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Replication fork stalling in WRN-deficient cells is overcome by prompt activation of a MUS81-dependent pathway.

Franchitto A, Pirzio LM, Prosperi E, Sapora O, Bignami M, Pichierri P - J. Cell Biol. (2008)

Bottom Line: Knockdown of the MUS81 endonuclease in WRN-deficient cells completely prevents the accumulation of DSBs after fork stalling.Also, MUS81 knockdown in WS cells results in reduced chromatin recruitment of recombination enzymes, decreased yield of sister chromatid exchanges, and reduced survival after replication arrest.Thus, we provide novel evidence that WRN is required to avoid accumulation of DSBs and fork collapse after replication perturbation, and that prompt MUS81-dependent generation of DSBs is instrumental for recovery from hydroxyurea-mediated replication arrest under such pathological conditions.

View Article: PubMed Central - PubMed

Affiliation: Section of Experimental and Computational Carcinogenesis, Istituto Superiore di Sanità, 00161 Rome, Italy.

ABSTRACT
Failure to stabilize and properly process stalled replication forks results in chromosome instability, which is a hallmark of cancer cells and several human genetic conditions that are characterized by cancer predisposition. Loss of WRN, a RecQ-like enzyme mutated in the cancer-prone disease Werner syndrome (WS), leads to rapid accumulation of double-strand breaks (DSBs) and proliferating cell nuclear antigen removal from chromatin upon DNA replication arrest. Knockdown of the MUS81 endonuclease in WRN-deficient cells completely prevents the accumulation of DSBs after fork stalling. Also, MUS81 knockdown in WS cells results in reduced chromatin recruitment of recombination enzymes, decreased yield of sister chromatid exchanges, and reduced survival after replication arrest. Thus, we provide novel evidence that WRN is required to avoid accumulation of DSBs and fork collapse after replication perturbation, and that prompt MUS81-dependent generation of DSBs is instrumental for recovery from hydroxyurea-mediated replication arrest under such pathological conditions.

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Replication arrest induces dispersal of PCNA from S-phase chromatin in WRN-deficient cells. (A) Levels of chromatin-bound PCNA in wild-type and WS fibroblasts treated with 2 mM HU for the indicated times or with 50 μM etoposide for 6 h (Etop.). H3 histone was used as a loading control. The residual amount of PCNA in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the H3 histone content. (B) Levels of chromatin-bound PCNA in S-phase cells after replication inhibition. Wild-type and WS fibroblasts were exposed to 2 mM HU for the indicated times or to 50 μM etoposide for 6 h (Etop.), before being processed for biparametric PCNA/DNA flow cytometry. Representative cytograms for each condition are presented. The values shown represent the fluorescence intensity of the PCNA staining relative to an S-phase DNA content, expressed as percentage of the untreated control. The gates indicate the area considered for the evaluation of PCNA fluorescence intensity. *, statistically significant compared with the wild type; P < 0.01 (analysis of variance test). (C) Levels of chromatin-bound PCNA in HeLa cells transfected with control (GFP) or WRN siRNAs and treated with 2 mM HU for the indicated times. H3 histone was used as a loading control. The residual amount of PCNA in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the H3 histone content. (D) Levels of chromatin-bound PCNA in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs treated with 2 mM HU for the indicated times. H3 histone was determined as a loading control. RNAi-labeled samples represent cells treated with MUS81 siRNAs; if not otherwise specified, samples were treated with control siRNAs against GFP.
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fig4: Replication arrest induces dispersal of PCNA from S-phase chromatin in WRN-deficient cells. (A) Levels of chromatin-bound PCNA in wild-type and WS fibroblasts treated with 2 mM HU for the indicated times or with 50 μM etoposide for 6 h (Etop.). H3 histone was used as a loading control. The residual amount of PCNA in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the H3 histone content. (B) Levels of chromatin-bound PCNA in S-phase cells after replication inhibition. Wild-type and WS fibroblasts were exposed to 2 mM HU for the indicated times or to 50 μM etoposide for 6 h (Etop.), before being processed for biparametric PCNA/DNA flow cytometry. Representative cytograms for each condition are presented. The values shown represent the fluorescence intensity of the PCNA staining relative to an S-phase DNA content, expressed as percentage of the untreated control. The gates indicate the area considered for the evaluation of PCNA fluorescence intensity. *, statistically significant compared with the wild type; P < 0.01 (analysis of variance test). (C) Levels of chromatin-bound PCNA in HeLa cells transfected with control (GFP) or WRN siRNAs and treated with 2 mM HU for the indicated times. H3 histone was used as a loading control. The residual amount of PCNA in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the H3 histone content. (D) Levels of chromatin-bound PCNA in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs treated with 2 mM HU for the indicated times. H3 histone was determined as a loading control. RNAi-labeled samples represent cells treated with MUS81 siRNAs; if not otherwise specified, samples were treated with control siRNAs against GFP.

Mentions: To validate our approach, we treated cells with 50 μM etoposide, a condition found to determine >80% of replisome disassembly as visualized at the single-cell level by PCNA staining (Montecucco et al., 2001). Our assays proved to be able to detect etoposide-induced replisome disassembly, and, thus, we consider it equivalent to the in situ assay used by Montecucco et al., (2001) and useful to investigate replisome collapse after HU treatment. Analysis of chromatin fractions revealed that HU-induced replication fork stalling did not induce an overt dissociation of PCNA from chromatin in wild-type cells, whereas WRN deficiency determined a much more substantial decrease (Fig. 4 A). In contrast, etoposide treatment, which directly causes DSBs irrespective of the WRN status of the cells, dropped the amount of chromatin-associated PCNA in both wild-type and WS cells (Fig. 4 A). Flow cytometry analysis confirmed that a significant fraction of PCNA dissociates from chromatin in WS cells treated with HU, and evidenced that loss of PCNA occurred specifically in S-phase cells (Fig. 4 B).


Replication fork stalling in WRN-deficient cells is overcome by prompt activation of a MUS81-dependent pathway.

Franchitto A, Pirzio LM, Prosperi E, Sapora O, Bignami M, Pichierri P - J. Cell Biol. (2008)

Replication arrest induces dispersal of PCNA from S-phase chromatin in WRN-deficient cells. (A) Levels of chromatin-bound PCNA in wild-type and WS fibroblasts treated with 2 mM HU for the indicated times or with 50 μM etoposide for 6 h (Etop.). H3 histone was used as a loading control. The residual amount of PCNA in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the H3 histone content. (B) Levels of chromatin-bound PCNA in S-phase cells after replication inhibition. Wild-type and WS fibroblasts were exposed to 2 mM HU for the indicated times or to 50 μM etoposide for 6 h (Etop.), before being processed for biparametric PCNA/DNA flow cytometry. Representative cytograms for each condition are presented. The values shown represent the fluorescence intensity of the PCNA staining relative to an S-phase DNA content, expressed as percentage of the untreated control. The gates indicate the area considered for the evaluation of PCNA fluorescence intensity. *, statistically significant compared with the wild type; P < 0.01 (analysis of variance test). (C) Levels of chromatin-bound PCNA in HeLa cells transfected with control (GFP) or WRN siRNAs and treated with 2 mM HU for the indicated times. H3 histone was used as a loading control. The residual amount of PCNA in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the H3 histone content. (D) Levels of chromatin-bound PCNA in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs treated with 2 mM HU for the indicated times. H3 histone was determined as a loading control. RNAi-labeled samples represent cells treated with MUS81 siRNAs; if not otherwise specified, samples were treated with control siRNAs against GFP.
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Related In: Results  -  Collection

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fig4: Replication arrest induces dispersal of PCNA from S-phase chromatin in WRN-deficient cells. (A) Levels of chromatin-bound PCNA in wild-type and WS fibroblasts treated with 2 mM HU for the indicated times or with 50 μM etoposide for 6 h (Etop.). H3 histone was used as a loading control. The residual amount of PCNA in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the H3 histone content. (B) Levels of chromatin-bound PCNA in S-phase cells after replication inhibition. Wild-type and WS fibroblasts were exposed to 2 mM HU for the indicated times or to 50 μM etoposide for 6 h (Etop.), before being processed for biparametric PCNA/DNA flow cytometry. Representative cytograms for each condition are presented. The values shown represent the fluorescence intensity of the PCNA staining relative to an S-phase DNA content, expressed as percentage of the untreated control. The gates indicate the area considered for the evaluation of PCNA fluorescence intensity. *, statistically significant compared with the wild type; P < 0.01 (analysis of variance test). (C) Levels of chromatin-bound PCNA in HeLa cells transfected with control (GFP) or WRN siRNAs and treated with 2 mM HU for the indicated times. H3 histone was used as a loading control. The residual amount of PCNA in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the H3 histone content. (D) Levels of chromatin-bound PCNA in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs treated with 2 mM HU for the indicated times. H3 histone was determined as a loading control. RNAi-labeled samples represent cells treated with MUS81 siRNAs; if not otherwise specified, samples were treated with control siRNAs against GFP.
Mentions: To validate our approach, we treated cells with 50 μM etoposide, a condition found to determine >80% of replisome disassembly as visualized at the single-cell level by PCNA staining (Montecucco et al., 2001). Our assays proved to be able to detect etoposide-induced replisome disassembly, and, thus, we consider it equivalent to the in situ assay used by Montecucco et al., (2001) and useful to investigate replisome collapse after HU treatment. Analysis of chromatin fractions revealed that HU-induced replication fork stalling did not induce an overt dissociation of PCNA from chromatin in wild-type cells, whereas WRN deficiency determined a much more substantial decrease (Fig. 4 A). In contrast, etoposide treatment, which directly causes DSBs irrespective of the WRN status of the cells, dropped the amount of chromatin-associated PCNA in both wild-type and WS cells (Fig. 4 A). Flow cytometry analysis confirmed that a significant fraction of PCNA dissociates from chromatin in WS cells treated with HU, and evidenced that loss of PCNA occurred specifically in S-phase cells (Fig. 4 B).

Bottom Line: Knockdown of the MUS81 endonuclease in WRN-deficient cells completely prevents the accumulation of DSBs after fork stalling.Also, MUS81 knockdown in WS cells results in reduced chromatin recruitment of recombination enzymes, decreased yield of sister chromatid exchanges, and reduced survival after replication arrest.Thus, we provide novel evidence that WRN is required to avoid accumulation of DSBs and fork collapse after replication perturbation, and that prompt MUS81-dependent generation of DSBs is instrumental for recovery from hydroxyurea-mediated replication arrest under such pathological conditions.

View Article: PubMed Central - PubMed

Affiliation: Section of Experimental and Computational Carcinogenesis, Istituto Superiore di Sanità, 00161 Rome, Italy.

ABSTRACT
Failure to stabilize and properly process stalled replication forks results in chromosome instability, which is a hallmark of cancer cells and several human genetic conditions that are characterized by cancer predisposition. Loss of WRN, a RecQ-like enzyme mutated in the cancer-prone disease Werner syndrome (WS), leads to rapid accumulation of double-strand breaks (DSBs) and proliferating cell nuclear antigen removal from chromatin upon DNA replication arrest. Knockdown of the MUS81 endonuclease in WRN-deficient cells completely prevents the accumulation of DSBs after fork stalling. Also, MUS81 knockdown in WS cells results in reduced chromatin recruitment of recombination enzymes, decreased yield of sister chromatid exchanges, and reduced survival after replication arrest. Thus, we provide novel evidence that WRN is required to avoid accumulation of DSBs and fork collapse after replication perturbation, and that prompt MUS81-dependent generation of DSBs is instrumental for recovery from hydroxyurea-mediated replication arrest under such pathological conditions.

Show MeSH
Related in: MedlinePlus