Research ArticleMOLECULAR BIOLOGY

Mus81-Eme1–dependent aberrant processing of DNA replication intermediates in mitosis impairs genome integrity

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Science Advances  09 Dec 2020:
Vol. 6, no. 50, eabc8257
DOI: 10.1126/sciadv.abc8257
  • Fig. 1 Chk1 loss triggers replication catastrophe and chromosome mis-segregation by independent pathways.

    (A) Western blot of γH2AX, phospho-RPA32 Ser4/8, phospho-KAP1 Ser824, KAP1, Chk1, and CDC45 in U2OS cells, 48 hours after transfection. The left panel shows the chromatin fraction, obtained after an extraction with CSK buffer; the right panel shows whole-cell extracts. H2B and actin were used as loading controls. (B) Percentage (mean ± SD) and representative images of U2OS cells with pan-nuclear γH2AX staining. More than 2000 cells per sample were analyzed in three independent experiments. Scale bar, 20 μm. As in all graphs in the manuscript, different letters indicate significant differences (see Materials and Methods). (C) Quantification by neutral comet assay of DSB accumulation in U2OS cells (A.U., arbitrary units). The right panel shows representative images of DNA comets. Three hundred cells per sample were analyzed in three independent experiments. The bars on top of the distribution clouds indicate the median. Scale bar, 10 μm. (D) Percentage (mean ± SD) and representative Z-stack images of U2OS anaphase cells with aberrations. About 150 anaphases per sample were analyzed in three independent experiments. The total percentage of aberrant anaphases (bridges plus lagging chromosomes) was used to calculate the statistics. Scale bar, 5 μm. (E) Percentage (mean ± SD) and representative images of binucleated U2OS cells with micronuclei. About 750 binucleated cells per sample were analyzed in three independent experiments. Scale bar, 10 μm. (F) Excess CDC45 in Chk1-deficient cells provokes DSBs and acute replication stress but does not cause chromosome mis-segregation.

  • Fig. 2 Mus81-Eme1 triggers CIN in Chk1-deficient cells.

    (A) Quantitative real-time PCR of Eme1 and Eme2 normalized to GAPDH in U2OS cells, 48 hours after transfection; error bars represent the SD of two technical replicates. (B) Percentage of U2OS cells with pan-nuclear γH2AX staining (mean ± SD). More than 1600 cells per sample were analyzed in two independent experiments. (C) Quantification by neutral comet assay of DSB accumulation in U2OS cells. Three hundred cells per sample were analyzed in three independent experiments. The bars on top of the distribution clouds indicate the median. (D) Percentage of U2OS anaphase cells with aberrations (mean ± SD). About 100 anaphases per sample were analyzed in two independent experiments. The total percentage of aberrant anaphases (bridges plus lagging chromosomes) was used to calculate the statistics. (E) Percentage of binucleated U2OS cells with micronuclei (mean ± SD). About 600 binucleated cells per sample were analyzed in three independent experiments.

  • Fig. 3 Mus81-Eme1 triggers mitotic DSBs in Chk1-deficient cells.

    (A) Percentage of metaphase chromosomes with breaks/gaps (mean ± SD) and representative images of intact or broken chromosomes and whole metaphase spreads. HCT116 cells were transfected with the indicated siRNAs and transduced 5 hours later with nontargeting shRNA [shScramble (shScr)] or shRNA targeting Chk1 (shChk1). About 4500 chromosomes (from 100 metaphases) per sample were analyzed in two independent experiments. Scale bars, 1 μm. (B) Percentage (mean ± SD) and representative Z-stack images of mitotic U2OS cells with >10 γH2AX foci. About 150 metaphases per sample were analyzed in three independent experiments. Scale bar, 5 μm. (C) Percentage of mitotic U2OS cells with >10 γH2AX foci (mean ± SD). About 120 metaphases per sample were analyzed in three independent experiments. (D) Model in which Mus81-Eme1–dependent DSBs in mitosis trigger CIN in Chk1-deficient cells, independently of Mus81-Eme2–dependent DSBs in S phase.

  • Fig. 4 DNA synthesis in mitosis precedes Mus81-Eme1–dependent cleavage and CIN in Chk1-deficient cells.

    (A) Percentage (mean ± SD) and representative Z-stack images of mitotic U2OS cells with EdU/FANCD2 spots. About 100 metaphases per sample were analyzed in two independent experiments. Scale bar, 5 μm. (B) Percentage (mean ± SD) and representative images of HCT116 chromosomes (DAPI, red) with semiconservative, conservative, or complex patterns of EdU incorporation (green). APH (0.2 μM) was added as a control, 24 hours before EdU incorporation. Only shChk1-transduced and APH-treated samples are shown, because shScr-transduced and DMSO-treated samples did not exhibit EdU incorporation. Four hundred EdU-positive events per sample were analyzed in two independent experiments. Scale bar, 1 μm. (C) DAPI-negative breaks (white arrows) at sites of EdU incorporation in metaphase chromosomes from shChk1-transduced HCT116 cells. No breaks were detected in EdU-negative DNA. Scale bar, 1 μm. (D) Percentage of EdU-positive events with breaks (mean ± SD) in HCT116 metaphase chromosomes. Four hundred EdU-positive events per sample were analyzed in two independent experiments. The representative images depict Eme1-dependent chromosome breakage at sites of shChk1-induced EdU incorporation. Scale bar, 1 μm. (E) Quantitative real-time PCR of PolD3 and Rad52 normalized to GAPDH in U2OS cells, 48 hours after transfection; error bars represent the SD of two technical replicates. (F) Percentage of mitotic U2OS cells with EdU spots (mean ± SD). About 120 metaphases per sample were analyzed in three independent experiments. (G) Percentage of mitotic U2OS cells with >10 γH2AX foci (mean ± SD). About 100 metaphases per sample were analyzed in three independent experiments. (H) Percentage of U2OS anaphase cells with aberrations (mean ± SD). About 100 anaphases per sample were analyzed in two independent experiments. Total percentage of aberrant anaphases was used to perform the statistics. (I) Percentage of binucleated U2OS cells with micronuclei (mean ± SD). About 400 binucleated cells per sample were analyzed in two independent experiments.

  • Fig. 5 Nucleotide deficiency during MiDAS leads to CIN in Chk1-deficient cells.

    (A) Percentage of mitotic U2OS cells with EdU spots (mean ± SD). Nucleosides (Ns) were added 24 hours before fixation. About 120 metaphases per sample were analyzed in three independent experiments. (B) Percentage of mitotic U2OS cells with >10 γH2AX foci (mean ± SD). Cells were treated as in (A). About 100 metaphases per sample were analyzed in three independent experiments. (C) Percentage of U2OS anaphase cells with aberrations (mean ± SD). Cells were treated as in (A). About 100 anaphases per sample were analyzed in two independent experiments. The total percentage of aberrant anaphases (bridges plus lagging chromosomes) was used to calculate the statistics. (D) Percentage of binucleated U2OS cells with micronuclei (mean ± SD). Cells were treated as in (A). About 400 binucleated cells per sample were analyzed in two independent experiments. (E) Model in which limited nucleotide availability restrains the completion of DNA synthesis in mitosis and propels CIN.

  • Fig. 6 CIN arising from nucleotide shortage during mitosis does not compromise survival of Chk1-deficient cells.

    (A) Sensitivity of U2OS cells to Chk1 depletion and CDC45 or Rad52 depletion or nucleoside supplementation. Cell number was determined 6 days after transfection. Data represent the mean (±SD) of three independent experiments. The right panel shows representative images of the data. Scale bar, 500 μm. (B) Graph showing that chromosome mis-segregation and cell death are uncorrelated in Chk1-deficient cells. Chromosome mis-segregation correlates with the accumulation of γH2AX foci in mitosis, whereas cell death correlates with the accumulation of pan-nuclear γH2AX in S phase. Data correspond to Figs. 1E, 4I, and 5D (micronuclei); Fig. 6A (cell death); Figs. 3B, 4G, and 5B (γH2AX foci in mitosis); and Fig. 1B and fig. S7A (pan-nuclear γH2AX). The data on pan-nuclear γH2AX upon Ns addition shown here are not presented in any preceding figure. (C) Model in which Chk1 loss triggers chromosome segregation defects and cell death by independent pathways. During S phase, Chk1 deficiency prompts surplus origin firing, reduced and asymmetric fork elongation, and Mus81-Eme2–dependent DSBs, culminating in cell death (a). Independently of these S phase events, Chk1-deficient cells enter mitosis with UR-DNA, whose duplication is completed in mitosis only if extra DNA precursors are supplied (b). Otherwise, most replication intermediates in mitosis are cleaved by Mus81-Eme1, leading to chromosome mis-segregation (c). Restraining MiDAS results in persistent UR-DNA, manifested as UFBs and 53BP1-NBs in G1 (d).

Supplementary Materials

  • Supplementary Materials

    Mus81-Eme1–dependent aberrant processing of DNA replication intermediates in mitosis impairs genome integrity

    Nicolás Luis Calzetta, Marina Alejandra González Besteiro, Vanesa Gottifredi

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