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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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MUS81 down-regulation reduces the recovery of WS cells from replication arrest, leading to the accumulation of chromosome damage. (A) Analysis of the inhibition of DNA synthesis in wild-type and WS cells. Wild-type and WS cells transfected with control (GFP) or MUS81 siRNAs (left) and Hela cells transfected with control (GFP), WRN, or WRN/MUS81 siRNAs (right) were treated with 2 mM HU for 14 h, and recovered in drug-free medium for the indicated times. The graphs show the percentage of BrdU-positive nuclei compared with the untreated control. Data are means ± SE from three independent experiments. (B) Analysis of aberrant mitoses induced by replication arrest. Wild-type and WS cells transfected with control (GFP) or MUS81 siRNAs were exposed to 2 mM HU for 18 h before being recovered in drug-free medium for the indicated times. Images show mitoses from wild-type and WS cells in which MUS81 was abrogated. Bars, 20 μM. (C) Survival of wild-type, WS, or HeLa cells transfected with the indicated siRNAs in response to increasing doses of HU. Data are means ± SE from three independent experiments. (D) Schematic model showing the pathways responsible for replication fork recovery in wild-type and WS cells.
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fig7: MUS81 down-regulation reduces the recovery of WS cells from replication arrest, leading to the accumulation of chromosome damage. (A) Analysis of the inhibition of DNA synthesis in wild-type and WS cells. Wild-type and WS cells transfected with control (GFP) or MUS81 siRNAs (left) and Hela cells transfected with control (GFP), WRN, or WRN/MUS81 siRNAs (right) were treated with 2 mM HU for 14 h, and recovered in drug-free medium for the indicated times. The graphs show the percentage of BrdU-positive nuclei compared with the untreated control. Data are means ± SE from three independent experiments. (B) Analysis of aberrant mitoses induced by replication arrest. Wild-type and WS cells transfected with control (GFP) or MUS81 siRNAs were exposed to 2 mM HU for 18 h before being recovered in drug-free medium for the indicated times. Images show mitoses from wild-type and WS cells in which MUS81 was abrogated. Bars, 20 μM. (C) Survival of wild-type, WS, or HeLa cells transfected with the indicated siRNAs in response to increasing doses of HU. Data are means ± SE from three independent experiments. (D) Schematic model showing the pathways responsible for replication fork recovery in wild-type and WS cells.

Mentions: To test this hypothesis, WS fibroblasts and HeLa cells RNA depleted of WRN alone or in combination with MUS81 were exposed to HU for 16 h, then recovered in drug-free medium for different times. 30 min before harvesting, cells were pulsed with BrdU to evaluate DNA synthesis. Alternatively, wild-type and WS fibroblasts were transfected with MUS81 siRNAs and analyzed for the presence of chromosomal damage after recovery from HU. Loss of WRN function alone did not influence arrest and recovery of DNA synthesis after HU (Figs. S3 and S4). In contrast, concomitant MUS81 inhibition by RNAi in WS fibroblasts or in HeLa cells knocked down for WRN significantly impaired the ability of HU-arrested cells to resume DNA synthesis (Fig. 7 A). Furthermore, MUS81 RNAi in WS cells that are recovered from HU-mediated S-phase arrest resulted in the appearance of aberrant metaphases with fuzzy chromosomes and in micronuclei (Fig. 7 B and not depicted).


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)

MUS81 down-regulation reduces the recovery of WS cells from replication arrest, leading to the accumulation of chromosome damage. (A) Analysis of the inhibition of DNA synthesis in wild-type and WS cells. Wild-type and WS cells transfected with control (GFP) or MUS81 siRNAs (left) and Hela cells transfected with control (GFP), WRN, or WRN/MUS81 siRNAs (right) were treated with 2 mM HU for 14 h, and recovered in drug-free medium for the indicated times. The graphs show the percentage of BrdU-positive nuclei compared with the untreated control. Data are means ± SE from three independent experiments. (B) Analysis of aberrant mitoses induced by replication arrest. Wild-type and WS cells transfected with control (GFP) or MUS81 siRNAs were exposed to 2 mM HU for 18 h before being recovered in drug-free medium for the indicated times. Images show mitoses from wild-type and WS cells in which MUS81 was abrogated. Bars, 20 μM. (C) Survival of wild-type, WS, or HeLa cells transfected with the indicated siRNAs in response to increasing doses of HU. Data are means ± SE from three independent experiments. (D) Schematic model showing the pathways responsible for replication fork recovery in wild-type and WS cells.
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fig7: MUS81 down-regulation reduces the recovery of WS cells from replication arrest, leading to the accumulation of chromosome damage. (A) Analysis of the inhibition of DNA synthesis in wild-type and WS cells. Wild-type and WS cells transfected with control (GFP) or MUS81 siRNAs (left) and Hela cells transfected with control (GFP), WRN, or WRN/MUS81 siRNAs (right) were treated with 2 mM HU for 14 h, and recovered in drug-free medium for the indicated times. The graphs show the percentage of BrdU-positive nuclei compared with the untreated control. Data are means ± SE from three independent experiments. (B) Analysis of aberrant mitoses induced by replication arrest. Wild-type and WS cells transfected with control (GFP) or MUS81 siRNAs were exposed to 2 mM HU for 18 h before being recovered in drug-free medium for the indicated times. Images show mitoses from wild-type and WS cells in which MUS81 was abrogated. Bars, 20 μM. (C) Survival of wild-type, WS, or HeLa cells transfected with the indicated siRNAs in response to increasing doses of HU. Data are means ± SE from three independent experiments. (D) Schematic model showing the pathways responsible for replication fork recovery in wild-type and WS cells.
Mentions: To test this hypothesis, WS fibroblasts and HeLa cells RNA depleted of WRN alone or in combination with MUS81 were exposed to HU for 16 h, then recovered in drug-free medium for different times. 30 min before harvesting, cells were pulsed with BrdU to evaluate DNA synthesis. Alternatively, wild-type and WS fibroblasts were transfected with MUS81 siRNAs and analyzed for the presence of chromosomal damage after recovery from HU. Loss of WRN function alone did not influence arrest and recovery of DNA synthesis after HU (Figs. S3 and S4). In contrast, concomitant MUS81 inhibition by RNAi in WS fibroblasts or in HeLa cells knocked down for WRN significantly impaired the ability of HU-arrested cells to resume DNA synthesis (Fig. 7 A). Furthermore, MUS81 RNAi in WS cells that are recovered from HU-mediated S-phase arrest resulted in the appearance of aberrant metaphases with fuzzy chromosomes and in micronuclei (Fig. 7 B and not depicted).

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