continued) the γH2AX labeling of the bridge. For (D) all mitotic stages together and for (E) cells segregated by phase (EA, early anaphase; LA, late anaphase; ET, early telophase; LT, late telophase). Displacement is calculated as the distance between the bridge kinetochores and the centroid of non-bridge kinetochores (in orange in the scheme) and normalized to the distance between centroids of the cluster of non-bridge kinetochores (in pale purple in the scheme). The mean and SD are indicated. Asterisks indicate statistical differences between unbroken and broken bridges (Mann–Whitney test, ****p < 0.0001, **p < 0.01, ns p > 0.05; n = 212 from two replicates). (F,G) Distance between bridge kinetochores (dark gray line in the diagram) classified according to the γH2AX labeling of the bridge in broken (γH2AX-positive) and unbroken (γH2AX-negative) bridges for (F) all mitotic stages together and for (G) segregated phases. The mean and SD are indicated. Asterisks indicate statistical differences between γH2AX-negative and γH2AX-positive bridges (Mann–Whitney test, ****p < 0.0001, ***p < 0.001, **p < 0.01, ns p > 0.05; n = 212 from two replicates). Dashed line at 5.96 µm indicates the minimum distance from which bridges begin to break. (H) Graph displaying the distances between the kinetochores (KT) involved in the bridge (distances between kinetochores of γH2AX-negative bridges are represented in blue and those of γH2AX-positive bridges in red). Each horizontal line represents a cell. Cells are ordered according to the distance between bridge kinetochores. Pale purple dots represent the distance between the non-bridge kinetochores of each cell. All images and analyses correspond to chromosome bridges induced by the CRISPR/Cas9 Chr4 system and cells synchronized with RO3306 and fixed after 43 and 50 min of release (n = 212 from two different experiments). it might require some time for RPA32 to be visible after DNA triggered at the mitotic DNA ends. Collectively, these findings breakage. When present, the RPA32 signal coincided with the suggest that the DNA repair machinery cannot be fully launched chromatin discontinuity, and it was frequently flanked by γH2AX in those bridges broken during mitosis. Thus, bridges may signals (41.30%; n = 46 chromosome bridges). Therefore, our remain unrepaired while the ends of the broken chromosome results indicate that bridge rupture in mitosis does not always are pulled apart. proceed through DSB formation. Sometimes, nicks can occur that affect only one strand of DNA with the subsequent formation of Breakage of Bridges in Mitotic Cells Is long RPA32-labeled single-stranded DNA fragments. Altogether, we conclude that chromosome bridges severed during mitosis Associated With their Stretching exhibit apical aspects of the DDR, as they are marked with During the last stages of mitosis, microtubules of the mitotic γH2AX and MDC1 and sometimes with RPA. spindle stretch the bridging chromatin toward the opposite poles of the cell. To determine whether the increasing distance between the poles of the mitotic cell is associated with bridge DNA Ends in Mitotic Chromatin Bridges rupture, we detected pericentrin, a component of centrosomes, Are Not Processed for Repair in the RPE1 CRISPR/Cas9 Chr4 experimental model. We Having established that mitotic cells detect and signal the found no association between the distance between cell poles broken ends of DNA bridges, we next examined the behavior and the breakage of bridges in mitotic cells (signaled with of 53BP1 (p53-binding protein 1), BRCA1 (breast cancer type γH2AX) (t-test; p = 0.46; Figure 7). In concordance with 1 susceptibility protein), and CtIP (CtBP-interacting protein), this result, we found no differences between cells with broken three proteins involved in the second stage of the DDR. At a and unbroken bridges when measuring the average distance molecular level, the balance between 53BP1 and BRCA1 plays between non-bridge kinetochores by using a CREST antibody an important role in the choice of a DSB repair pathway, and (Mann–Whitney test; p = 0.62; Figures 8A–C). Therefore, the CtIP is responsible for DNA end-resection initiation (Sartori separation between cell poles does not determine chromosome et al., 2007; Escribano-Díaz et al., 2013; Ceccaldi et al., 2016). bridge breakage. By using an anti-53BP1 antibody in the CRISPR/Cas9 Chr4 Although there was no association between bridge breakage experimental system and in irradiated MCF10A cells, we found and cell pole distance, we frequently observed that the that, in marked contrast to interphase cells, 53BP1 was excluded bridge kinetochores were displaced from the cluster of non- from mitotic chromatin (average number of 53BP1 foci per bridge kinetochores (Figure 8A, panel i). When we measured cell was 2.43 and 2.56 in RPE1 and MCF10A interphase cells, the distance of this displacement (Figure 8C) we found respectively, and 0 in mitotic cells). It is important to note that, that it was longer for unbroken chromosome bridges than whereas in interphase cells most 53BP1 foci colocalized with for the broken ones (Mann–Whitney test, p < 0.0001; γH2AX foci (RPE1: 90.33%, 243/269 foci; MCF10A: 94.55%, Figure 8D), thus indicating that unbroken bridges were 191/202 foci; Figure 6A upper panel), in broken mitotic bridges under tension, which was released by breakage. In fact, γH2AX did not recruit 53BP1 (RPE1: 0/71 foci; MCF10A: 0/47 the displacement was only significant on unbroken bridges foci; Figure 6A lower panel). A similar pattern was found of cells at the EA and LA stages of mitosis (Mann– when we detected BRCA1 (Figure 6B). Whereas focal structures Whitney test; p < 0.01; Figure 8E), which is precisely when of BRCA1 on γH2AX-decorated chromatin were observed in mitotic spindle microtubules generate maximum tension on interphase cells (RPE1: 69.40% colocalization in BRCA1 positive segregating chromosomes. cells in interphase, 195/281 foci; MCF10A: 56.50%, 113/200 To confirm the role of mechanical stress in bridge resolution, foci), colocalization of this factor with γH2AX was absent in we then measured the distance between the bridge kinetochores chromatin bridges of mitotic cells (RPE1: 0/71 foci; MCF10A: and found that there were significant differences between broken 0/53 foci). CtIP was also missing in those broken chromosome and unbroken bridges: the longer the distance between the bridges marked with γH2AX (RPE1: 0/66 foci; MCF10A: 0/41 bridge kinetochores, the higher the probability of bridge breakage foci; Figure 6C), thus indicating that DNA end-resection was not (Mann–Whitney test, p < 0.0001; Figure 8F). It is important Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis to note that this association was only observed in EA and most of the chromosome bridges broke during the anaphase LA, but not in telophase (Mann–Whitney test; p < 0.01; and telophase stages. Thus, we conclude that chromosome Figure 8G). Furthermore, when cells were classified according bridges may break during the last stages of cell division. to the distance between bridge kinetochores, we found that in Our conclusion is based on several observations: firstly, this experimental system (RPE1 CRISPR/Cas9 Chr4) a minimum on immunofluorescent analysis of mitotic cells, in which distance of 5.96 µm between centromeres was needed for the discontinuous chromatin fiber of bridges is frequently bridges to break (Figures 8F,G). Once the bridge kinetochores labeled with intense γH2AX staining on both sides of had exceeded this distance, we found broken bridges, but the discontinuity. Secondly, on the time-lapse recording of also unbroken bridges, which could indicate that in addition mitotic cells expressing a fluorescent form of the DDR factor to tensile forces there may be other factors that influence MDC1, as we observed chromosome bridge end retraction the breakage of bridges during mitosis (Figure 8H). Similar after MDC1 recruitment to DNA. Finally, confirmation of results were observed for bridges induced in U2OS cells with bridge breakage during mitosis was obtained by the STRIDE the LacO system (Supplementary Figure 4) giving support assay, as 30 -OH free ends were detected in the bridges of to our conclusion. mitotic cells. However, bridge rupture in mitosis does not In order to further corroborate this finding, we used the always proceed through DSB formation. Sometimes, nicks system RPE1 CRISPR/Cas9 with a sgRNA targeting the q-arm can occur in mitosis that affect only one strand of DNA subtelomeric region of Chr2 as it allowed us to generate with the subsequent formation of long RPA32-labeled single- bridges with a different distance in base pairs between bridge stranded DNA fragments. centromeres (Supplementary Figure 1). While subtelomeric target sequences in Chr4 are at 139.4 and 50.7 Mb from the centromere, for Chr2 there is only one target sequence, and it The Ends of Broken Chromosome is at 147.6 Mb from the centromere; thus, the intercentromeric Bridges in Mitotic Cells Are Detected but distance is longer for Chr2 than for Chr4 bridges. Interestingly, Not Repaired we found that for bridges induced with the Chr2 CRISPR/Cas9 Although genome stability needs to be maintained during cycles system, the minimum distance between bridge kinetochores for cells to pass on their hereditary material to the next from which bridges begin to break is 8.2 µm (Supplementary generation, cells suppress DNA repair during mitosis as changes Figure 5), longer than the 5.96 µm obtained for bridges in chromatin structure necessary for repair would interfere induced with the Chr4 RNA guides. This result reveals a with the correct segregation of chromosomes during mitosis relationship between the centromere distance in base pairs of (Blackford and Stucki, 2020). Thus, cells prioritize completion the dicentric chromosome and the minimum separation in of mitosis over activation of a full DDR and repair of DNA micrometers between bridge kinetochores from which bridge damage. According to this notion, our data show that the breakage occurs. recruitment to broken bridges of key downstream factors that Finally, as the guide RNA for RPE1 Cas9 Chr4 targets regulate DSB repair pathway choice such as 53BP1, BRCA1 and a sequence located at the end of both, the p- and the CtIP is blocked during mitosis. However, the cellular response to q-arm (Supplementary Figure 1C), in some mitotic cells we DNA breaks is only partially disrupted in broken bridges during identified bridging rings. This particular kind of bridges are mitosis as upstream events such as H2AX phosphorylation and formed when a break in the p-arm plus a break in the MDC1 recruitment still occur. A similar specialized response q-arm of chromosome 4 are induced and fusion between to DNA breaks in mitosis was previously described for DNA sister chromatids leads to one short and one long bridge breaks induced by exposure to ionizing radiation (Giunta (Figure 8A, panel iii). In these double bridges, we frequently et al., 2010) or laser microirradiation (Alcaraz Silva et al., found one broken and one unbroken bridge sharing only a 2014). Furthermore, 53BP1 was found to be actively removed pair of kinetochores, confirming that with the same kinetochore from mitotic chromatin as it dissociates from endogenously separation two bridges of different intercentromeric base pair arising DNA DSBs at the G2/M boundary (FitzGerald et al., length may respond differently to the tension exerted by the 2009; Nelson et al., 2009). Thus, the truncated nature of the microtubules of the mitotic spindle. Although we cannot rule DDR in mitotic cells does not depend on how DNA injury out the contribution of other causes to the resolution of was induced or whether injury was induced before or after chromatin bridges, our results suggest that the mechanical entering mitosis. stress to which bridges are subjected may contribute to It has been suggested that the activation of early DDR events their resolution. in mitotic cells may facilitate recognition of DNA damage and its repair during the following cell cycle (Giunta et al., 2010). Although this is plausible for breaks induced in most DISCUSSION regions of the genome, it cannot be conceived of when the DNA ends are pulled apart, such as in the case of a broken Our results show that there is no single cell cycle stage at chromosome bridge. It is unlikely that the two DNA ends which chromosome bridges are broken. Although we found derived from a broken bridge will be rejoined in the daughter that some bridges persisted beyond telophase and formed cells, as repair proteins cannot overcome the tension of the nucleoplasmic connections between the two daughter cell nuclei, mitotic spindle that pulls the two ends toward opposite poles Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 September 2021 | Volume 9 | Article 745195 Rodriguez-Muñoz et al. Chromosome Bridge Breakage in Mitosis (Doherty and Jackson, 2001; Blackford and Stucki, 2020). DATA AVAILABILITY STATEMENT Therefore, once produced, the breakage of the chromosome bridge is likely permanent. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be Intense Stretching and Constriction of directed to the corresponding author/s. the Condensed Chromosome of Bridges in Dividing Cells We found an association between the distance between bridge AUTHOR CONTRIBUTIONS kinetochores and the probability of breakage of the chromosome bridge. These results indicate that the mechanical tension to AG and TA: conceptualization and supervision. MR-M: design which the chromosome bridge is subjected during the last stages of guide RNAs. MR-M and MS: methodology and results. of mitosis would contribute to its breakage. However, we cannot DS: specialized technical support. TA, MR-M, and MS: exclude the possibility that additional mechanisms, such as those formal analysis. AG: writing—original draft preparation, project based on biochemical digestion or mechanical compression, administration and funding acquisition. TA, AG, and MR-M: could contribute to the breakage of chromosome bridges. writing—reviewing and editing. All authors have read and agreed Apart from the particular characteristics of each bridge, to the published version of the manuscript. at least two additional components need to be considered to understand the behavior of chromosome bridges during mitosis: the complexity of mitotic chromosomal decondensation and the FUNDING magnitude of the forces applied to the bridging chromosome. It could be speculated that, because the mitotic chromosome This study was funded by La Marató de TV3 Foundation is highly condensed, it would decondense to prevent breakage (201918-30). The research of the Genome Integrity Group at when it is stretched to opposite poles. However, the mitotic the Universitat Autònoma de Barcelona is supported by the chromosome must be able to withstand mechanical stress and Consejo de Seguridad Nuclear and the Generalitat de Catalunya viscous resistance as premature decondensation of chromatin (2017-SGR-503). MR-M holds a postgraduate grant funded by the would cause the sister chromatids to become entangled Universitat Autònoma de Barcelona. and missegregated. According to this notion, chromosome segregation and decondensation are tightly orchestrated with cytokinesis at the exit of mitosis (Kitagawa and Lee, 2015). As for the forces applied on the chromosome bridge, spindle ACKNOWLEDGMENTS microtubules can generate pushing or pulling forces by adding or losing subunits from their ends. At the single-molecule level, We would like to gratefully acknowledge IntoDNA for cytoskeletal proteins generate small forces. However, during cell performing the STRIDE assay on our samples. We also thank the division these proteins function cooperatively to generate forces Scientific and Technical Services from the Universitat Autònoma in the range of nanonewtons and serve to accurately move de Barcelona (SCAC and UTPR) and Joan Repullés from the separated chromatids over distances of micrometers (Scholey IDIBELL Optical Microscopy platform. Finally, we especially et al., 2003). Our results suggest that the force generated by thank those laboratories that have provided us with plasmids, hundreds to thousands of cytoskeletal force generators acting cell lines, and DNA probes that have been essential for this cooperatively must be able to break chromosome bridges work (David Pellman and Neil Umbreit’s Laboratory, Ulrike during anaphase. Kutay’s Laboratory, Manuel Stuki’s Laboratory, Iain Cheeseman’s Considering that previous studies identified mechanical Laboratory, and the Genetics of Male Fertility Research Group at stress or biochemical digestion as possible causes of bridge Universitat Autònoma de Barcelona). breakage in interphase cells, a multifactorial model emerges for the breakage of chromosome bridges that, according to our results, can occur at different stages of the cell SUPPLEMENTARY MATERIAL cycle and can obey different mechanisms. Therefore, we offer support for the validity of the BFB model as The Supplementary Material for this article can be found a mechanism capable of self-feeding to generate new online at: https://www.frontiersin.org/articles/10.3389/fcell.2021. changes in the genome. 745195/full#supplementary-material REFERENCES Blackford, A. N., and Stucki, M. (2020). How cells respond to DNA breaks in mitosis. Trends Biochem. 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Conflict of Interest: The authors declare that the research was conducted in the Biol. 3:14. doi: 10.3389/fcell.2015.00014 absence of any commercial or financial relationships that could be construed as a Kordon, M. M., Zar˛ebski, M., Solarczyk, K., Ma, H., Pederson, T., and Dobrucki, potential conflict of interest. J. W. (2019). STRIDE—a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells. Publisher’s Note: All claims expressed in this article are solely those of the authors Nucleic Acids Res. 48:e14. doi: 10.1093/nar/gkz1118 and do not necessarily represent those of their affiliated organizations, or those of Leimbacher, P. A., Jones, S. E., Shorrocks, A. M. K., de Marco Zompit, M., Day, the publisher, the editors and the reviewers. Any product that may be evaluated in M., Blaauwendraad, J., et al. (2019). MDC1 Interacts with TOPBP1 to Maintain this article, or claim that may be made by its manufacturer, is not guaranteed or Chromosomal Stability during Mitosis. Mol. Cell 74, 571.e8–583.e8. doi: 10. endorsed by the publisher. 1016/j.molcel.2019.02.014 Maciejowski, J., Chatzipli, A., Dananberg, A., Chu, K., Toufektchan, E., Copyright © 2021 Rodriguez-Muñoz, Serrat, Soler, Genescà and Anglada. This is an Klimczak, L. J., et al. (2020). APOBEC3-dependent kataegis and TREX1-driven open-access article distributed under the terms of the Creative Commons Attribution chromothripsis during telomere crisis. Nat. Genet. 52, 884–890. doi: 10.1038/ License (CC BY). The use, distribution or reproduction in other forums is permitted, s41588-020-0667-5 provided the original author(s) and the copyright owner(s) are credited and that the Maciejowski, J., Li, Y., Bosco, N., Campbell, P. J., and De Lange, T. (2015). original publication in this journal is cited, in accordance with accepted academic Chromothripsis and kataegis induced by telomere crisis. Cell 163, 1641–1654. practice. No use, distribution or reproduction is permitted which does not comply doi: 10.1016/j.cell.2015.11.054 with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 14 September 2021 | Volume 9 | Article 745195