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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 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.
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pgen-1003910-g001: 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.

Mentions: In human cells, inactivation of replication checkpoint factors results in formation of DSBs [4]. To determine whether these DSBs are derived from MUS81-mediated endonucleolytic cleavage at stalled forks, we performed a neutral Comet assay after depletion of different checkpoint proteins. To this end, hTERT-immortalised human primary fibroblasts were transfected with siRNAs directed against selected checkpoint factors, by themselves or in combination with siRNAs against MUS81 (Figure 1A). In addition, we down-regulated expression of CHK2, a checkpoint kinase that is not activated after perturbed replication, and used siRNAs as internal controls. The efficiency of RNAi was evaluated by Western blotting 48 h post-transfection, and protein level of the targets was reduced by at least 80% in comparison to cells transfected with GFP siRNAs (siCtrl; Figure 1A). After transfection, cells were treated with 2 mM HU for 6 h, a condition sufficient to induce DSBs only under pathological condition [14], and subjected to neutral Comet assay. Depletion of each of the selected checkpoint factors resulted in DSBs formation when replication was not perturbed, and further enhanced after HU treatment (Figure 1B). The DSBs level was highest after CHK1 or ATR depletion, whereas RAD9 or TOPBP1 down-regulation resulted in a limited DSBs enhancement. No increase was observed in cells depleted of MUS81, TIPIN or CHK2. Interestingly, MUS81 depletion reduced DSBs levels, albeit to a different extent, in all the checkpoint-deficient cells, either untreated or treated with HU. The only exceptions were TIPIN or CHK2 knock-down cells (Figure 1B). Consistent results were also obtained analysing the formation of pan-nuclear staining of the phosphorylated H2AX. In this case, checkpoint impairment after ATR or CHK1 depletion gave rise to a strong accumulation of nuclei showing intense γH2AX staining, which was reduced by concomitant MUS81 knock-down (Figure S1A and B).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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pgen-1003910-g001: 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.
Mentions: In human cells, inactivation of replication checkpoint factors results in formation of DSBs [4]. To determine whether these DSBs are derived from MUS81-mediated endonucleolytic cleavage at stalled forks, we performed a neutral Comet assay after depletion of different checkpoint proteins. To this end, hTERT-immortalised human primary fibroblasts were transfected with siRNAs directed against selected checkpoint factors, by themselves or in combination with siRNAs against MUS81 (Figure 1A). In addition, we down-regulated expression of CHK2, a checkpoint kinase that is not activated after perturbed replication, and used siRNAs as internal controls. The efficiency of RNAi was evaluated by Western blotting 48 h post-transfection, and protein level of the targets was reduced by at least 80% in comparison to cells transfected with GFP siRNAs (siCtrl; Figure 1A). After transfection, cells were treated with 2 mM HU for 6 h, a condition sufficient to induce DSBs only under pathological condition [14], and subjected to neutral Comet assay. Depletion of each of the selected checkpoint factors resulted in DSBs formation when replication was not perturbed, and further enhanced after HU treatment (Figure 1B). The DSBs level was highest after CHK1 or ATR depletion, whereas RAD9 or TOPBP1 down-regulation resulted in a limited DSBs enhancement. No increase was observed in cells depleted of MUS81, TIPIN or CHK2. Interestingly, MUS81 depletion reduced DSBs levels, albeit to a different extent, in all the checkpoint-deficient cells, either untreated or treated with HU. The only exceptions were TIPIN or CHK2 knock-down cells (Figure 1B). Consistent results were also obtained analysing the formation of pan-nuclear staining of the phosphorylated H2AX. In this case, checkpoint impairment after ATR or CHK1 depletion gave rise to a strong accumulation of nuclei showing intense γH2AX staining, which was reduced by concomitant MUS81 knock-down (Figure S1A and B).

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