Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Immunopurification of the human MUS81/EME1 complex from 293T cells.One-twentieth of human MUS81/EME1 complex immunopurified using anti-Myc-agarose/GSH agarose (see Materials and Methods) was resolved onto an SDS-PAGE gel and revealed by Coomassie blue-stain (CBB). (B) In vitro MUS81/EME1 cleavage of model D-loop substrates. The D-loops were produced by the RAD52-mediated annealing, by the RAD51-mediated strand invasion, or by the heat-mediated annealing as described in “Materials and Methods” and schematically depicted over the gel. MUS81/EME1-mediated cleavage results in the loss of superhelicity and, upon deproteination of the products, in the displacement of the radioactively-labeled oligonucleotide from the plasmid. Thus, the D-loop loss is an indicator of MUS81/EME1-dependent D-loop cleavage. The D-loops were separated from the unincorporated and displaced oligonucleotides on the agarose gel. The table above the gel summarizes the constituencies and conditions of each reaction. The band corresponding to the D-loop migration is marked on the side of the gel. The graph under the gel shows the gel quantification. (C) In vitro MUS81/EME1 cleavage of a 3′-flap substrate. The substrate was assembled as described in “Materials and Methods” and schematically depicted side the gel. MUS81/EME1-mediated cleavage results in formation of a nicked product, which was separated from the intact substrate and the not-assembled, single-stranded, substrate on the agarose gel. The graph shows the gel quantification. (D) RAD52 pulled-down MUS81 from nuclear extracts. Five µg of purified 6xHis-tagged RAD52 was incubated with 1 mg of benzonase-treated nuclear extract. After incubation with anti-His antibody-coupled magnetic beads, RAD52 protein complexes were released in 1x Laemmli sample buffer, subjected to SDS-PAGE and Western blotting using the indicated antibodies. Data are presented as a mean of replicate experiments, SEs were <10% of the mean. * = p<0.05 Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814295&req=5

pgen-1003910-g004: (A) Immunopurification of the human MUS81/EME1 complex from 293T cells.One-twentieth of human MUS81/EME1 complex immunopurified using anti-Myc-agarose/GSH agarose (see Materials and Methods) was resolved onto an SDS-PAGE gel and revealed by Coomassie blue-stain (CBB). (B) In vitro MUS81/EME1 cleavage of model D-loop substrates. The D-loops were produced by the RAD52-mediated annealing, by the RAD51-mediated strand invasion, or by the heat-mediated annealing as described in “Materials and Methods” and schematically depicted over the gel. MUS81/EME1-mediated cleavage results in the loss of superhelicity and, upon deproteination of the products, in the displacement of the radioactively-labeled oligonucleotide from the plasmid. Thus, the D-loop loss is an indicator of MUS81/EME1-dependent D-loop cleavage. The D-loops were separated from the unincorporated and displaced oligonucleotides on the agarose gel. The table above the gel summarizes the constituencies and conditions of each reaction. The band corresponding to the D-loop migration is marked on the side of the gel. The graph under the gel shows the gel quantification. (C) In vitro MUS81/EME1 cleavage of a 3′-flap substrate. The substrate was assembled as described in “Materials and Methods” and schematically depicted side the gel. MUS81/EME1-mediated cleavage results in formation of a nicked product, which was separated from the intact substrate and the not-assembled, single-stranded, substrate on the agarose gel. The graph shows the gel quantification. (D) RAD52 pulled-down MUS81 from nuclear extracts. Five µg of purified 6xHis-tagged RAD52 was incubated with 1 mg of benzonase-treated nuclear extract. After incubation with anti-His antibody-coupled magnetic beads, RAD52 protein complexes were released in 1x Laemmli sample buffer, subjected to SDS-PAGE and Western blotting using the indicated antibodies. Data are presented as a mean of replicate experiments, SEs were <10% of the mean. * = p<0.05 Student's t-test.

Mentions: One of the putative MUS81 substrates that may be formed at the HU-stalled or collapsed forks is a D-loop [7]. To verify this possibility, we evaluated the ability of a purified MUS81/EME1 complex to cleave a model D-loop assembled in vitro by either RAD52 or RAD51. The human MUS81/EME1 complex was immunopurified from 293T cells transiently transfected with plasmids expressing Myc-tagged MUS81 and GST-tagged EME1 (Figure 4A). Purified RAD52 or RAD51 were pre-incubated with P32-labelled oligonucleotides complementary to a region of the φX174 plasmid. The resulting nucleoprotein complexes mediated formation of the D-loops comprised by φX174 RFI supercoiled dsDNA and the P32-labelled oligonucleotides. The resulting D-loops were incubated with increasing amounts of the MUS81/EME1 complex. Cleaving or nicking the D-loop should result in the loss of superhelicity, displacement of the oligonucleotide, and disappearance of the D-loop. As shown in Figure 4B, MUS81/EME1 cleaved the D-loop produced by RAD52 in a concentration-dependent manner, as demonstrated by the reduction in the amount of the substrate. In contrast, the D-loop produced by RAD51-mediated strand invasion appeared resistant to endonucleolytic cleavage (Figure 4B). Notably, cleavage of this type of a D-loop required the presence of both MUS81/EME1 nuclease and RAD52 since MUS81/EME1 was extremely less efficient in cleaving the protein-free D-loops produced in the control experiment by heat-mediated annealing. To investigate if RAD52 stimulation of MUS81 activity was specific for the D-loop, we prepared a Cy5-labelled 3′-flap substrate, which represents one of the acknowledged and preferred MUS81 substrates. As shown in Figure 4C, MUS81 cleaved the 3′-flap substrates, giving rise in the generation of the nicked product. As expected, incubation of RAD52 alone did not result in any cleavage but, surprisingly, it prevented almost completely MUS81 from cutting the ssDNA flap.


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)

(A) Immunopurification of the human MUS81/EME1 complex from 293T cells.One-twentieth of human MUS81/EME1 complex immunopurified using anti-Myc-agarose/GSH agarose (see Materials and Methods) was resolved onto an SDS-PAGE gel and revealed by Coomassie blue-stain (CBB). (B) In vitro MUS81/EME1 cleavage of model D-loop substrates. The D-loops were produced by the RAD52-mediated annealing, by the RAD51-mediated strand invasion, or by the heat-mediated annealing as described in “Materials and Methods” and schematically depicted over the gel. MUS81/EME1-mediated cleavage results in the loss of superhelicity and, upon deproteination of the products, in the displacement of the radioactively-labeled oligonucleotide from the plasmid. Thus, the D-loop loss is an indicator of MUS81/EME1-dependent D-loop cleavage. The D-loops were separated from the unincorporated and displaced oligonucleotides on the agarose gel. The table above the gel summarizes the constituencies and conditions of each reaction. The band corresponding to the D-loop migration is marked on the side of the gel. The graph under the gel shows the gel quantification. (C) In vitro MUS81/EME1 cleavage of a 3′-flap substrate. The substrate was assembled as described in “Materials and Methods” and schematically depicted side the gel. MUS81/EME1-mediated cleavage results in formation of a nicked product, which was separated from the intact substrate and the not-assembled, single-stranded, substrate on the agarose gel. The graph shows the gel quantification. (D) RAD52 pulled-down MUS81 from nuclear extracts. Five µg of purified 6xHis-tagged RAD52 was incubated with 1 mg of benzonase-treated nuclear extract. After incubation with anti-His antibody-coupled magnetic beads, RAD52 protein complexes were released in 1x Laemmli sample buffer, subjected to SDS-PAGE and Western blotting using the indicated antibodies. Data are presented as a mean of replicate experiments, SEs were <10% of the mean. * = p<0.05 Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003910-g004: (A) Immunopurification of the human MUS81/EME1 complex from 293T cells.One-twentieth of human MUS81/EME1 complex immunopurified using anti-Myc-agarose/GSH agarose (see Materials and Methods) was resolved onto an SDS-PAGE gel and revealed by Coomassie blue-stain (CBB). (B) In vitro MUS81/EME1 cleavage of model D-loop substrates. The D-loops were produced by the RAD52-mediated annealing, by the RAD51-mediated strand invasion, or by the heat-mediated annealing as described in “Materials and Methods” and schematically depicted over the gel. MUS81/EME1-mediated cleavage results in the loss of superhelicity and, upon deproteination of the products, in the displacement of the radioactively-labeled oligonucleotide from the plasmid. Thus, the D-loop loss is an indicator of MUS81/EME1-dependent D-loop cleavage. The D-loops were separated from the unincorporated and displaced oligonucleotides on the agarose gel. The table above the gel summarizes the constituencies and conditions of each reaction. The band corresponding to the D-loop migration is marked on the side of the gel. The graph under the gel shows the gel quantification. (C) In vitro MUS81/EME1 cleavage of a 3′-flap substrate. The substrate was assembled as described in “Materials and Methods” and schematically depicted side the gel. MUS81/EME1-mediated cleavage results in formation of a nicked product, which was separated from the intact substrate and the not-assembled, single-stranded, substrate on the agarose gel. The graph shows the gel quantification. (D) RAD52 pulled-down MUS81 from nuclear extracts. Five µg of purified 6xHis-tagged RAD52 was incubated with 1 mg of benzonase-treated nuclear extract. After incubation with anti-His antibody-coupled magnetic beads, RAD52 protein complexes were released in 1x Laemmli sample buffer, subjected to SDS-PAGE and Western blotting using the indicated antibodies. Data are presented as a mean of replicate experiments, SEs were <10% of the mean. * = p<0.05 Student's t-test.
Mentions: One of the putative MUS81 substrates that may be formed at the HU-stalled or collapsed forks is a D-loop [7]. To verify this possibility, we evaluated the ability of a purified MUS81/EME1 complex to cleave a model D-loop assembled in vitro by either RAD52 or RAD51. The human MUS81/EME1 complex was immunopurified from 293T cells transiently transfected with plasmids expressing Myc-tagged MUS81 and GST-tagged EME1 (Figure 4A). Purified RAD52 or RAD51 were pre-incubated with P32-labelled oligonucleotides complementary to a region of the φX174 plasmid. The resulting nucleoprotein complexes mediated formation of the D-loops comprised by φX174 RFI supercoiled dsDNA and the P32-labelled oligonucleotides. The resulting D-loops were incubated with increasing amounts of the MUS81/EME1 complex. Cleaving or nicking the D-loop should result in the loss of superhelicity, displacement of the oligonucleotide, and disappearance of the D-loop. As shown in Figure 4B, MUS81/EME1 cleaved the D-loop produced by RAD52 in a concentration-dependent manner, as demonstrated by the reduction in the amount of the substrate. In contrast, the D-loop produced by RAD51-mediated strand invasion appeared resistant to endonucleolytic cleavage (Figure 4B). Notably, cleavage of this type of a D-loop required the presence of both MUS81/EME1 nuclease and RAD52 since MUS81/EME1 was extremely less efficient in cleaving the protein-free D-loops produced in the control experiment by heat-mediated annealing. To investigate if RAD52 stimulation of MUS81 activity was specific for the D-loop, we prepared a Cy5-labelled 3′-flap substrate, which represents one of the acknowledged and preferred MUS81 substrates. As shown in Figure 4C, MUS81 cleaved the 3′-flap substrates, giving rise in the generation of the nicked product. As expected, incubation of RAD52 alone did not result in any cleavage but, surprisingly, it prevented almost completely MUS81 from cutting the ssDNA flap.

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