RESUMO
The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Dano ao DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes de Troca , Genes cdc , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Treonina/genéticaRESUMO
During cell proliferation, DNA damage inflicted by intrinsic or extrinsic genotoxic stresses impose a threat to DNA replication. The stability of the DNA replication forks that encounter DNA damage is crucial for genomic integrity. Both the ATR-regulated checkpoint pathway and the translesion DNA synthesis mediated by the ubiquitinated PCNA are important for continuous replication of damaged DNA. We have recently shown that Chk1, a key effector kinase of ATR in checkpoint response, is required for efficient PCNA ubiquitination after DNA damage. Surprisingly, the ubiquitination of PCNA is independent of ATR, but regulated by Claspin, a replication protein that mediates the activation of Chk1 by ATR. Like Claspin, Timeless and Rad17, two other Chk1 regulators at stressed replication forks, are also implicated in PCNA ubiquitination. These findings suggest that while ATR signaling and PCNA ubiquitination are two independent processes, they are mediated by a common group of proteins including Chk1 and it regulators at replication forks. Furthermore, these data raise the possibility that Chk1 and its regulators may constitute a functional module at replication forks to enable multiple stress responses.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Quinases/metabolismo , Ubiquitinação/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem , HumanosRESUMO
Chk1 is a kinase crucial for genomic integrity and an effector of ATR (ATM and Rad3-related) in DNA damage response. Here, we show that Chk1 regulates the DNA damage-induced ubiquitination of proliferating cell nuclear antigen (PCNA), which facilitates the continuous replication of damaged DNA. Surprisingly, this Chk1 function requires the DNA replication protein Claspin but not ATR. Claspin, which is stabilized by Chk1, regulates the binding of the ubiquitin ligase Rad18 to chromatin. Timeless, a Claspin-associating protein, is also required for efficient PCNA ubiquitination. Thus, Chk1 and the Claspin-Timeless module of replication forks not only participate in ATR signaling, but also protect stressed forks independently of ATR.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Replicação do DNA/genética , Células HeLa , Humanos , Hidroxiureia/farmacologia , Morfolinas/farmacologia , Plasmídeos/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pironas/farmacologia , RNA Interferente Pequeno/genética , Transfecção , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/efeitos da radiação , Raios UltravioletaAssuntos
Proteína BRCA1/química , Quebras de DNA , Ubiquitina/química , Motivos de Aminoácidos , Proteína BRCA1/metabolismo , Ciclo Celular , Genoma , Histonas/química , Humanos , Modelos Biológicos , Modelos Genéticos , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Especificidade por SubstratoRESUMO
The DNA damage and replication checkpoints are signaling mechanisms that regulate and coordinate cellular responses to genotoxic conditions. The activation of checkpoints not only attenuates cell cycle progression, but also facilitates DNA repair and recovery of faulty replication forks, thereby preventing DNA lesions from being converted to inheritable mutations. It has become increasingly clear that the activation and signaling of the checkpoint are intimately linked to the cellular processes directly involved in chromosomal metabolism, such as DNA replication and DNA repair. Thus, the checkpoint pathway is not just a surveillance system that monitors genomic integrity and regulates cell proliferation, but also an integral part of the processes that work directly on chromosomes to maintain genomic stability. In this article, we discuss the current models of DNA damage and replication checkpoints, and highlight recent advances in the field.
