RESUMO
Regulation of homologous recombination (HR) represents the best-characterized DNA repair function of p53. The role of p53 phosphorylation in DNA repair is largely unknown. Here, we show that wild-type p53 repressed repair of DNA double-strand breaks (DSBs) by HR in a manner partially requiring the ATM/ATR phosphorylation site, serine 15. Cdk-mediated phosphorylation of serine 315 was dispensable for this anti-recombinogenic effect. However, without targeted cleavage of the HR substrate, serine 315 phosphorylation was necessary for the activation of topoisomerase I-dependent HR by p53. Moreover, overexpression of cyclin A1, which mimics the situation in tumors, inappropriately stimulated DSB-induced HR in the presence of oncogenic p53 mutants (not Wtp53). This effect required cyclin A1/cdk-mediated phosphorylation for stable complex formation with topoisomerase I. We conclude that p53 mutants have lost the balance between activation and repression of HR, which results in a net increase of potentially mutagenic DNA rearrangements. Our data provide new insight into the mechanism underlying gain-of-function of mutant p53 in genomic instability.
Assuntos
Reparo do DNA , Genes p53 , Mutação , Recombinação Genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/antagonistas & inibidores , Ciclina A/metabolismo , Ciclina A1 , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Humanos , Células K562 , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae , Serina/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and gamma-irradiation which was mediated by p53. cyclin A1-/- cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1-/- cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina A/metabolismo , Reparo do DNA , DNA/metabolismo , DNA/efeitos da radiação , Regulação da Expressão Gênica , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Quinases relacionadas a CDC2 e CDC28/genética , Células Cultivadas , Ciclina A/genética , Ciclina A1 , Quinase 2 Dependente de Ciclina , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Raios gama , Células-Tronco Hematopoéticas , Humanos , Autoantígeno Ku , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Regiões Promotoras Genéticas , Distribuição Aleatória , Recombinação Genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Raios UltravioletaRESUMO
Phosphorylation of p53 on serine 15 by ATM or ATR is a frequent modification and initiates a cascade of post-translational modifications. To identify possible mechanisms that modulate p53 functions in recombination surveillance, we compared the nuclear localization of p53 phosphorylated on serine 15 (p53pSer15) and the key enzymes of homologous recombination (HR) after replication fork stalling. We demonstrate an almost mutually exclusive subcompartmentalization with Rad52, while p53pSer15 was colocalizing with 40-60% of the Rad51 and Mre11 foci. Therefore, possible sites of p53pSer15-dependent regulation seem to be sites of Rad51- rather than Rad52-dependent HR processes. Remarkably, the association of p53pSer15 with repair complexes containing Rad51 or Mre11 was transient, because less than 20% of the Rad51 and Mre11 foci overlapped with p53pSer15 after 6 h. When we examined colocalization and co-immunoprecipitation of p53pSer15 and the RecQ helicase BLM with recombination surveillance and proapoptotic functions, we observed colocalization within a fraction of approximately 70% of the BLM foci and stable physical interactions until 6 h after replication arrest. Our data suggest that p53pSer15 plays a dual role in the functional interactions with early complexes of Rad51-dependent recombination and with BLM-associated surveillance and signalling complexes within distinct nuclear subcompartments.
Assuntos
Fosfosserina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Afidicolina/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Cinética , Fosforilação , Ligação Proteica , RecQ Helicases , Recombinação Genética , Proteína Supressora de Tumor p53/genéticaRESUMO
In addition to vitamin C (and other vitamins/antioxidants), clinical ecologists (functional medicine) recommend selenium supplementation as a fundamental therapeutic remedy for the treatment of environment associated health disorders. This recommendation is based on the postulation that the trace element selenium inhibits oxidative stress generated during endogenous detoxification of xenobiotics (phase 1) by increasing selenium-dependent glutathione peroxidase activity, and that it counteracts heavy metal toxicity by forming inert metal complexes. The objective of this review was to investigate whether there are any valid studies providing reliable evidence of the therapeutic benefits of selenium supplementation in potentially environment associated health disorders. A systematic review was conducted based on the rigorous and well-defined methods developed by the Cochrane Collaboration. To achieve the demanding standards for systematic review set by the Cochrane Collaboration, study selection, quality assessment and data abstraction were performed independently and in duplicate using a standardized protocol. Overall, 1290 studies were identified as being eligible for inclusion. Twelve of these met the inclusion criteria and their quality was evaluated individually. None of the studies included in the analysis provided evidence of the therapeutic benefits of selenium supplementation in environment associated health disorders.
Assuntos
Antioxidantes/farmacologia , Suplementos Nutricionais , Doença Ambiental/prevenção & controle , Selênio/farmacologia , Idoso , Criança , Ensaios Clínicos como Assunto , Geriatria , Humanos , Doenças Musculoesqueléticas/etiologia , Doenças Musculoesqueléticas/prevenção & controle , Medicina Preventiva , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Chromosomal translocations and retroviral integration events at breakpoint cluster regions (bcrs) have been associated with leukaemias. To directly compare the effect of different cis-regulatory sequences on recombination, we adapted our SV40 based model system to the analysis of correspondingly selected bcrs from the TAL1, LMO2, retinoic acid receptor alpha (RARalpha) and MLL genes. We show that a 399 bp fragment from the MLL bcr is sufficient to cause a 3-4-fold stimulation of spontaneously occurring DNA exchange and to respond to etoposide by up to 10-fold further elevated frequencies, i.e. to mimic the fragility of the 8.3 kb bcr during chemotherapy. To analyse the regulatory role of p53 in recombination involving leukaemia-related sequences, we stably expressed wtp53 and a transactivation negative mutant. Consistent with the proposed role of p53 as a suppressor of error-prone recombination, both p53 proteins down-regulated recombination with most of the sequences tested, even with the MLL bcr after etoposide treatment. Surprisingly, however, p53 stimulated recombination, in constructs carrying the RARalpha bcr fragment. This is the first study, which provides evidence for a stimulatory role of p53 in homologous recombination. Our data further indicate that inhibition of topoisomerase I can mimic the effects of p53 on stimulating recombination on the RARalpha bcr. Thus, these data also firstly describe a biological role of the biochemical interactions between p53 and topoisomerase I that may have implications for a gain-of-function phenotype of certain p53 mutants in genetic destabilization.