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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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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.
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pgen-1003910-g003: 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.

Mentions: Neutral Comet assays were performed in cells in which CHK1 was chemically inhibited and RAD52 depleted by RNAi alone or in combination with MUS81. Comparable reduction in protein levels was verified by Western blotting (Figure 3A). RAD52 down-regulation barely affected the level of DSBs after single treatments with HU or UCN-01 (Figure 3B). Upon combined treatment, however, RAD52 down-regulation efficiently suppressed DSBs, and this reduction was comparable to that observed following MUS81 depletion. In contrast, MUS81/RAD52 co-depletion resulted in the reappearance of DSBs, at levels similar to that of treatment with UCN-01 alone (Figure 3B). Even though either MUS81 or RAD52 down-regulation suppressed DSBs formation after CHK1 inhibition, MUS81 depletion strongly increased the mean tail moment observed by alkaline Comet assay, which are reversed by RAD52 but not RAD51 knock-down (Figure S7). Since the alkaline Comet assay detects both ssDNA or DNA gaps and DSBs, in the single depleted MUS81 or RAD52 cells, where DSBs are almost absent, only ssDNA regions are likely formed. In contrast, the reduction of the mean tail moment observed in the double MUS81/RAD52-depleted cells implies that only DSBs are formed.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814295&req=5

pgen-1003910-g003: 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.
Mentions: Neutral Comet assays were performed in cells in which CHK1 was chemically inhibited and RAD52 depleted by RNAi alone or in combination with MUS81. Comparable reduction in protein levels was verified by Western blotting (Figure 3A). RAD52 down-regulation barely affected the level of DSBs after single treatments with HU or UCN-01 (Figure 3B). Upon combined treatment, however, RAD52 down-regulation efficiently suppressed DSBs, and this reduction was comparable to that observed following MUS81 depletion. In contrast, MUS81/RAD52 co-depletion resulted in the reappearance of DSBs, at levels similar to that of treatment with UCN-01 alone (Figure 3B). Even though either MUS81 or RAD52 down-regulation suppressed DSBs formation after CHK1 inhibition, MUS81 depletion strongly increased the mean tail moment observed by alkaline Comet assay, which are reversed by RAD52 but not RAD51 knock-down (Figure S7). Since the alkaline Comet assay detects both ssDNA or DNA gaps and DSBs, in the single depleted MUS81 or RAD52 cells, where DSBs are almost absent, only ssDNA regions are likely formed. In contrast, the reduction of the mean tail moment observed in the double MUS81/RAD52-depleted cells implies that only DSBs are formed.

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