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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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Related in: MedlinePlus

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.
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pgen-1003910-g007: 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.

Mentions: We have previously shown that loss of MUS81 increases chromosomal damage in WRN-deficient cells, whereas it decreases chromosome abnormalities upon oncogene-induced replication stress [14], [18]. In both cases, however, MUS81 down-regulation increases cell death as we observed in replication checkpoint-deficient cells. Thus, we investigated whether MUS81 down-regulation enhanced chromosomal instability in CHK1-inhibited cells. To this end, we induced replication stress by concomitant CHK1 inhibition and HU treatment, and analysed chromosomal damage in metaphase cells after recovery in the absence of UCN-01 to limit cell death. Given that double knock-down cells were extremely sick, we limited the analysis of chromosome damage in Ctrl, MUS81 or RAD52 RNAi-treated cells (Figure 7A–C).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003910-g007: 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.
Mentions: We have previously shown that loss of MUS81 increases chromosomal damage in WRN-deficient cells, whereas it decreases chromosome abnormalities upon oncogene-induced replication stress [14], [18]. In both cases, however, MUS81 down-regulation increases cell death as we observed in replication checkpoint-deficient cells. Thus, we investigated whether MUS81 down-regulation enhanced chromosomal instability in CHK1-inhibited cells. To this end, we induced replication stress by concomitant CHK1 inhibition and HU treatment, and analysed chromosomal damage in metaphase cells after recovery in the absence of UCN-01 to limit cell death. Given that double knock-down cells were extremely sick, we limited the analysis of chromosome damage in Ctrl, MUS81 or RAD52 RNAi-treated cells (Figure 7A–C).

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