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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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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.
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pgen-1003910-g002: 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.

Mentions: Disruption of replication checkpoint function can lead to loss of CHK1 phosphorylation [4]. We observed the highest levels of DNA breakage after ATR or CHK1 silencing, while depletion of TIPIN, which may or may not affect CHK1 activation, did not induce MUS81-dependent DSBs. Thus, we investigated whether formation of DSBs by MUS81 was directly related to a defective CHK1 phosphorylation in checkpoint-deficient cells. To this aim, we down-regulated ATR, RAD9 or TIPIN, then we analysed CHK1 phosphorylation by Western blotting using phospho-specific antibodies. Down-regulation of ATR or RAD9, but not of TIPIN, impaired CHK1 phosphorylation at S345 (Figure 2A). It is worth noting that the residual level of CHK1 phosphorylation seems to be inversely correlated with the amount of MUS81-dependent DSBs. In fact, residual CHK1 phosphorylation in RAD9 RNAi cells corresponded to less DSBs than in ATR knock-down cells (see Figure 1B). CHK1 phosphorylation is a pre-requisite to kinase activation [20], and CHK1-mediated phosphorylation of downstream targets may contribute to preventing replication fork collapse via MUS81-dependent cleavage. Thus, cells in which MUS81 was down-regulated by RNAi were treated with UCN-01 to inhibit CHK1, alone or in combination with 2 mM HU, and then processed by neutral Comet assay. Inhibition of CHK1 by UCN-01 recapitulated the phenotype of CHK1 RNAi-treated cells, albeit with a reduced accumulation of DSBs (Figure 2B). However, MUS81 down-regulation decreased the number of DSBs formed as a consequence of UCN-01 treatment as efficiently as observed after CHK1 RNAi (Figure 2B and 1B). Chemical inhibition of CHK1 also allowed the analysis of time-dependent formation of DSBs at perturbed forks, as well as their genetic dependency on MUS81. UCN-01 triggered DSBs already after 4 h of HU treatment, and increased substantially at 6 h, when DSBs are detectable also in cells treated with UCN-01 or HU alone (Figure S4A). Similarly to what observed after 6 h of the combined UCN-01 and HU treatment, the DSBs detected at 4 h were MUS81-dependent (Figure S4B). Generation of DSBs by MUS81 could be secondary to accumulation of single-stranded DNA (ssDNA) regions or gaps at the leading or lagging strand, caused by the checkpoint inhibition. Using alkaline Comet assay, we analysed the formation of ssDNA regions or gaps after CHK1 inhibition at forks stalled by HU. As expected, in the HU-treated cells, ssDNA or gaps start to accumulate already at 1 h after CHK1 inhibition, and greatly increased at 4–6 h (Figure S4C), when DSBs (see Figure S4A) are also detected by alkaline Comet assay. Thus, formation of ssDNA regions or DNA gaps precedes MUS81-dependent cleavage at perturbed replication forks.


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)

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.
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pgen-1003910-g002: 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.
Mentions: Disruption of replication checkpoint function can lead to loss of CHK1 phosphorylation [4]. We observed the highest levels of DNA breakage after ATR or CHK1 silencing, while depletion of TIPIN, which may or may not affect CHK1 activation, did not induce MUS81-dependent DSBs. Thus, we investigated whether formation of DSBs by MUS81 was directly related to a defective CHK1 phosphorylation in checkpoint-deficient cells. To this aim, we down-regulated ATR, RAD9 or TIPIN, then we analysed CHK1 phosphorylation by Western blotting using phospho-specific antibodies. Down-regulation of ATR or RAD9, but not of TIPIN, impaired CHK1 phosphorylation at S345 (Figure 2A). It is worth noting that the residual level of CHK1 phosphorylation seems to be inversely correlated with the amount of MUS81-dependent DSBs. In fact, residual CHK1 phosphorylation in RAD9 RNAi cells corresponded to less DSBs than in ATR knock-down cells (see Figure 1B). CHK1 phosphorylation is a pre-requisite to kinase activation [20], and CHK1-mediated phosphorylation of downstream targets may contribute to preventing replication fork collapse via MUS81-dependent cleavage. Thus, cells in which MUS81 was down-regulated by RNAi were treated with UCN-01 to inhibit CHK1, alone or in combination with 2 mM HU, and then processed by neutral Comet assay. Inhibition of CHK1 by UCN-01 recapitulated the phenotype of CHK1 RNAi-treated cells, albeit with a reduced accumulation of DSBs (Figure 2B). However, MUS81 down-regulation decreased the number of DSBs formed as a consequence of UCN-01 treatment as efficiently as observed after CHK1 RNAi (Figure 2B and 1B). Chemical inhibition of CHK1 also allowed the analysis of time-dependent formation of DSBs at perturbed forks, as well as their genetic dependency on MUS81. UCN-01 triggered DSBs already after 4 h of HU treatment, and increased substantially at 6 h, when DSBs are detectable also in cells treated with UCN-01 or HU alone (Figure S4A). Similarly to what observed after 6 h of the combined UCN-01 and HU treatment, the DSBs detected at 4 h were MUS81-dependent (Figure S4B). Generation of DSBs by MUS81 could be secondary to accumulation of single-stranded DNA (ssDNA) regions or gaps at the leading or lagging strand, caused by the checkpoint inhibition. Using alkaline Comet assay, we analysed the formation of ssDNA regions or gaps after CHK1 inhibition at forks stalled by HU. As expected, in the HU-treated cells, ssDNA or gaps start to accumulate already at 1 h after CHK1 inhibition, and greatly increased at 4–6 h (Figure S4C), when DSBs (see Figure S4A) are also detected by alkaline Comet assay. Thus, formation of ssDNA regions or DNA gaps precedes MUS81-dependent cleavage at perturbed replication forks.

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