RESUMEN
Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.
Asunto(s)
Antígenos Nucleares/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Proteínas de Unión al ADN/genética , Fase S/genética , Animales , Daño del ADN/genética , Reparación del ADN/genética , Fibroblastos/metabolismo , Células HCT116 , Recombinación Homóloga , Humanos , Autoantígeno Ku , Ratones , Transducción de SeñalRESUMEN
Translesion synthesis (TLS) enables DNA replication through damaged bases, increases cellular DNA damage tolerance, and maintains genomic stability. The sliding clamp PCNA and the adaptor polymerase Rev1 coordinate polymerase switching during TLS. The polymerases Pol η, ι, and κ insert nucleotides opposite damaged bases. Pol ζ, consisting of the catalytic subunit Rev3 and the regulatory subunit Rev7, then extends DNA synthesis past the lesion. Here, we show that Rev7 binds to the transcription factor TFII-I in human cells. TFII-I is required for TLS and DNA damage tolerance. The TLS function of TFII-I appears to be independent of its role in transcription, but requires homodimerization and binding to PCNA. We propose that TFII-I bridges PCNA and Pol ζ to promote TLS. Our findings extend the general principle of component sharing among divergent nuclear processes and implicate TLS deficiency as a possible contributing factor in Williams-Beuren syndrome.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Proteínas Mad2/metabolismo , Factores de Transcripción TFII/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/biosíntesis , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Proteínas Mad2/biosíntesis , Proteínas Mad2/genética , Proteínas Nucleares/biosíntesis , Nucleotidiltransferasas/biosíntesis , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Antígeno Nuclear de Célula en Proliferación/metabolismo , Factores de Transcripción TFII/biosíntesis , Factores de Transcripción TFII/metabolismoRESUMEN
Akt phosphorylation has previously been described to be involved in mediating DNA damage repair through the nonhomologous end-joining (NHEJ) repair pathway. Yet the mechanism how Akt stimulates DNA-protein kinase catalytic subunit (DNA-PKcs)-dependent DNA double-strand break (DNA-DSB) repair has not been described so far. In the present study, we investigated the mechanism by which Akt can interact with DNA-PKcs and promote its function during the NHEJ repair process. The results obtained indicate a prominent role of Akt, especially Akt1 in the regulation of NHEJ mechanism for DNA-DSB repair. As shown by pull-down assay of DNA-PKcs, Akt1 through its C-terminal domain interacts with DNA-PKcs. After exposure of cells to ionizing radiation (IR), Akt1 and DNA-PKcs form a functional complex in a first initiating step of DNA-DSB repair. Thereafter, Akt plays a pivotal role in the recruitment of AKT1/DNA-PKcs complex to DNA duplex ends marked by Ku dimers. Moreover, in the formed complex, Akt1 promotes DNA-PKcs kinase activity, which is the necessary step for progression of DNA-DSB repair. Akt1-dependent DNA-PKcs kinase activity stimulates autophosphorylation of DNA-PKcs at S2056 that is needed for efficient DNA-DSB repair and the release of DNA-PKcs from the damage site. Thus, targeting of Akt results in radiosensitization of DNA-PKcs and Ku80 expressing, but not of cells deficient for, either of these proteins. The data showed indicate for the first time that Akt through an immediate complex formation with DNA-PKcs can stimulate the accumulation of DNA-PKcs at DNA-DSBs and promote DNA-PKcs activity for efficient NHEJ DNA-DSB repair.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Proteína Quinasa Activada por ADN , Proteínas Proto-Oncogénicas c-akt , Radiación Ionizante , Cromonas/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Células HCT116 , Humanos , Morfolinas/farmacología , Complejos Multiproteicos/efectos de los fármacos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
DNA double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The DNA cell cycle stage and resection of the DSB ends are two key mechanisms which are believed to push DSB repair to the HR pathway. Here, we show that the NHEJ factor Ku80 associates with DSBs in S phase, when HR is thought to be the preferred repair pathway, and its dynamics/kinetics at DSBs is similar to those observed for Ku80 in non-S phase in mammalian cells. A Ku homolog from Mycobacterium tuberculosis binds to and is retained at DSBs in S phase and was used as a tool to determine if blocking DNA ends affects end resection and HR in mammalian cells. A decrease in DNA end resection, as marked by IR-induced RPA, BrdU, and Rad51 focus formation, and HR are observed when Ku deficient rodent cells are complemented with Mt-Ku. Together, this data suggests that Ku70/80 binds to DSBs in all cell cycle stages and is likely actively displaced from DSB ends to free the DNA ends for DNA end resection and thus HR to occur.
Asunto(s)
Antígenos Nucleares/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Recombinación Homóloga/genética , Animales , Células CHO , Cricetinae , Cricetulus , Reparación del ADN , Humanos , Autoantígeno Ku , Ratones , Mycobacterium tuberculosis/metabolismo , Unión Proteica , Transporte de Proteínas , Recombinasa Rad51/metabolismo , Fase SRESUMEN
BACKGROUND AND PURPOSE: In the present study effect of erbB2 as well as DNA-PKcs on ionizing radiation (IR)- and erbB1 ligand-induced phosphorylation of Akt at S473 in cytoplasmic and nuclear fractions was investigated. MATERIALS AND METHODS: DNA-PKcs proficient and deficient syngeneic colon carcinoma sublines of HCT116 and the glioblastoma cell lines MO59K and MO59J as well as the lung carcinoma cell line A549 were used. Akt-S473 phosphorylation was investigated in cells pre-treated with pharmacological inhibitors or transfected with siRNA by immunoprecipitation, Western blotting and confocal microscopy after different stimuli, i.e., ligands and IR. RESULTS: IR-induced phosphorylation of Akt in both MO59K and MO59J cell lines but not in HCT116 cells was DNA-PKcs dependent. In A549 cells, IR-induced phosphorylation of nuclear Akt-S473 was dependent on erbB1, erbB2, and DNA-PKcs. EGF induced phosphorylation of nuclear Akt-S473 in a DNA-PKcs and erbB2 independent manner. CONCLUSION: Data indicate that the function of DNA-PKcs on IR-induced Akt-S473 phosphorylation is cell line specific. IR-induced, but not EGF-induced phosphorylation of cytoplasmic and/or nuclear Akt-S473 is erbB2 dependent.
Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación/efectos de la radiación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Células Tumorales Cultivadas/efectos de la radiación , Western Blotting , Núcleo Celular/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/radioterapia , Citoplasma/metabolismo , Proteína Quinasa Activada por ADN/efectos de la radiación , Fluorescencia , Glioblastoma/genética , Glioblastoma/radioterapia , Humanos , Ligandos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Proteínas Nucleares/efectos de la radiación , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Radiación Ionizante , Receptor ErbB-2/efectos de la radiación , Sensibilidad y Especificidad , Transducción de Señal , Células Tumorales Cultivadas/metabolismoRESUMEN
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an important role in DNA double-strand break (DSB) repair as the underlying mechanism of the non-homologous end joining pathway. When DSBs occur, DNA-PKcs is rapidly phosphorylated at both the Thr-2609 and Ser-2056 residues, and such phosphorylations are critical for DSB repair. In this study we report that, in addition to responding to DSBs, DNA-PKcs is activated and phosphorylated in normal cell cycle progression through mitosis. Mitotic induction of DNA-PKcs phosphorylation is closely associated with the spindle apparatus at centrosomes and kinetochores. Furthermore, depletion of DNA-PKcs protein levels or inhibition of DNA-PKcs kinase activity results in the delay of mitotic transition because of chromosome misalignment. These results demonstrate for the first time that DNA-PKcs, in addition to its role in DSB repair, is a critical regulator of mitosis and could modulate microtubule dynamics in chromosome segregation.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Proteína Quinasa Activada por ADN/metabolismo , Mitosis/efectos de los fármacos , Western Blotting , Células Cultivadas , Segregación Cromosómica/genética , Segregación Cromosómica/fisiología , Proteína Quinasa Activada por ADN/genética , Citometría de Flujo , Células HCT116 , Células HeLa , Humanos , Immunoblotting , Microtúbulos/metabolismo , Mitosis/genética , Nocodazol/farmacología , Fosforilación/efectos de los fármacosRESUMEN
The DNA-dependent protein kinase (DNA-PK) complex is a serine/threonine protein kinase comprised of a 469-kDa catalytic subunit (DNA-PK(cs)) and the DNA binding regulatory heterodimeric (Ku70/Ku86) complex Ku. DNA-PK functions in the nonhomologous end-joining pathway for the repair of DNA double-stranded breaks (DSBs) introduced by either exogenous DNA damage or endogenous processes, such as lymphoid V(D)J recombination. Not surprisingly, mutations in Ku70, Ku86, or DNA-PK(cs) result in animals that are sensitive to agents that cause DSBs and that are also immune deficient. While these phenotypes have been validated in several model systems, an extension of them to humans has been missing due to the lack of patients with mutations in any one of the three DNA-PK subunits. The worldwide lack of patients suggests that during mammalian evolution this complex has become uniquely essential in primates. This hypothesis was substantiated by the demonstration that functional inactivation of either Ku70 or Ku86 in human somatic cell lines is lethal. Here we report on the functional inactivation of DNA-PK(cs) in human somatic cells. Surprisingly, DNA-PK(cs) does not appear to be essential, although the cell line lacking this gene has profound proliferation and genomic stability deficits not observed for other mammalian systems.
Asunto(s)
Dominio Catalítico , Proteína Quinasa Activada por ADN/metabolismo , Inestabilidad Genómica , Telómero/metabolismo , Biomarcadores/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Daño del ADN , Proteína Quinasa Activada por ADN/deficiencia , Etopósido/farmacología , Marcación de Gen , Inestabilidad Genómica/efectos de los fármacos , Células HCT116 , Heterocigoto , Homocigoto , HumanosRESUMEN
Gene targeting has two important applications. One is the inactivation of genes ("knockouts"), and the second is the correction of a mutated allele back to wild-type ("gene therapy"). Central to these processes is the efficient introduction of the targeting DNA into the cells of interest. In humans, this targeting is often accomplished through the use of recombinant adeno-associated virus (rAAV). rAAV is presumed to use a pathway of DNA double-strand break (DSB) repair termed homologous recombination (HR) to mediate correct targeting; however, the specifics of this mechanism remain unknown. In this work, we attempted to generate Ku70-null human somatic cells by using a rAAV-based gene knockout strategy. Ku70 is the heterodimeric partner of Ku86, and together they constitute an end-binding activity that is required for a pathway [nonhomologous end joining (NHEJ)] of DSB repair that is believed to compete with HR. Our data demonstrated that Ku70 is an essential gene in human somatic cells. More importantly, however, in Ku70(+/-) cells, the frequency of gene targeting was 5- to 10-fold higher than in wild-type cells. RNA interference and short-hairpinned RNA strategies to deplete Ku70 phenocopied these results in wild-type cells and greatly accentuated them in Ku70(+/-) cell lines. Thus, Ku70 protein levels significantly influenced the frequency of rAAV-mediated gene targeting in human somatic cells. Our data suggest that gene-targeting frequencies can be significantly improved in human cells by impairing the NHEJ pathway, and we propose that Ku70 depletion can be used to facilitate both knockout and gene therapy approaches.
Asunto(s)
Antígenos Nucleares/genética , Proteínas de Unión al ADN/genética , Dependovirus/genética , Marcación de Gen , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/metabolismo , Células HCT116 , Humanos , Autoantígeno Ku , Recombinación Genética , TransfecciónRESUMEN
NHEJ (non-homologous end joining) is the predominant mechanism for repairing DNA double-stranded breaks in human cells. One essential NHEJ factor is the Ku heterodimer, which is composed of Ku70 and Ku86. Here we have generated heterozygous loss-of-function mutations for each of these genes in two different human somatic cell lines, HCT116 and NALM-6, using gene targeting. Previous work had suggested that phenotypic differences might exist between the genes and/or between the cell lines. By providing a side-by-each comparison of the four cell lines, we demonstrate that there are indeed subtle differences between loss-of-function mutations for Ku70 versus Ku86, which is accentuated by whether the mutations were derived in the HCT116 or NALM-6 genetic background. Overall, however, the phenotypes of the four lines are quite similar and they provide a compelling argument for the hypothesis that Ku loss-of-function mutations in human somatic cells result in demonstrable haploinsufficiencies. Collectively, these studies demonstrate the importance of proper biallelic expression of these genes for NHEJ and telomere maintenance and they provide insights into why these genes are uniquely essential for primates.