RESUMO
Proliferating cell nuclear antigen (PCNA) lies at the center of the faithful duplication of eukaryotic genomes. With its distinctive doughnut-shaped molecular structure, PCNA was originally studied for its role in stimulating DNA polymerases. However, we now know that PCNA does much more than promote processive DNA synthesis. Because of the complexity of the events involved, cellular DNA replication poses major threats to genomic integrity. Whatever predicament lies ahead for the replication fork, PCNA is there to orchestrate the events necessary to handle it. Through its many protein interactions and various post-translational modifications, PCNA has far-reaching impacts on a myriad of cellular functions.
Assuntos
Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S-phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT-type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA We show that HUWE1-knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono-ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.
Assuntos
Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Estresse Fisiológico , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Dano ao DNA , Reparo do DNA , Técnicas de Inativação de Genes , Instabilidade Genômica , Histonas/metabolismo , Humanos , Fenótipo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Supressoras de Tumor , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Genomic instability, a major hallmark of cancer cells, is caused by incorrect or ineffective DNA repair. Many DNA repair mechanisms cooperate in cells to fight DNA damage, and are generally regulated by post-translational modification of key factors. Poly-ADP-ribosylation, catalyzed by PARP1, is a post-translational modification playing a prominent role in DNA repair, but much less is known about mono-ADP-ribosylation. Here we report that mono-ADP-ribosylation plays an important role in homologous recombination DNA repair, a mechanism essential for replication fork stability and double strand break repair. We show that the mono-ADP-ribosyltransferase PARP14 interacts with the DNA replication machinery component PCNA and promotes replication of DNA lesions and common fragile sites. PARP14 depletion results in reduced homologous recombination, persistent RAD51 foci, hypersensitivity to DNA damaging agents and accumulation of DNA strand breaks. Our work uncovered PARP14 as a novel factor required for mitigating replication stress and promoting genomic stability.
Assuntos
Replicação do DNA , Recombinação Homóloga , Poli(ADP-Ribose) Polimerases/metabolismo , Linhagem Celular , Sítios Frágeis do Cromossomo , Quebras de DNA , Dano ao DNA , Reparo do DNA , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Interferente Pequeno/genética , Fase SRESUMO
All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation.
Assuntos
Dano ao DNA , Instabilidade Genômica , Poli(ADP-Ribose) Polimerases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Sumoilação , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Células HeLa , Humanos , Poli(ADP-Ribose) Polimerases/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
Mitotic cells attenuate the DNA damage response (DDR) by phosphorylating 53BP1, a critical DDR mediator, to prevent its localization to damaged chromatin. Timely dephosphorylation of 53BP1 is critical for genome integrity, as premature recruitment of 53BP1 to DNA lesions impairs mitotic fidelity. Protein phosphatase 4 (PP4) dephosphorylates 53BP1 in late mitosis to allow its recruitment to DNA lesions in G1. How cells appropriately dephosphorylate 53BP1, thereby restoring DDR, is unclear. Here, we elucidate the underlying mechanism of kinetic control of 53BP1 dephosphorylation in mitosis. We demonstrate that CDK5, a kinase primarily functional in post-mitotic neurons, is active in late mitotic phases in non-neuronal cells and directly phosphorylates PP4R3ß, the PP4 regulatory subunit that recognizes 53BP1. Specific inhibition of CDK5 in mitosis abrogates PP4R3ß phosphorylation and abolishes its recognition and dephosphorylation of 53BP1, ultimately preventing the localization of 53BP1 to damaged chromatin. Our results establish CDK5 as a regulator of 53BP1 recruitment.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Reparo do DNA/genética , Fase G1/genética , Fosfoproteínas Fosfatases/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/genética , Células HEK293 , Células HeLa , Humanos , Mitose/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
Two structurally related protein kinase families, the Rho kinases (ROCK) and the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCK) are required for migration and invasion of cancer cells. We hypothesized that simultaneous targeting of these two kinase families might represent a novel therapeutic strategy to block the migration and invasion of metastatic cancers. To this end, we developed DJ4 as a novel small molecule inhibitor of these kinases. DJ4 potently inhibited activities of ROCK and MRCK in an ATP competitive manner. In cellular functional assays, DJ4 treatment significantly blocked stress fiber formation and inhibited migration and invasion of multiple cancer cell lines in a concentration dependent manner. Our results strongly indicate that DJ4 may be further developed as a novel anti-metastatic chemotherapeutic agent for multiple cancers.