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Replication stress activates DNA repair synthesis in mitosis

Abstract

Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis1. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs)2,3,4,5. CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS ‘expression’), particularly when cells have been exposed to replicative stress6. The MUS81–EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress7,8,9. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN+) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

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Figure 1: Replicative stress activates DNA synthesis at CFSs in mitosis.
Figure 2: MUS81, SLX4, SMC2 and WAPL promote DNA synthesis at CFSs in mitosis.
Figure 3: DNA synthesis at CFSs in mitosis suppresses non-disjunction.
Figure 4: POLD3 promotes DNA synthesis in mitosis.

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Acknowledgements

We thank T. Palmai-Pallag, L. Che, B. Tian, M. Xing, X. Geng and T. Wass for technical help. This study was supported by The European Research Council, Danish National Research Foundation and Nordea Foundation (H.W.M., Y.L. and I.D.H.), the National 1000 Talents Program, the National Natural Science Foundation of China (31370901, 81422031), Zhejiang Provincial Natural Science Foundation of China (LR14H160001) (S.Y.), National Key Scientific and Technology Support Program: Collaborative innovation of Clinical Research for chronic obstructive pulmonary disease and lung cancer, no. 2013BAI09B09 (H.S. and S.Y.), and The Danish Medical Research Council (S.M.). I.D.H. is a Qiushi Guest Professor at Zhejiang University.

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Authors and Affiliations

Authors

Contributions

S.M., S.Y., V.A.B., S.B., A.A. and W.W. carried out experiments. S.M., S.Y., H.W.M., H.S., Y.L. and I.D.H. designed experiments and interpreted results. S.M., H.W.M., Y.L. and I.D.H. wrote the manuscript, and all authors edited it.

Corresponding authors

Correspondence to Huahao Shen, Ying Liu or Ian D. Hickson.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Replicative stress activates DNA synthesis in mitosis.

a, b, Experimental workflow (a) and representative images (b) showing asynchronously growing U2OS cells pre-treated with low-dose APH (+APH) or without APH (−APH) followed by EdU labelling for 30 min. Nuclei were stained with DAPI (blue). S-phase cells are indicated as being positive for cyclin A (red) and EdU incorporation (green). Cells in G2 (cyclin A positive) and G1 daughter cells (cyclin A negative) show negligible EdU staining. c, Uncropped images from Fig. 1a showing EdU foci (red) formation in condensed mitotic nuclei stained using DAPI (blue). Scale bars (b, c), 10 μm. d, Still images from Supplementary Video 1 showing representative individual frames from a live cell imaging experiment (n = 3 live cell imaging experiments) at various time points. The yellow arrow (frame 3, from the left) shows the initiation of nuclear envelope breakdown (NEBD) and characteristic invaginations in the nuclear lamina in a late prophase cell with partially condensed chromosomes. Selected areas (white boxes) are enlarged below. Scale bars, 10 μm. e, Experimental workflows used to follow cells through mitosis and into the next cell cycle and as indicated. f, FACS analysis of U2OS and HeLa cells as indicated above. g, Western blot analysis for the mitosis-specific MPM2 epitope-containing antigens (top panel) and PH3S10 (bottom panel). Actin was used as a loading control.

Extended Data Figure 2 Assessment of mitotic progression and EdU incorporation following a reversible arrest of cells in very late G2 using the RO3306 CDK1 small molecule inhibitor.

ac, Experimental workflow used (a) and representative images (b) and quantification (c) showing the progression of cells through mitosis using PH3S10 (green) as a marker of entry into mitosis and chromosome condensation following a reversible G2 arrest. Nuclei and chromosomes are stained using DAPI (blue). d, Representative images showing the progressive disappearance of the nuclear lamina (lamin A/C, green) as cells progress into mitosis while condensing their nuclear genome. Yellow arrows indicate invaginations in the nuclear lamina upon entry into mitosis. Un/condensed nuclei were stained using DAPI (blue). e, f, Experimental workflows used (e) and representative images (f) showing DNA synthesis (EdU foci, red) in late G2 phase arrested cells or in cells released from this arrest into prometaphase or metaphase and as indicated. Low-dose APH (+APH) and/or EdU were added to cells where indicated. Intact or partially condensed nuclei are stained using DAPI (blue). g, Quantification of EdU positive (EdU+) or EdU negative (EdU) metaphase cells following a release from the nocodazole arrest (e, right column). Scale bars (b, d, f), 10 μm. n = number of cells quantified in each case as indicated. Data are means of at least three independent experiments. Error bars represent s.e.m.

Extended Data Figure 3 Replicative stress activates DNA synthesis at common fragile sites (CFSs) in mitosis.

a, b, Experimental workflow used (a) and representative images (FRA3B cropped from Fig 1d). (b) showing examples of chromosome breaks/gaps harbouring known CFS loci (ideograms, black arrows) and that are positive for EdU (red). Chromosomes were stained using DAPI (blue). Images were captured using a 60× objective. c, Representative images showing EdU FISH analysis to assess the co-localization (yellow arrowheads) between EdU foci (red) and biotin-labelled FRA3B or FRA16D FISH signals (green) in individual metaphase spreads. Chromosomes were stained using DAPI (blue). Selected regions of interest are magnified and shown in numbered panels. d, Quantification of co-localized EdU and FRA16D foci from panel c in cells treated with (APH) or without (control) low-dose APH. e, f, Representative images (e) and quantification (f) of EdU foci (red) at centromeres or telomeres (green) in DAPI-stained mitotic nuclei (blue). Selected regions of interest are magnified and shown in numbered panels below. Scale bars (c, e), 10 μm. Data are means of at least three independent experiments. Error bars represent s.e.m.

Extended Data Figure 4 Catalytically active MUS81, SLX4, WAPL and SMC2 are collectively required for sister chromatid disjunction in anaphase.

a, Representative western blots of the indicated proteins (left) after siRNA treatments (shown above). Actin and histone H3 were used as whole-cell lysate and nuclear loading controls, respectively, and PH3S10 indicates mitotic chromatin. b, c, Representative images (b) and quantification (c) of co-localized SLX4 foci (red) and FANCD2 twin foci (green) in mitotic cells (DAPI, blue) after pre-treatment of cells with low-dose APH (+APH). Selected regions of interest are magnified and shown in numbered panels. d, Same as panel c, except following the indicated RNA interference (RNAi). eg, Experimental workflow used (e), representative images (f) and quantification (g) of anaphase cells stained for PICH (red) and FANCD2 (green) after depletion of the proteins indicated on the left. Selected regions of interest are magnified and shown in numbered panels. Yellow arrows show lagging chromatin. DNA was stained with DAPI (blue). h, Representative images showing DAPI-positive bulky chromatin bridges in anaphase cells after depletion of indicated proteins and pre-treatment of cells with low-dose APH. In the upper panel, MUS81 RNAi-treated cells were reconstituted with either wild-type MUS81 (MUS81 WT) or a nuclease-dead MUS81 (MUS81(D338A/D339A)) version, as indicated in red. Scale bars (b, f, h), 10 μm. Data are means of at least three independent experiments. Error bars represent s.e.m.

Extended Data Figure 5 Early mitotic events promote DNA synthesis at CFSs in early mitosis and reduce the transmission of errors to daughter cells.

ac, Experimental workflow used (a), representative images (b) and quantification (c) of 53BP1 nuclear bodies (green) in G1 daughter cells (DAPI, blue) after the indicated siRNA treatments (shown above). df, Experimental workflow used (d), representative metaphase chromosome spreads (stained using DAPI) (e) and quantification (f) of sister chromatid cohesion release after depletion of the proteins indicated or following inhibition of PLK1 using the small molecule inhibitor BI-2536 (PLK1i) in mitosis. g, h, Experimental workflow used (g) and quantification (h) of FANCD2 DNA damage foci in EdU+ S phase cells after RNAi and APH pre-treatment (+APH) as indicated. i, j, Experimental workflow used (i) and quantification (j) of EdU-positive (EdU+) or EdU-negative (EdU) FANCD2 twin foci in prometaphase cells after exposure to PLK1i in mitosis. Scale bars (b, e), 10 μm. Data are means of at least three independent experiments. Error bars represent s.e.m.

Extended Data Figure 6 Administration of high-dose APH in mitosis abolishes DNA synthesis in mitosis and decreases cell survival.

a, b, Representative images (a) and quantification (b) of EdU incorporation at breaks/gaps on metaphase chromosomes (DAPI, blue) after low-dose aphidicolin pre-treatment in S phase. cf, Experimental workflow (c), representative FACS analysis profiles (d), representative EdU immunofluorescence images (e), and quantification (f) of a lack of co-localization between sites of EdU incorporation (labelled in the previous mitosis; red) and 53BP1 nuclear bodies (green) in G1 daughter cells. Selected regions of interest are magnified and shown in numbered panels. g, Representative FISH images showing localization of FRA16D (red signals) and FRA3B (green signals) on metaphase chromosomes (DAPI, blue). h, Representative images showing CFS-associated FANCD2 twin foci (green) and PICH staining (red) in prometaphase and anaphase MRC5 cells treated with or without high-dose APH (±2 μM APH) in mitosis and as indicated. White and yellow arrows indicate lagging chromosomes and anaphase bridges, respectively. i, j, Representative images (i) and quantification (j) showing FISH analysis using directly labelled ‘paint’ probes for chromosome 3 (green) or chromosome 16 (green) in cytochalasin B arrested binucleated cells (DAPI; blue) treated or not with low-dose aphidicolin (+ APH) in S phase. k, l, Experimental workflow used (k, top), representative crystal violet stained colonies (k, bottom) and quantification (l) of colony formation assay used to assess cell viability as indicated in HCT-116 or MRC5 cell lines. Scale bars, 10 μm unless indicated otherwise. Data are means of at least three independent experiments. Error bars represent s.e.m.

Extended Data Figure 7 DNA repair proteins recruited to regions synthesizing DNA in early mitosis after replicative stress.

a, b, Experimental workflows (a) and representative extended western blots of the indicated proteins (left, b) and in the indicated cell lines (top) from Fig. 4a. Actin and histone H3 were used as loading controls for soluble (cytosolic and nuclear) and insoluble (chromatin bound) fractions, respectively. PH3S10 indicates mitotic chromatin fractions. Three cell populations are shown: asynchronous (AS), G2-arrested (G2) and prometaphase (M). cf, Experimental workflows used (c, d) and western blots (e, f) showing POLD3 or POLD4 levels after either POLD3 (e) or POLD4 (f) siRNA treatments (shown above) in the indicated cell lines. Histone H3 was used as a nuclear loading control. g, FACS traces of asynchronous (left) and prometaphase (right) cells following RNAi as indicated. h, i, Representative images (h) and quantification (i) of FANCD2 DNA damage foci in EdU+ S-phase cells or EdU G2 phase cells after RNAi and APH treatment (+APH).

Extended Data Figure 8 POLD3 promotes faithful chromosome segregation and maintains genome stability.

a, Experimental workflow used. b, Representative western blot analysis of the proteins as indicated (left) from the whole-cell lysates obtained from two POLD3-siRNA-resistent wild-type POLD3 U2OS stable clones following the indicated RNAi treatments. c, d, Representative FISH images of isolated metaphase chromosomes (c) and quantification (d) of chromosome breaks/gaps at the FRA16D CFS locus (red). e, f, Representative images (e) and quantification (f) of UFBs stained using PICH (red) and FANCD2 foci at their bridge termini (green) in anaphase cells (stained using DAPI) (blue) and following the siRNA treatments as indicated (left). Selected regions are magnified and shown in separate numbered panels. g, h, Representative images (g) and quantification (h) of 53BP1 nuclear bodies (green) in early G1 daughter cells (stained using DAPI) (blue) following the siRNA treatments as indicated (left). ik, Representative western blots (i) and their quantification (j, k) of the indicated proteins (left) in chromatin-bound nuclear fractions from asynchronous or prometaphase (mitotic) cells following the RNAi treatment as indicated above. Histone H3 was used as a chromatin loading control and PH3S10 indicates mitotic chromatin. All scale bars, 10 μm. Data are means of at least three independent experiments. Error bars represent s.e.m.

Extended Data Figure 9 Cell lines analysed, and proposed models summarizing and extending the main findings of this study.

a, A list of cultured human cell lines analysed in this study. b, DNA-level model showing how a stalled replication fork at a CFS might be repaired after replication stress. This would first require cleavage of the stalled replication fork, which we propose utilizes the MUS81–EME1 endonuclease and/or other nucleases. The subsequent formation of a D-loop structure would initiate POLD3-dependent conservative DNA synthesis, akin to BIR, in early mitosis. c, A model at the chromosome level to indicate how MUS81- and POLD3-dependent DNA synthesis is triggered at CFSs (marked by SLX4 and FANCD2 twin foci), and how it is dependent on the collective action of chromosome condensation and WAPL-mediated cohesin release in prophase (diagrams in top panel). DNA synthesized in this pathway manifests as uncondensed regions that nevertheless facilitate chromosome disjunction (diagrams in top panel). In contrast, if WAPL-dependent cohesion dissolution in prophase or chromosome condensation is defective, or when MUS81 or POLD3 function is compromised, a reduction in CFS expression is observed, associated with an increase in chromosome non-disjunction/mis-segregation events, which can activate cell death and increase the frequency of aneuploidy (diagrams in bottom panel).

Extended Data Figure 10 Aneuploid/CIN+ cancer cells show elevated levels of DNA synthesis at CFSs in condensed mitotic nuclei.

a, Experimental workflow used to assess the DNA damage response in human cells after replicative stress (+APH). b, c, Representative images of asynchronous human cells (b) and quantification (c) showing FACS (top panel) and immunofluorescence analysis for FANCD2 DNA damage foci (green) in EdU-positive (red) S-phase nuclei (bottom two panels). d, Experimental workflow used to assess mitotic DNA synthesis at CFS-associated FANCD2 twin foci in human cells after replicative stress (+APH). e, f, Representative images (e) and quantification (f) showing FACS (top panel) and immunofluorescence analysis for EdU foci (red) and CFS-associated FANCD2 twin foci (green) (bottom two panels) in cells arrested in prometaphase. g, Total number and normalized values of co-localized EdU foci and FANCD2 twin foci. In each cell line studied, values per 100 cells scored were normalized to a diploid chromosome number. Scale bars (b, e), 10 μm. Data are means of at least three independent experiments. Error bars represent s.e.m.

Supplementary information

Chromosome condensation in prophase precedes nuclear envelope breakdown (NEBD)

A representative video (n = 3 live cell imaging experiments) demonstrating the progress of cells during a time-lapse microscopy experiment. The video shows a representative cell captured using DIC (differential imaging contrast) as it progresses from G2 (uncondensed nuclei) through mitosis (condensed chromosomes) and into the G1 phase (decondensed daughter cells) of the following cell cycle. Time is shown hours:minutes. The initiation of mitotic chromosome condensation occurs at 56 mins and characteristic invaginations in the nuclear lamina associated with the commencement of NEBD occur at 59 mins. (MOV 17660 kb)

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Minocherhomji, S., Ying, S., Bjerregaard, V. et al. Replication stress activates DNA repair synthesis in mitosis. Nature 528, 286–290 (2015). https://doi.org/10.1038/nature16139

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