RESUMO
The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling.
Assuntos
Dano ao DNA , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Quinases/metabolismo , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Imunoprecipitação da Cromatina , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Ligação Proteica/efeitos da radiação , Proteínas Quinases/genética , Interferência de RNA , Raios UltravioletaRESUMO
Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models.
Assuntos
Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Marcadores Genéticos , Células HeLa , Humanos , Mitose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Although p21 upregulation is required to block cell-cycle progression following many types of genotoxic insult, UV irradiation triggers p21 proteolysis. The significance of the increased p21 turnover is unclear and might be associated with DNA repair. While the role of p21 in nucleotide excision repair (NER) remains controversial, recent reports have explored its effect on translesion DNA synthesis (TLS), a process that avoids replication blockage during S phase. Herein, we analyze the effect of p21 on different PCNA-driven processes including DNA replication, NER and TLS. Whereas only the CDK-binding domain of p21 is required for cell-cycle arrest in unstressed cells, neither the CDK-binding nor the PCNA-binding domain of p21 is able to block early and late steps of NER. Intriguingly, through its PCNA-binding domain, p21 inhibits the interaction of the TLS polymerase, pol eta (pol eta), with PCNA and impairs the assembly of pol eta foci after UV. Moreover, this obstruction correlates with accumulation of phosphorylated H2AX and increased apoptosis. By showing that p21 is a negative regulator of PCNA-pol eta interaction, our data unveil a link between efficient TLS and UV-induced degradation of p21.