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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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Loss of WRN results in enhanced association of MUS81 with chromatin after replication fork stalling. Levels of chromatin-bound and total MUS81 in HeLa cells transfected with control (GFP), WRN, or MUS81 siRNAs and treated with 2 mM HU for indicated times. ORC2 was used as a loading control. The amount of MUS81 in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the ORC2 content. (bottom) Total content of MUS81 in RNAi-treated HeLa cells was evaluated by Western immunoblotting after treatment with 2 mM HU, as in Fig. 2.
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fig3: Loss of WRN results in enhanced association of MUS81 with chromatin after replication fork stalling. Levels of chromatin-bound and total MUS81 in HeLa cells transfected with control (GFP), WRN, or MUS81 siRNAs and treated with 2 mM HU for indicated times. ORC2 was used as a loading control. The amount of MUS81 in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the ORC2 content. (bottom) Total content of MUS81 in RNAi-treated HeLa cells was evaluated by Western immunoblotting after treatment with 2 mM HU, as in Fig. 2.

Mentions: Mutations in vertebrate and mammalian genes associated with the replication stress response, such as ataxia telangiectasia and Rad3–related (ATR), determine the appearance of DNA breaks in replicating cells (Cliby et al., 1998; Casper et al., 2002; Lomonosov et al., 2003; Trenz et al., 2006). Thus, to investigate whether loss of WRN could influence DSB formation during DNA replication, we inhibited DNA synthesis in wild-type and WS cells with HU. We evaluated DSB induction in the whole genome using pulsed-field gel electrophoresis (PFGE) and, at the single-cell level, anti–phospho-H2AX (γ-H2AX) immunostaining. Formation of chromosomal breaks was analyzed early after HU-mediated replication arrest and up to 14 h, a time-point corresponding to accumulation of DSBs in the wild type (Saintigny et al., 2001). WRN deficiency resulted in enhanced accumulation of DSBs under unstressed conditions and at early time points after HU treatment. In contrast, wild-type cells accumulated DSBs only at the 14 h time point (Fig. 1 A). To confirm correlation between loss of WRN function and accumulation of DSBs after replication arrest in a common genetic background, we transfected WRN siRNAs in HeLa cells and then analyzed DSB formation after HU by PFGE. RNAi efficiently depleted WRN protein from HeLa cells (see Fig. 3 B), conferring a WS-like phenotype of hypersensitivity to camptothecin (unpublished data). Down-regulation of WRN by RNAi determined a time-dependent increase in the accumulation of DSBs in response to HU treatment, whereas chromosomal fragmentation was barely detectable after transfection with control siRNAs (Fig. 1 B). Morphological inspection of HU-treated cultures did not provide evidence of cell death, which excludes the possibility that chromosomal fragmentation derived from the induction of apoptosis (unpublished data).


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)

Loss of WRN results in enhanced association of MUS81 with chromatin after replication fork stalling. Levels of chromatin-bound and total MUS81 in HeLa cells transfected with control (GFP), WRN, or MUS81 siRNAs and treated with 2 mM HU for indicated times. ORC2 was used as a loading control. The amount of MUS81 in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the ORC2 content. (bottom) Total content of MUS81 in RNAi-treated HeLa cells was evaluated by Western immunoblotting after treatment with 2 mM HU, as in Fig. 2.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2568021&req=5

fig3: Loss of WRN results in enhanced association of MUS81 with chromatin after replication fork stalling. Levels of chromatin-bound and total MUS81 in HeLa cells transfected with control (GFP), WRN, or MUS81 siRNAs and treated with 2 mM HU for indicated times. ORC2 was used as a loading control. The amount of MUS81 in the chromatin fraction is expressed as the percentage of the amount in the untreated control normalized against the ORC2 content. (bottom) Total content of MUS81 in RNAi-treated HeLa cells was evaluated by Western immunoblotting after treatment with 2 mM HU, as in Fig. 2.
Mentions: Mutations in vertebrate and mammalian genes associated with the replication stress response, such as ataxia telangiectasia and Rad3–related (ATR), determine the appearance of DNA breaks in replicating cells (Cliby et al., 1998; Casper et al., 2002; Lomonosov et al., 2003; Trenz et al., 2006). Thus, to investigate whether loss of WRN could influence DSB formation during DNA replication, we inhibited DNA synthesis in wild-type and WS cells with HU. We evaluated DSB induction in the whole genome using pulsed-field gel electrophoresis (PFGE) and, at the single-cell level, anti–phospho-H2AX (γ-H2AX) immunostaining. Formation of chromosomal breaks was analyzed early after HU-mediated replication arrest and up to 14 h, a time-point corresponding to accumulation of DSBs in the wild type (Saintigny et al., 2001). WRN deficiency resulted in enhanced accumulation of DSBs under unstressed conditions and at early time points after HU treatment. In contrast, wild-type cells accumulated DSBs only at the 14 h time point (Fig. 1 A). To confirm correlation between loss of WRN function and accumulation of DSBs after replication arrest in a common genetic background, we transfected WRN siRNAs in HeLa cells and then analyzed DSB formation after HU by PFGE. RNAi efficiently depleted WRN protein from HeLa cells (see Fig. 3 B), conferring a WS-like phenotype of hypersensitivity to camptothecin (unpublished data). Down-regulation of WRN by RNAi determined a time-dependent increase in the accumulation of DSBs in response to HU treatment, whereas chromosomal fragmentation was barely detectable after transfection with control siRNAs (Fig. 1 B). Morphological inspection of HU-treated cultures did not provide evidence of cell death, which excludes the possibility that chromosomal fragmentation derived from the induction of apoptosis (unpublished data).

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