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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

Model for processing of stalled forks in replication checkpoint-deficient cells.Inactivation of CHK1 determines destabilisation of stalled replication forks and accumulation of ssDNA gaps, likely at both the leading and the lagging strand. Stalled forks with ssDNA gaps (1) may undergo extensive extrusion of the newly-synthesized strands by fork regression (2) leading to a preferential engagement of RAD52 (A). RAD52, through its ssDNA annealing activity, would produce a D-loop intermediate (3) and possibly helps recruiting MUS81/EME1 complex by protein-protein interaction. Alternatively, RAD52 may assemble a D-loop intermediate from the ssDNA gap, either at the leading or the lagging strand behind the stalled fork (1.1). The D-loop intermediate is targeted by MUS81 resulting in DSBs and fork collapse. The BIR event that follows may involve subsequent requirement for viability of another SSE, GEN1. In the absence of a functional checkpoint (i.e. inactive CHK1), the RAD52-dependent pathway is a favourite, but inefficient, way of ensuring proliferation at the expense of genome stability. In the absence of RAD52, a RAD51-dependent mechanism (B) may be forcedly engaged. Viability of RAD52-deficient cells would require MUS81 and GEN1 to process the branched intermediates generated. This latter option, would limit genome instability at the cost of reduced survival, and would result in excessive lethality if MUS81 is also depleted. In contrast, MUS81 down-regulation, would stimulate a RAD51-mediated mechanism (C), but at the expense of both reduced cell viability and genome stability. Further details are discussed in the text.
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pgen-1003910-g008: Model for processing of stalled forks in replication checkpoint-deficient cells.Inactivation of CHK1 determines destabilisation of stalled replication forks and accumulation of ssDNA gaps, likely at both the leading and the lagging strand. Stalled forks with ssDNA gaps (1) may undergo extensive extrusion of the newly-synthesized strands by fork regression (2) leading to a preferential engagement of RAD52 (A). RAD52, through its ssDNA annealing activity, would produce a D-loop intermediate (3) and possibly helps recruiting MUS81/EME1 complex by protein-protein interaction. Alternatively, RAD52 may assemble a D-loop intermediate from the ssDNA gap, either at the leading or the lagging strand behind the stalled fork (1.1). The D-loop intermediate is targeted by MUS81 resulting in DSBs and fork collapse. The BIR event that follows may involve subsequent requirement for viability of another SSE, GEN1. In the absence of a functional checkpoint (i.e. inactive CHK1), the RAD52-dependent pathway is a favourite, but inefficient, way of ensuring proliferation at the expense of genome stability. In the absence of RAD52, a RAD51-dependent mechanism (B) may be forcedly engaged. Viability of RAD52-deficient cells would require MUS81 and GEN1 to process the branched intermediates generated. This latter option, would limit genome instability at the cost of reduced survival, and would result in excessive lethality if MUS81 is also depleted. In contrast, MUS81 down-regulation, would stimulate a RAD51-mediated mechanism (C), but at the expense of both reduced cell viability and genome stability. Further details are discussed in the text.

Mentions: It is generally thought that MUS81 may cleave RAD51-dependent recombination intermediates, such as HJs, mainly outside DNA replication. The identity of the MUS81 substrate and how it is generated at perturbed forks, however, remains unresolved [10], [12], [14], [45], [46], [47], [48]. Our data suggest that, upon fork collapse, MUS81 does not target a RAD51-dependent recombination intermediate, as similarly reported in WRN-deficient cells [21]. In fact, RAD51 depletion does not stimulate or revert formation of MUS81-dependent DSBs upon CHK1 inhibition, suggesting that MUS81 either targets the stalled forks directly, or processes other intermediates that form independently of RAD51. Since we observe that CHK1 inhibition and HU treatment stimulate RAD52 binding to chromatin, and that RAD52 depletion abrogates MUS81-dependent DSBs, we conclude that MUS81 does not cleave collapsed forks directly, but rather after the formation of a RAD52-dependent intermediate. It is worth noting that remodelling of collapsed forks prior to MUS81-dependent cleavage, might explain why DSBs are not formed immediately after replication arrest. Interestingly, MUS81-dependent DSBs accumulate after that CHK1 inhibition has induced a large amount of ssDNA, suggesting that MUS81 is cleaving an intermediate assembled from unreplicated leading or lagging strand. One of the substrate that could be generated by the ssDNA annealing activity of RAD52, perhaps through assistance of an helicase, is a D-loop, which is an ideal substrate for MUS81. Indeed, our in vitro studies support this hypothesis, and also demonstrate that MUS81 specifically targets D-loops assembled by RAD52. The apparent inability of MUS81/EME1 to cleave D-loops produced by RAD51, provides a mechanistic understanding of the RAD51 independency showed by DSBs formed by MUS81 in vivo. This conclusion is further reinforced by data in yeast, showing that MUS81 may act on RAD52-dependent D-loops produced at collapsed forks [12]. Such a D-loop might result from either pairing of the extruded leading or lagging strand after fork regression, or by the attempt to repair a ssDNA gap behind the replication fork (see Figure 8).


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)

Model for processing of stalled forks in replication checkpoint-deficient cells.Inactivation of CHK1 determines destabilisation of stalled replication forks and accumulation of ssDNA gaps, likely at both the leading and the lagging strand. Stalled forks with ssDNA gaps (1) may undergo extensive extrusion of the newly-synthesized strands by fork regression (2) leading to a preferential engagement of RAD52 (A). RAD52, through its ssDNA annealing activity, would produce a D-loop intermediate (3) and possibly helps recruiting MUS81/EME1 complex by protein-protein interaction. Alternatively, RAD52 may assemble a D-loop intermediate from the ssDNA gap, either at the leading or the lagging strand behind the stalled fork (1.1). The D-loop intermediate is targeted by MUS81 resulting in DSBs and fork collapse. The BIR event that follows may involve subsequent requirement for viability of another SSE, GEN1. In the absence of a functional checkpoint (i.e. inactive CHK1), the RAD52-dependent pathway is a favourite, but inefficient, way of ensuring proliferation at the expense of genome stability. In the absence of RAD52, a RAD51-dependent mechanism (B) may be forcedly engaged. Viability of RAD52-deficient cells would require MUS81 and GEN1 to process the branched intermediates generated. This latter option, would limit genome instability at the cost of reduced survival, and would result in excessive lethality if MUS81 is also depleted. In contrast, MUS81 down-regulation, would stimulate a RAD51-mediated mechanism (C), but at the expense of both reduced cell viability and genome stability. Further details are discussed in the text.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003910-g008: Model for processing of stalled forks in replication checkpoint-deficient cells.Inactivation of CHK1 determines destabilisation of stalled replication forks and accumulation of ssDNA gaps, likely at both the leading and the lagging strand. Stalled forks with ssDNA gaps (1) may undergo extensive extrusion of the newly-synthesized strands by fork regression (2) leading to a preferential engagement of RAD52 (A). RAD52, through its ssDNA annealing activity, would produce a D-loop intermediate (3) and possibly helps recruiting MUS81/EME1 complex by protein-protein interaction. Alternatively, RAD52 may assemble a D-loop intermediate from the ssDNA gap, either at the leading or the lagging strand behind the stalled fork (1.1). The D-loop intermediate is targeted by MUS81 resulting in DSBs and fork collapse. The BIR event that follows may involve subsequent requirement for viability of another SSE, GEN1. In the absence of a functional checkpoint (i.e. inactive CHK1), the RAD52-dependent pathway is a favourite, but inefficient, way of ensuring proliferation at the expense of genome stability. In the absence of RAD52, a RAD51-dependent mechanism (B) may be forcedly engaged. Viability of RAD52-deficient cells would require MUS81 and GEN1 to process the branched intermediates generated. This latter option, would limit genome instability at the cost of reduced survival, and would result in excessive lethality if MUS81 is also depleted. In contrast, MUS81 down-regulation, would stimulate a RAD51-mediated mechanism (C), but at the expense of both reduced cell viability and genome stability. Further details are discussed in the text.
Mentions: It is generally thought that MUS81 may cleave RAD51-dependent recombination intermediates, such as HJs, mainly outside DNA replication. The identity of the MUS81 substrate and how it is generated at perturbed forks, however, remains unresolved [10], [12], [14], [45], [46], [47], [48]. Our data suggest that, upon fork collapse, MUS81 does not target a RAD51-dependent recombination intermediate, as similarly reported in WRN-deficient cells [21]. In fact, RAD51 depletion does not stimulate or revert formation of MUS81-dependent DSBs upon CHK1 inhibition, suggesting that MUS81 either targets the stalled forks directly, or processes other intermediates that form independently of RAD51. Since we observe that CHK1 inhibition and HU treatment stimulate RAD52 binding to chromatin, and that RAD52 depletion abrogates MUS81-dependent DSBs, we conclude that MUS81 does not cleave collapsed forks directly, but rather after the formation of a RAD52-dependent intermediate. It is worth noting that remodelling of collapsed forks prior to MUS81-dependent cleavage, might explain why DSBs are not formed immediately after replication arrest. Interestingly, MUS81-dependent DSBs accumulate after that CHK1 inhibition has induced a large amount of ssDNA, suggesting that MUS81 is cleaving an intermediate assembled from unreplicated leading or lagging strand. One of the substrate that could be generated by the ssDNA annealing activity of RAD52, perhaps through assistance of an helicase, is a D-loop, which is an ideal substrate for MUS81. Indeed, our in vitro studies support this hypothesis, and also demonstrate that MUS81 specifically targets D-loops assembled by RAD52. The apparent inability of MUS81/EME1 to cleave D-loops produced by RAD51, provides a mechanistic understanding of the RAD51 independency showed by DSBs formed by MUS81 in vivo. This conclusion is further reinforced by data in yeast, showing that MUS81 may act on RAD52-dependent D-loops produced at collapsed forks [12]. Such a D-loop might result from either pairing of the extruded leading or lagging strand after fork regression, or by the attempt to repair a ssDNA gap behind the replication fork (see Figure 8).

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