RESUMO
Progression of DNA replication is tightly controlled by replication checkpoints to ensure the accurate and rapid duplication of genetic information. Upon replication stress, the replication checkpoint slows global DNA replication by inhibiting the late-firing origins and by slowing replication fork progression. Activation of the replication checkpoint has been studied in depth; however, little is known about the termination of the replication checkpoint. Here, we show that Src family kinases promote the recovery from replication checkpoints. shRNA knockdown of a Src family kinase, Lyn, and acute chemical inhibition of Src kinases prevented inactivation of Chk1 after removal of replication stress. Consistently, Src inhibition slowed resumption of DNA replication, after the removal of replication blocks. The effect of Src inhibition was not observed in the presence of an ATM/ATR inhibitor caffeine. These data indicate that Src kinases promote the resumption of DNA replication by suppressing ATR-dependent replication checkpoints. Surprisingly, the resumption of replication was delayed by caffeine. In addition, Src inhibition delayed recovery from replication fork collapse. We propose that Src kinases maintain the balance between replication stress and the activity of the replication checkpoint.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Replicação do DNA/fisiologia , Quinases da Família src/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Cafeína/farmacologia , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Reparo do DNA , Células HeLa , Humanos , Transdução de Sinais , Quinases da Família src/antagonistas & inibidoresRESUMO
DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Pontos de Checagem do Ciclo Celular/genética , Transdução de Sinais/genética , Quinases da Família src/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Doxorrubicina/farmacologia , Regulação da Expressão Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Fosforilação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Proteína 28 com Motivo Tripartido , Quinases da Família src/metabolismoRESUMO
ATR-dependent DNA damage checkpoint is crucial to maintain genomic stability. Recently, we showed that Src family kinases suppress ATR-dependent checkpoint signaling in termination of DNA damage checkpoint. However, the precise molecular mechanism is unclear. Therefore, we examined the role of oncogenic v-Src on ATR-Chk1 signaling. We show that v-Src suppresses thymidine-induced Chk1 phosphorylation and induces replication fork collapse. v-Src inhibits interaction between Rad17 and Rad9 in chromatin fraction. By contrast, v-Src does not inhibit RPA32 phosphorylation, ATR autophosphorylation, or TopBP1-Rad9 interaction. These data suggest that v-Src attenuates ATR-Chk1 signaling through the inhibition of Rad17-Rad9 interaction.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Proteína Oncogênica pp60(v-src)/metabolismo , Quinase 1 do Ponto de Checagem , Células HeLa , Humanos , Ligação Proteica , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The DNA damage checkpoint arrests cell cycle progression to allow time for repair. Once DNA repair is completed, checkpoint signaling is terminated. Currently little is known about the mechanism by which checkpoint signaling is terminated, and the disappearance of DNA lesions is considered to induce the end of checkpoint signaling; however, here we show that the termination of checkpoint signaling is an active process promoted by Src family tyrosine kinases. Inhibition of Src activity delays recovery from the G2 phase DNA damage checkpoint following DNA repair. Src activity is required for the termination of checkpoint signaling, and inhibition of Src activity induces persistent activation of ataxia telangiectasia mutated (ATM)- and Rad3-related (ATR) and Chk1 kinases. Src-dependent nuclear protein tyrosine phosphorylation and v-Src expression suppress the ATR-mediated Chk1 and Rad17 phosphorylation induced by DNA double strand breaks or DNA replication stress. Thus, Src family kinases promote checkpoint recovery through termination of ATR- and Chk1-dependent G2 DNA damage checkpoint. These results suggest a model according to which Src family kinases send a termination signal between the completion of DNA repair and the initiation of checkpoint termination.
