MUS81 nuclease activity is essential for replication stress tolerance and chromosome segregation in BRCA2-deficient cells

Abstract

Failure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity.

Figure 1. MUS81 facilitates replication and sustains proliferation in cells lacking BRCA2.

(a) H1299 cells carrying a doxycycline (DOX)-inducible BRCA2 shRNA were transfected with control or MUS81 siRNAs. Stable cells lines expressing either wild type (WT) or catalytically inactive (CI) human MUS81 were similarly processed. Cells were processed 24 h later for DNA fibre analysis as outlined in the inset, followed by quantification of CldU+IdU track length. Fork velocity was calculated using a conversion factor of 1 μm=2.59 kb min⁻¹. Red bars indicate mean (n=3). ****P<0.0001 (two-tailed Mann–Whitney test). (b) Cells treated as in a were processed 24 h later for proliferation assays. siRNAs were re-transfected at an interval of 4 days. Cell extracts prepared at indicated time points were immunoblotted as shown. SMC1 was used as a loading control. Graph shown is representative of three independent experiments. Error bars represent s.d. of triplicate values obtained from a single experiment. (c) H1299 cells expressing DOX-inducible BRCA2 shRNA were transfected with control or SLX4 siRNAs and processed as in b. The graph shown is representative of two independent experiments. Error bars represent s.d. of triplicate values obtained from a single experiment. (d) Cells treated as in a were plated 24 h later for clonogenic survival assays. Colonies were stained after 10–14 days. Error bars represent s.d. (n=4). ****P<0.0001 (unpaired two-tailed t-test). (e) HeLa or Capan-1 cells were transfected with control or MUS81 siRNAs and 48 h later plated for clonogenic survival assays. Colonies were stained after 10–14 days. Error bars represent s.d. (n=4). ***P<0.001 (unpaired two-tailed t-test).

Figure 2. MUS81 promotes DNA synthesis during mitosis in BRCA2-deficient cells.

(a) H1299 cells carrying a DOX-inducible BRCA2 shRNA were transfected with control, MUS81 and/or POLD3 siRNAs. Cells were pulsed with EdU 72 h later and processed for detection of EdU (green) and immunofluorescence staining with anti-FANCD2 antibody (red). DNA was counterstained with DAPI. (b) Quantification of the frequency of EdU-positive mitotic cells treated as in a. Error bars represent s.d. (n=3). *P<0.05; ***P<0.001 (unpaired two-tailed t-test). (c) Western blot analysis of chromatin-bound fractions of indicated cell lines at indicated cell cycle stages. Histone H3 was used as a loading control for the chromatin fraction. AS, asynchronous; G2, G2-arrested; M, mitotic (prometaphase). (d) Quantification of the average number of FANCD2 foci co-localizing with EdU in mitotic cells treated as in a. Error bars represent s.d. (n=3). *P<0.05; ***P<0.001 (unpaired two-tailed t-test). (e) Cell extracts prepared from samples analysed in d were immunoblotted as indicated. SMC1 and GAPDH were used as loading controls.

Figure 3. MUS81 resolves mitotic chromosome interlinks in BRCA2-deficient cells.

(a) H1299 cells carrying a DOX-inducible BRCA2 shRNA were transfected with control or MUS81 siRNAs and processed 72 h later for anaphase bridge analysis. Stable cells lines expressing either WT or CI human MUS81 were similarly processed. Representative images of DAPI-stained anaphase cells are shown. Yellow arrows indicate multiple DAPI bridges. (b) Quantification of the frequency of anaphase cells containing multiple DAPI bridges. Error bars represent s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (unpaired two-tailed t-test).

Figure 4. MUS81 inhibition leads to multinucleation and supernumerary centrosomes in cells lacking BRCA2.

(a) H1299 cells carrying a DOX-inducible BRCA2 shRNA were transfected with control or MUS81 siRNAs. Representative images of cells stained with an α-tubulin antibody (green) 72 h after transfection are shown. DNA was counterstained with DAPI. Scale bar, 10 μm. (b) Quantification of the frequency of multinucleated cells in asynchronous cultures treated as in a. Similar analyses were conducted using stable cells lines expressing either WT or CI human MUS81. Error bars represent s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001 (unpaired two-tailed t-test). (c) Cells treated as in a were processed for immunostaining with γ-tubulin (green) 72 h after transfection. Insets show selected regions containing centrosomes at a higher magnification. Scale bar, 10 μm. (d) Quantification of the frequency of cells with multiple centrosomes treated as in c. Similar analyses were conducted using stable cells lines expressing either WT or CI human MUS81. Error bars represent s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001 (unpaired two-tailed t-test).

Figure 5. MUS81 inhibition in BRCA2-deficient cells causes accumulation of 53BP1 nuclear bodies and G1 arrest.

(a) H1299 cells carrying a DOX-inducible BRCA2 shRNA were transfected with control or MUS81 siRNAs. Representative images of cells processed 72 h later for immunostaining with anti-53BP1 (green) and anti-cyclin A (red) antibodies. DNA was counterstained with DAPI. Scale bar, 10 μm. (b) Quantification of the frequency of cyclin A-negative G1 cells containing >5 53BP1 nuclear bodies in cells treated as in a. Similar analyses were conducted using stable cells lines expressing either WT or CI human MUS81. Error bars represent s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001 (unpaired two-tailed t-test). (c) Quantification of G1, S and G2 cell populations (boxed) in asynchronous cultures of EdU-labelled cells treated as in a. PI, propidium iodide.

Figure 6. Model for concerted action of MUS81 and BRCA2 during mitosis.

BRCA2-proficient cells require MUS81 for cleavage of UFBs formed during mitosis at under-replicated CFS. In cells lacking BRCA2, incomplete DNA replication at multiple sites leads to DAPI-stained bridges detectable in anaphase as chromosome interlinks. MUS81 is required to resolve these bridges and to promote mitotic DNA synthesis, ultimately facilitating chromosome segregation. MUS81 inactivation in BRCA2-deficient cells leads to persistent chromosome interlinks, multinucleation, supernumerary centrosomes and cell death. Blue, sister chromatids; yellow, centrosomes; light blue, microtubules.