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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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Accumulation of DSBs after replication arrest observed in the absence of WRN depends on the presence of the MUS81 endonuclease. (A) MUS81 down-regulation by transfection of MUS81 siRNAs in wild-type and WS cells. (B) MUS81 and/or WRN down-regulation by transfection of MUS81 and/or WRN siRNAs in HeLa cells was verified by immunoblotting 48 h after transfection. (C) Formation of DSBs in wild-type and WS fibroblasts transfected with MUS81 siRNAs was evaluated by PFGE after 6 h of treatment with 2 mM HU or 1 μM etoposide (Etop.). Data are presented as mean ± SE from three independent experiments. (D) Formation of DSBs in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs and treated with 2 mM HU for the indicated times was evaluated by PFGE. Data are presented as mean ± SE from three independent experiments. (E) Analysis of DSB accumulation by anti–γ-H2AX immunofluorescence in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs and treated with 2 mM HU for 6 h. Representative images are shown. The percentage of γ-H2AX–positive nuclei from three independent experiments is shown. Data are presented as mean ± SE. Bars, 10 μM.
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fig2: Accumulation of DSBs after replication arrest observed in the absence of WRN depends on the presence of the MUS81 endonuclease. (A) MUS81 down-regulation by transfection of MUS81 siRNAs in wild-type and WS cells. (B) MUS81 and/or WRN down-regulation by transfection of MUS81 and/or WRN siRNAs in HeLa cells was verified by immunoblotting 48 h after transfection. (C) Formation of DSBs in wild-type and WS fibroblasts transfected with MUS81 siRNAs was evaluated by PFGE after 6 h of treatment with 2 mM HU or 1 μM etoposide (Etop.). Data are presented as mean ± SE from three independent experiments. (D) Formation of DSBs in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs and treated with 2 mM HU for the indicated times was evaluated by PFGE. Data are presented as mean ± SE from three independent experiments. (E) Analysis of DSB accumulation by anti–γ-H2AX immunofluorescence in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs and treated with 2 mM HU for 6 h. Representative images are shown. The percentage of γ-H2AX–positive nuclei from three independent experiments is shown. Data are presented as mean ± SE. Bars, 10 μM.

Mentions: Having demonstrated that WS cells accumulate DSBs after HU-induced replication arrest, we asked whether those DSBs could be the end result of the action of a backup pathway processing replication intermediates in the absence of WRN. In yeast, mutations in the RecQ helicases, Sgs1 or Rqh1, are synthetic lethal, with a mutation inactivating the Mus81–Eme1 complex, which indicates that RecQ helicases and the Mus81 endonuclease can define two competing pathways processing branched DNA intermediates at stalled replisome (Boddy et al., 2001; Doe et al., 2002; Ciccia et al., 2003; Whitby et al., 2003; Cotta-Ramusino et al., 2005; Fricke et al., 2005). Furthermore, a recent paper suggests that the MUS81 function could be required to process forks stalled at interstrand DNA crosslink sites forming DSBs (Hanada et al., 2006). We investigated whether the genetic interaction observed in yeast was conserved in humans by testing the hypothesis that MUS81 could be involved in DSB formation in a WRN-deficient background. To this end, we used siRNA-mediated RNAi to down-regulate MUS81 expression in either WS or HeLa cells. Under our experimental conditions, RNAi reduced MUS81 expression of >80% in wild-type, WS, and HeLa cells (Fig. 2, A and B), and did not alter cell cycle progression (Fig. S3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200803173/DC1). 48 h after transfection, replication fork arrest was induced with HU, and DSBs were evaluated by PFGE. In wild-type fibroblasts or HeLa cells, MUS81 RNAi did not significantly affect DSB formation after HU treatment, at least up to 14 h of replication arrest (Fig. 2, C and D). In contrast, depletion of MUS81 in WRN-deficient fibroblasts or concomitant knockdown of WRN and MUS81 in HeLa cells decreased the amount of DSBs induced by replication fork stalling close to that observed in untreated controls (Fig. 2, C and D). Notably, MUS81 down regulation also reduced the spontaneous level of DNA breaks observed in WRN-deficient cells (Fig. 2 C), even though it failed to modulate the breakage in untreated WRN RNAi HeLa cells (Fig. 2 D). This apparent discrepancy could depend on the enhanced level of MUS81-related DSBs accumulating as a consequence of chronic versus acute WRN depletion and the PFGE threshold of detection, could be related to a more efficient MUS81 silencing in WS cells, or could be combination of both. However, analysis of the induction of γ-H2AX foci in HeLa cells treated with WRN or WRN/MUS81 siRNAs revealed a drastic reduction of γ-H2AX immunostaining in HU-treated cells and untreated samples as well (Fig. 2 E). A consistent reduction of DSBs evaluated by γ-H2AX immunofluorescence was found also in HU-treated WS cells upon transfection with MUS81 siRNAs (unpublished data). It is worthwhile to note that accumulation of DSBs after treatment with the topoisomerase II–poison etoposide was independent of the presence of WRN and MUS81 (Fig. 2 C), which suggests that MUS81 cleavage is not involved in DSB formation when DSBs are formed directly, as occurs after inhibition of topoisomerase II by etoposide.


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)

Accumulation of DSBs after replication arrest observed in the absence of WRN depends on the presence of the MUS81 endonuclease. (A) MUS81 down-regulation by transfection of MUS81 siRNAs in wild-type and WS cells. (B) MUS81 and/or WRN down-regulation by transfection of MUS81 and/or WRN siRNAs in HeLa cells was verified by immunoblotting 48 h after transfection. (C) Formation of DSBs in wild-type and WS fibroblasts transfected with MUS81 siRNAs was evaluated by PFGE after 6 h of treatment with 2 mM HU or 1 μM etoposide (Etop.). Data are presented as mean ± SE from three independent experiments. (D) Formation of DSBs in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs and treated with 2 mM HU for the indicated times was evaluated by PFGE. Data are presented as mean ± SE from three independent experiments. (E) Analysis of DSB accumulation by anti–γ-H2AX immunofluorescence in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs and treated with 2 mM HU for 6 h. Representative images are shown. The percentage of γ-H2AX–positive nuclei from three independent experiments is shown. Data are presented as mean ± SE. Bars, 10 μM.
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Related In: Results  -  Collection

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fig2: Accumulation of DSBs after replication arrest observed in the absence of WRN depends on the presence of the MUS81 endonuclease. (A) MUS81 down-regulation by transfection of MUS81 siRNAs in wild-type and WS cells. (B) MUS81 and/or WRN down-regulation by transfection of MUS81 and/or WRN siRNAs in HeLa cells was verified by immunoblotting 48 h after transfection. (C) Formation of DSBs in wild-type and WS fibroblasts transfected with MUS81 siRNAs was evaluated by PFGE after 6 h of treatment with 2 mM HU or 1 μM etoposide (Etop.). Data are presented as mean ± SE from three independent experiments. (D) Formation of DSBs in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs and treated with 2 mM HU for the indicated times was evaluated by PFGE. Data are presented as mean ± SE from three independent experiments. (E) Analysis of DSB accumulation by anti–γ-H2AX immunofluorescence in HeLa cells transfected with control (GFP), MUS81, and/or WRN siRNAs and treated with 2 mM HU for 6 h. Representative images are shown. The percentage of γ-H2AX–positive nuclei from three independent experiments is shown. Data are presented as mean ± SE. Bars, 10 μM.
Mentions: Having demonstrated that WS cells accumulate DSBs after HU-induced replication arrest, we asked whether those DSBs could be the end result of the action of a backup pathway processing replication intermediates in the absence of WRN. In yeast, mutations in the RecQ helicases, Sgs1 or Rqh1, are synthetic lethal, with a mutation inactivating the Mus81–Eme1 complex, which indicates that RecQ helicases and the Mus81 endonuclease can define two competing pathways processing branched DNA intermediates at stalled replisome (Boddy et al., 2001; Doe et al., 2002; Ciccia et al., 2003; Whitby et al., 2003; Cotta-Ramusino et al., 2005; Fricke et al., 2005). Furthermore, a recent paper suggests that the MUS81 function could be required to process forks stalled at interstrand DNA crosslink sites forming DSBs (Hanada et al., 2006). We investigated whether the genetic interaction observed in yeast was conserved in humans by testing the hypothesis that MUS81 could be involved in DSB formation in a WRN-deficient background. To this end, we used siRNA-mediated RNAi to down-regulate MUS81 expression in either WS or HeLa cells. Under our experimental conditions, RNAi reduced MUS81 expression of >80% in wild-type, WS, and HeLa cells (Fig. 2, A and B), and did not alter cell cycle progression (Fig. S3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200803173/DC1). 48 h after transfection, replication fork arrest was induced with HU, and DSBs were evaluated by PFGE. In wild-type fibroblasts or HeLa cells, MUS81 RNAi did not significantly affect DSB formation after HU treatment, at least up to 14 h of replication arrest (Fig. 2, C and D). In contrast, depletion of MUS81 in WRN-deficient fibroblasts or concomitant knockdown of WRN and MUS81 in HeLa cells decreased the amount of DSBs induced by replication fork stalling close to that observed in untreated controls (Fig. 2, C and D). Notably, MUS81 down regulation also reduced the spontaneous level of DNA breaks observed in WRN-deficient cells (Fig. 2 C), even though it failed to modulate the breakage in untreated WRN RNAi HeLa cells (Fig. 2 D). This apparent discrepancy could depend on the enhanced level of MUS81-related DSBs accumulating as a consequence of chronic versus acute WRN depletion and the PFGE threshold of detection, could be related to a more efficient MUS81 silencing in WS cells, or could be combination of both. However, analysis of the induction of γ-H2AX foci in HeLa cells treated with WRN or WRN/MUS81 siRNAs revealed a drastic reduction of γ-H2AX immunostaining in HU-treated cells and untreated samples as well (Fig. 2 E). A consistent reduction of DSBs evaluated by γ-H2AX immunofluorescence was found also in HU-treated WS cells upon transfection with MUS81 siRNAs (unpublished data). It is worthwhile to note that accumulation of DSBs after treatment with the topoisomerase II–poison etoposide was independent of the presence of WRN and MUS81 (Fig. 2 C), which suggests that MUS81 cleavage is not involved in DSB formation when DSBs are formed directly, as occurs after inhibition of topoisomerase II by etoposide.

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