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Checkpoint-dependent and independent roles of the Werner syndrome protein in preserving genome integrity in response to mild replication stress.

Basile G, Leuzzi G, Pichierri P, Franchitto A - Nucleic Acids Res. (2014)

Bottom Line: Analysis of replication fork dynamics shows that loss of WRN checkpoint mediator function as well as of WRN helicase activity hamper replication fork progression, and lead to new origin activation to allow recovery from replication slowing upon replication stress.Furthermore, bypass of WRN checkpoint mediator function through overexpression of a phospho-mimic form of CHK1 restores fork progression and chromosome stability to the wild-type levels.Together, these findings are the first demonstration that WRN regulates the ATR-checkpoint activation upon mild replication stress, preventing chromosome fragility.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Epidemiology, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Rome, Italy Genome Stability Group, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Rome, Italy.

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ATR-dependent WRN phosphorylation, but not defective WRN helicase activity, fails to activate CHK1 upon moderate replication stress. (A) WRN-deficient (WS) cells and WS cells complemented with wild-type (WSWRN) or expressing an unphosphorylable mutant form of WRN (WSWRN6A) were treated or not with Aph for the indicated times. CHK1 phosphorylation was analyzed by WB using phospho-specific antibodies (pS345). Total amount of CHK1 was determined with an anti-CHK1 antibody. Equal loading was confirmed probing with an anti-Lamin B1 antibody. Total WRN was used to assess the amount of wild-type or mutant form of WRN. (B) WS cells and WS cells complemented with wild-type (WSWRN) or expressing a mutant form of WRN affecting helicase (WRN-K577M) were treated or not with Aph for the indicated times. CHK1 phosphorylation was analyzed by WB using phospho-specific antibodies (pS345). Total amount of CHK1 was determined with an anti-CHK1 antibody. Lamin B1 was used as loading control. The ratio of phosphorylated protein to total protein and then normalized to the untreated wild type is listed below each lane. All experiments are representative images of at least three replicates.
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Figure 3: ATR-dependent WRN phosphorylation, but not defective WRN helicase activity, fails to activate CHK1 upon moderate replication stress. (A) WRN-deficient (WS) cells and WS cells complemented with wild-type (WSWRN) or expressing an unphosphorylable mutant form of WRN (WSWRN6A) were treated or not with Aph for the indicated times. CHK1 phosphorylation was analyzed by WB using phospho-specific antibodies (pS345). Total amount of CHK1 was determined with an anti-CHK1 antibody. Equal loading was confirmed probing with an anti-Lamin B1 antibody. Total WRN was used to assess the amount of wild-type or mutant form of WRN. (B) WS cells and WS cells complemented with wild-type (WSWRN) or expressing a mutant form of WRN affecting helicase (WRN-K577M) were treated or not with Aph for the indicated times. CHK1 phosphorylation was analyzed by WB using phospho-specific antibodies (pS345). Total amount of CHK1 was determined with an anti-CHK1 antibody. Lamin B1 was used as loading control. The ratio of phosphorylated protein to total protein and then normalized to the untreated wild type is listed below each lane. All experiments are representative images of at least three replicates.

Mentions: To verify whether WRN phosphorylation by ATR is a prerequisite for checkpoint activation after replication stress induced by Aph, we studied the ability of WS cells stably expressing the mutant form of WRN unphosphorylable by ATR (WSWRN6A) (12) to phosphorylate CHK1. WSWRN, WS and WSWRN6A cells were used and CHK1 phosphorylation evaluated. As expected, WSWRN cells were proficient in activation of CHK1 after treatment (Figure 3A). In contrast, WSWRN6A cells showed defective CHK1 phosphorylation as WS cells. Therefore, these data suggest that phosphorylation of WRN may play a role in the ATR-checkpoint activation in response to moderate replication stress.


Checkpoint-dependent and independent roles of the Werner syndrome protein in preserving genome integrity in response to mild replication stress.

Basile G, Leuzzi G, Pichierri P, Franchitto A - Nucleic Acids Res. (2014)

ATR-dependent WRN phosphorylation, but not defective WRN helicase activity, fails to activate CHK1 upon moderate replication stress. (A) WRN-deficient (WS) cells and WS cells complemented with wild-type (WSWRN) or expressing an unphosphorylable mutant form of WRN (WSWRN6A) were treated or not with Aph for the indicated times. CHK1 phosphorylation was analyzed by WB using phospho-specific antibodies (pS345). Total amount of CHK1 was determined with an anti-CHK1 antibody. Equal loading was confirmed probing with an anti-Lamin B1 antibody. Total WRN was used to assess the amount of wild-type or mutant form of WRN. (B) WS cells and WS cells complemented with wild-type (WSWRN) or expressing a mutant form of WRN affecting helicase (WRN-K577M) were treated or not with Aph for the indicated times. CHK1 phosphorylation was analyzed by WB using phospho-specific antibodies (pS345). Total amount of CHK1 was determined with an anti-CHK1 antibody. Lamin B1 was used as loading control. The ratio of phosphorylated protein to total protein and then normalized to the untreated wild type is listed below each lane. All experiments are representative images of at least three replicates.
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Related In: Results  -  Collection

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Figure 3: ATR-dependent WRN phosphorylation, but not defective WRN helicase activity, fails to activate CHK1 upon moderate replication stress. (A) WRN-deficient (WS) cells and WS cells complemented with wild-type (WSWRN) or expressing an unphosphorylable mutant form of WRN (WSWRN6A) were treated or not with Aph for the indicated times. CHK1 phosphorylation was analyzed by WB using phospho-specific antibodies (pS345). Total amount of CHK1 was determined with an anti-CHK1 antibody. Equal loading was confirmed probing with an anti-Lamin B1 antibody. Total WRN was used to assess the amount of wild-type or mutant form of WRN. (B) WS cells and WS cells complemented with wild-type (WSWRN) or expressing a mutant form of WRN affecting helicase (WRN-K577M) were treated or not with Aph for the indicated times. CHK1 phosphorylation was analyzed by WB using phospho-specific antibodies (pS345). Total amount of CHK1 was determined with an anti-CHK1 antibody. Lamin B1 was used as loading control. The ratio of phosphorylated protein to total protein and then normalized to the untreated wild type is listed below each lane. All experiments are representative images of at least three replicates.
Mentions: To verify whether WRN phosphorylation by ATR is a prerequisite for checkpoint activation after replication stress induced by Aph, we studied the ability of WS cells stably expressing the mutant form of WRN unphosphorylable by ATR (WSWRN6A) (12) to phosphorylate CHK1. WSWRN, WS and WSWRN6A cells were used and CHK1 phosphorylation evaluated. As expected, WSWRN cells were proficient in activation of CHK1 after treatment (Figure 3A). In contrast, WSWRN6A cells showed defective CHK1 phosphorylation as WS cells. Therefore, these data suggest that phosphorylation of WRN may play a role in the ATR-checkpoint activation in response to moderate replication stress.

Bottom Line: Analysis of replication fork dynamics shows that loss of WRN checkpoint mediator function as well as of WRN helicase activity hamper replication fork progression, and lead to new origin activation to allow recovery from replication slowing upon replication stress.Furthermore, bypass of WRN checkpoint mediator function through overexpression of a phospho-mimic form of CHK1 restores fork progression and chromosome stability to the wild-type levels.Together, these findings are the first demonstration that WRN regulates the ATR-checkpoint activation upon mild replication stress, preventing chromosome fragility.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Epidemiology, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Rome, Italy Genome Stability Group, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Rome, Italy.

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Related in: MedlinePlus