ABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Repair , DNA Replication , DNA-Binding Proteins/metabolism , Exodeoxyribonucleases/genetics , Protein Serine-Threonine Kinases/metabolism , RecQ Helicases/genetics , Tumor Suppressor Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins , Blotting, Western , Cell Cycle , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Nucleus/metabolism , DNA-Binding Proteins/genetics , Exodeoxyribonucleases/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Humans , Immunoprecipitation , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/genetics , RNA, Small Interfering/pharmacology , Rad51 Recombinase/antagonists & inhibitors , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , RecQ Helicases/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Proteins/genetics , Werner Syndrome HelicaseABSTRACT
The Werner syndrome protein (WRN) is a member of the human RecQ family DNA helicases implicated in the maintenance of genome stability. Loss of WRN gives rise to the Werner syndrome, a genetic disease characterised by premature aging and cancer predisposition. WRN plays a crucial role in the response to replication stress and significantly contributes to the recovery of stalled replication forks, although how this function is regulated is not fully appreciated. There is a growing body of evidence that WRN accomplishes its task in close connection with the replication checkpoint. In eukaryotic cells, the replication checkpoint response, which involves both the ATR and ATM kinase activities, is deputed to the maintenance of fork integrity and re-establishment of fork progression. Our recent findings indicate that ATR and ATM modulate WRN function at defined steps of the response to replication fork arrest. This review focuses on the novel evidence of a functional relationship between WRN and the replication checkpoint and how this cross-talk might contribute to prevent genome instability, a common feature of senescent and cancer cells.