Survival of the replication checkpoint deficient cells requires MUS81-RAD52 function

Abstract

In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress.

Figure 1. MUS81 promotes DSB formation and cell viability in response to replication checkpoint down-regulation.

(A) Analysis of protein depletion by Western blotting in GM01604 cells after transfection with control siRNAs directed against GFP (siCtrl) or siCHK1, siATR, siTOPBP1, siRAD9, siTIPIN and siCHK2, alone or in combination with siMUS81. Immunoblotting was assessed 48 h after transfection using the appropriate antibodies. PCNA was used as loading control. (B) DSBs accumulation by neutral Comet assay in GM01604 cells transfected as in (A) and treated with 2 mM HU for 6 h before subjecting to Comet assay. Graph shows data presented as mean tail moment +/− SE from three independent experiments. Error bars represent standard errors. Where not depicted, standard errors were <15% of the mean. In the panel representative images from selected samples are shown. (C) Evaluation of cell death after replication stress in GM01604 cells transfected with control siRNAs (siCtrl) or siCHK1, siATR and siTIPIN alone or in combination with siMUS81. Forty-eight hours after RNAi, CHK1 inhibitor (UCN-01), ATR inhibitor (ETP-46464) or solvent (DMSO) was added to media 1 h prior HU treatment. After 6 h of HU, cells were recovered overnight before being analysed. Cell viability was evaluated by LIVE/DEAD assay as described in “Materials and Methods”. Data are presented as percentage of dead cells and are mean values from three independent experiments. Error bars represent standard error. Where not depicted, standard errors were <15% of the mean. The panel shows representative images: live cells are green stained, while dead cells are red.

Figure 2. Loss of CHK1 function leads to MUS81-dependent DSBs production independently from RAD51 regulation.

(A) Analysis of CHK1 phosphorylation in GM01604 cells transfected with control siRNAs (siCtrl) or siATR, siTIPIN and siRAD9 and treated with 2 mM HU for 6 h. Lysates were subjected to SDS-PAGE and immunoblotted for pS345CHK1 and CHK1. (B) Evaluation of MUS81-dependent DSBs formation by neutral Comet assay in cells in which CHK1 function was chemically inhibited. GM01604 cells were transfected with control siRNAs (siCtrl) or siMUS81. Forty-eight hours thereafter, cells were treated or not with CHK1 inhibitor (UCN-01) for 1 h and with 2 mM HU for 6 h and then subjected to Comet assay. Data are presented as mean tail moment and are means of three independent experiments. Error bars represent standard errors. Where not depicted, standard errors were <15% of the mean. In the panel, representative images are shown. (C) Western blotting in GM01604 cells transfected with control siRNAs (siCtrl) or siRAD51 and/or siMUS81. Depletion of proteins was verified 48 h after transfection using the relevant antibodies. Tubulin was used as loading control. (D) Analysis of DSBs formation in the absence of RAD51. Cells in which RAD51 and/or MUS81 was down-regulated were treated or not with UCN-01 for 1 h and with 2 mM HU for 6 h, then cells were subjected to neutral Comet assay. Cells treated with UCN-01 were used as positive control. Graph shows data presented as mean tail moment +/− SE from three independent experiments. Error bars represent standard errors. Where not depicted, standard errors were <15% of the mean. (E) Experimental scheme for genetic knock-down and rescue experiments. GM01604 cells were transfected with siRNA oligos targeting the UTR of RAD51 or GFP (siCtrl). RAD51-depleted cells were nucleofected to express RNAi-resistant wild-type or phosphorylation mutant form of RAD51 (RAD51-T309A). (F) Depletion of RAD51 and expression of the ectopic wild-type or RAD51-T309A were verified by immunoblotting 48 h thereafter using the anti-RAD51 antibody. Tubulin was used as loading control. (G) Analysis of DSBs formation in cells with impaired RAD51 function. GM01604 cells were transfected with control siRNAs (siCtrl) or siMUS81 and/or siRAD51. Forty-eight hours thereafter, cells were transfected with the RAD51-T309A plasmid (see Text S1). Then cells were treated or not with UCN-01 for 1 h and exposed to 2 mM HU for 6 h before being subjected to neutral Comet assay. Sample treated with UCN-01 was used as positive control. Data are presented as mean tail moment and are means of three independent experiments. Error bars represent standard errors. Where not depicted, standard errors were <15% of the mean.

Figure 3. Role of RAD52 in MUS81-dependent DSBs formation.

(A) Assessment of protein depletion by Western blotting in GM01604 cells after transfection with control siRNAs (siCtrl) or siRAD52 and/or siMUS81. Immunoblotting was performed using the relevant antibodies. Tubulin was used as loading control. (B) Analysis of DSBs accumulation in RAD52 depleted cells by neutral Comet assay. GM01604 cells were transfected as in (A) and treated with 400 nM UCN-01 and/or 2 mM HU for 6 h and then subjected to Comet assay. Graph shows data presented as mean tail moment +/− SE from three independent experiments. Error bars represent standard errors. (C) Assessment of protein depletion by Western blotting in GM01604 cells after transfection with control siRNAs (siCtrl) or the indicated combination of siRNAs. Immunoblotting was performed using the relevant antibodies. Lamin B1 was used as loading control. (D) Analysis of DSBs accumulation in RAD52/MUS81-depleted cells by neutral Comet assay. GM01604 cells were transfected as in (C) and treated with 400 nM UCN-01 and/or 2 mM HU for 6 h and then subjected to Comet assay. Graph shows data presented as mean tail moment +/− SE from three independent experiments. Error bars represent standard errors. (E) Levels of chromatin-bound RAD52 in GM01604 cells transfected with control siRNAs (siCtrl) or siMUS81 and treated with UCN-01 for 1 h and then with HU for 6 h. The amount of RAD52 in the chromatin fraction was presented as fold increase compared with the matched untreated control, normalized against the amount of histone H3.

Figure 4. (A) Immunopurification of the human MUS81/EME1 complex from 293T cells.

One-twentieth of human MUS81/EME1 complex immunopurified using anti-Myc-agarose/GSH agarose (see Materials and Methods) was resolved onto an SDS-PAGE gel and revealed by Coomassie blue-stain (CBB). (B) In vitro MUS81/EME1 cleavage of model D-loop substrates. The D-loops were produced by the RAD52-mediated annealing, by the RAD51-mediated strand invasion, or by the heat-mediated annealing as described in “Materials and Methods” and schematically depicted over the gel. MUS81/EME1-mediated cleavage results in the loss of superhelicity and, upon deproteination of the products, in the displacement of the radioactively-labeled oligonucleotide from the plasmid. Thus, the D-loop loss is an indicator of MUS81/EME1-dependent D-loop cleavage. The D-loops were separated from the unincorporated and displaced oligonucleotides on the agarose gel. The table above the gel summarizes the constituencies and conditions of each reaction. The band corresponding to the D-loop migration is marked on the side of the gel. The graph under the gel shows the gel quantification. (C) In vitro MUS81/EME1 cleavage of a 3′-flap substrate. The substrate was assembled as described in “Materials and Methods” and schematically depicted side the gel. MUS81/EME1-mediated cleavage results in formation of a nicked product, which was separated from the intact substrate and the not-assembled, single-stranded, substrate on the agarose gel. The graph shows the gel quantification. (D) RAD52 pulled-down MUS81 from nuclear extracts. Five µg of purified 6xHis-tagged RAD52 was incubated with 1 mg of benzonase-treated nuclear extract. After incubation with anti-His antibody-coupled magnetic beads, RAD52 protein complexes were released in 1x Laemmli sample buffer, subjected to SDS-PAGE and Western blotting using the indicated antibodies. Data are presented as a mean of replicate experiments, SEs were <10% of the mean. * = p<0.05 Student's t-test.

Figure 5. MUS81 and RAD52 differently affect restart of stalled forks upon CHK1 inhibition.

(A) GM01604 cells were transfected with siRNAs directed against GFP (siCtrl), MUS81 (siMUS81) or RAD52 (siRAD52). Replication sites were first labeled with CldU (red signal), left untreated or treated as indicated, followed by recovery for 45 min in IdU (green signal). After immunostaining with antibodies specific for CldU and IdU, the overlapping foci were quantified in each isolated red-positive cell and results expressed as the percentage of CldU/IdU colocalising foci (0–20% of total CldU foci; 20–60% of total CldU foci; 60–100% of total CldU foci). Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. The images shown in the panel (B) are representative of labeling and of different classes of colocalising nuclei.

Figure 6. MUS81 and RAD52 promote survival also independently from each other. (A)

Effect of the down-regulation of RAD52 and/or MUS81 on cell viability. GM01604 cells were transfected with the indicate siRNAs and 48 h later treated with 400 nM UCN-01 and/or 2 mM HU for 6 h. Cell viability was evaluated by LIVE/DEAD assay as described in “Materials and Methods” after 18 h of recovery in HU-free medium, with or without continuous exposure to UCN-01. Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. Where not depicted, standard errors were <15% of the mean. In the panel representative images from samples treated with HU are reported: live cells are green stained while dead cells are red. (B) Analysis of replication checkpoint activation. Cells were treated with 400 nM UCN-01 and/or 2 mM HU for 6 h. Then cells were recovered for 4 h and immunoblotted for pS345CHK1 and CHK1. MUS81 was used to verified protein depletion and Lamin B1 as loading control. The graph shows the gel quantification. (C) Effect of the over-expression of RAD51-T309D on cell viability of cells experiencing replication stress in the absence of MUS81. GM01604 cells were transfected with the indicate siRNAs and 24 h later nucleofected with plasmids expressing either RAD51wt or RAD51-T309D. Twenty-four hours thereafter, cells were treated with 400 nM UCN-01 and/or 2 mM HU for 6 h. Cell viability was evaluated by LIVE/DEAD assay as described in “Materials and Methods” after 18 h of recovery in HU-free medium, with or without continuous exposure to UCN-01. Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. Where not depicted, standard errors were <15% of the mean. (D) Levels of chromatin-bound RAD51 in GM01604 cells transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were treated with UCN-01 for 1 h and then with HU for 6 h. The graph shows the amount of RAD51 in the chromatin fraction determined after densitometry of the representative gels and presented as arbitrary units normalized against the amount of Lamin B1. (E) Western blotting showing depletion of protein levels after transfection with the indicated siRNAs. PCNA was used as loading control. (F) Effect of the down-regulation of RAD51 in RAD52/MUS81-depleted cells on cell viability. GM01604 cells were transfected with control siRNAs (siCtrl) or siRAD52, siRAD51 and siMUS81. Forty-eight hours after interference, cells were treated with 400 nM UCN-01 and/or 2 mM HU for 6 h. Cell viability was evaluated by LIVE/DEAD assay 18 h after recovery in HU-free medium, as described in “Materials and Methods”. Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. Where not depicted, standard errors were <15% of the mean.

Figure 7. Effect of MUS81 or RAD52 depletion on chromosomal damage in response to replication checkpoint down-regulation.

(A) Western blotting showing MUS81 and RAD52 depletion verified 48 h after interference using the relevant antibodies. Lamin B1 was used as loading control. (B) Aberrations per cell in WI-38 SV40-transformed fibroblasts transfected with control siRNAs (siCtrl), siMUS81 or siRAD52. Cells were treated as described in “Materials and Methods”. Asterisks indicate that the result is statistically significant compared to the indicated experimental point; (** = P<0.05, Student's t test). (C) Representative Giemsa-stained metaphases from cells transfected with the indicated siRNAs and recovered in drug-free medium after replication checkpoint inhibition. Arrows indicate chromosomal aberrations.

Figure 8. Model for processing of stalled forks in replication checkpoint-deficient cells.

Inactivation of CHK1 determines destabilisation of stalled replication forks and accumulation of ssDNA gaps, likely at both the leading and the lagging strand. Stalled forks with ssDNA gaps (1) may undergo extensive extrusion of the newly-synthesized strands by fork regression (2) leading to a preferential engagement of RAD52 (A). RAD52, through its ssDNA annealing activity, would produce a D-loop intermediate (3) and possibly helps recruiting MUS81/EME1 complex by protein-protein interaction. Alternatively, RAD52 may assemble a D-loop intermediate from the ssDNA gap, either at the leading or the lagging strand behind the stalled fork (1.1). The D-loop intermediate is targeted by MUS81 resulting in DSBs and fork collapse. The BIR event that follows may involve subsequent requirement for viability of another SSE, GEN1. In the absence of a functional checkpoint (i.e. inactive CHK1), the RAD52-dependent pathway is a favourite, but inefficient, way of ensuring proliferation at the expense of genome stability. In the absence of RAD52, a RAD51-dependent mechanism (B) may be forcedly engaged. Viability of RAD52-deficient cells would require MUS81 and GEN1 to process the branched intermediates generated. This latter option, would limit genome instability at the cost of reduced survival, and would result in excessive lethality if MUS81 is also depleted. In contrast, MUS81 down-regulation, would stimulate a RAD51-mediated mechanism (C), but at the expense of both reduced cell viability and genome stability. Further details are discussed in the text.