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

Murfuni I, Basile G, Subramanyam S, Malacaria E, Bignami M, Spies M, Franchitto A, Pichierri P - PLoS Genet. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.

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.

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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.
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pgen-1003910-g006: 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.

Mentions: To address whether RAD52 might also play a MUS81-independent role in cells with a compromised CHK1 function, we evaluated cell death after recovery from replication stress in cells depleted of MUS81, RAD52 or both, with or without persistent CHK1 inhibition. We found that, in wild-type cells, combined exposure to HU and UCN-01 resulted in a 20% cell death. When cells were allowed to recover in the absence of the CHK1 inhibitor, only a minimal reduction in toxicity was observed (Figure 6A). After CHK1 inhibition, cell death of MUS81-depleted cells increased by two-fold, but decreased significantly when UCN-01 was left during recovery (Figure 6A). Among other enzymes involved in the resolution of intermediates thought to accumulate at collapsed forks, i.e. SLX4, GEN1 or BLM, only GEN1 depletion increased cell death in CHK1-deficient cells after HU treatment (Figure S9A). RAD52 down-regulation also resulted in enhanced cell death during recovery from UCN-01-induced replication stress. This phenotype, however, was unaffected by persistent CHK1 inhibition during recovery (Figure 6A). Interestingly, the simultaneous inactivation of RAD52/MUS81 was associated with extreme toxicity. Indeed, cells depleted of RAD52 and MUS81 showed about 60% cell death, independently of CHK1 activity during recovery (Figure 6A). Increased cell death of MUS81-depleted cells was also observed following down-regulation of GEN1 or SLX4, and at a lesser extent after BLM RNAi (Figure S9B), suggesting their involvement in processing intermediates formed at stalled forks in the absence of MUS81.


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

Murfuni I, Basile G, Subramanyam S, Malacaria E, Bignami M, Spies M, Franchitto A, Pichierri P - PLoS Genet. (2013)

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.
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Related In: Results  -  Collection

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Show All Figures
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pgen-1003910-g006: 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.
Mentions: To address whether RAD52 might also play a MUS81-independent role in cells with a compromised CHK1 function, we evaluated cell death after recovery from replication stress in cells depleted of MUS81, RAD52 or both, with or without persistent CHK1 inhibition. We found that, in wild-type cells, combined exposure to HU and UCN-01 resulted in a 20% cell death. When cells were allowed to recover in the absence of the CHK1 inhibitor, only a minimal reduction in toxicity was observed (Figure 6A). After CHK1 inhibition, cell death of MUS81-depleted cells increased by two-fold, but decreased significantly when UCN-01 was left during recovery (Figure 6A). Among other enzymes involved in the resolution of intermediates thought to accumulate at collapsed forks, i.e. SLX4, GEN1 or BLM, only GEN1 depletion increased cell death in CHK1-deficient cells after HU treatment (Figure S9A). RAD52 down-regulation also resulted in enhanced cell death during recovery from UCN-01-induced replication stress. This phenotype, however, was unaffected by persistent CHK1 inhibition during recovery (Figure 6A). Interestingly, the simultaneous inactivation of RAD52/MUS81 was associated with extreme toxicity. Indeed, cells depleted of RAD52 and MUS81 showed about 60% cell death, independently of CHK1 activity during recovery (Figure 6A). Increased cell death of MUS81-depleted cells was also observed following down-regulation of GEN1 or SLX4, and at a lesser extent after BLM RNAi (Figure S9B), suggesting their involvement in processing intermediates formed at stalled forks in the absence of MUS81.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.

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.

Show MeSH
Related in: MedlinePlus