RESUMO
Accurate response to replication arrest is crucial to preserve genome stability and requires both the ATR and ATM functions. The Werner syndrome protein (WRN) is implicated in the recovery of stalled replication forks, and although an ATR/ATM-dependent phosphorylation of WRN was observed after replication arrest, the function of such modifications during the response to perturbed replication is not yet appreciated. Here, we report that WRN is directly phosphorylated by ATR at multiple C-terminal S/TQ residues. Suppression of ATR-mediated phosphorylation of WRN prevents proper accumulation of WRN in nuclear foci, co-localisation with RPA and causes breakage of stalled forks. On the other hand, inhibition of ATM kinase activity or expression of an ATM-unphosphorylable WRN allele leads to retention of WRN in nuclear foci and impaired recruitment of RAD51 recombinase resulting in reduced viability after fork collapse. Altogether, our findings indicate that ATR and ATM promote recovery from perturbed replication by differently regulating WRN at defined moments of the response to replication fork arrest.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RecQ Helicases/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Imunoprecipitação , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Helicase da Síndrome de WernerRESUMO
WRN is a member of the RecQ family of DNA helicases implicated in the resolution of DNA structures leading to the stall of replication forks. Fragile sites have been proposed to be DNA regions particularly sensitive to replicative stress. Here, we establish that WRN is a key regulator of fragile site stability. We demonstrate that in response to mild doses of aphidicolin, WRN is efficiently relocalized in nuclear foci in replicating cells and that WRN deficiency is associated with accumulation of gaps and breaks at common fragile sites even under unperturbed conditions. By expressing WRN isoforms impaired in either helicase or exonuclease activity in defective cells, we identified WRN helicase activity as the function required for maintaining the stability of fragile sites. Finally, we find that WRN stabilizes fragile sites acting in a common pathway with the ataxia telangiectasia and Rad3 related replication checkpoint. These findings provide the first evidence of a crucial role for a helicase in protecting cells against chromosome breakage at normally occurring replication fork stalling sites.
Assuntos
Sítios Frágeis do Cromossomo , RecQ Helicases/metabolismo , Síndrome de Werner/enzimologia , Afidicolina/farmacologia , Códon sem Sentido , Replicação do DNA/efeitos dos fármacos , Exodesoxirribonucleases , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Humanos , RecQ Helicases/genética , Síndrome de Werner/metabolismo , Helicase da Síndrome de WernerRESUMO
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.