RESUMEN
p53 is thought to function in the maintenance of genomic stability by modulating transcription and interacting with cellular proteins to influence the cell cycle, DNA repair and apoptosis. p53 mutations occur in >50% of human cancers, and cells which lack wild type p53 accumulate karyotypic abnormalities such as amplifications, deletions, inversions and translocations. We propose that p53 hinders these promiscuous recombinational events by interacting with cellular recombination and repair machinery. We recently reported that p53 can directly bind in vivo to human Rad51 (hRad51) protein and in vitro to its bacterial homologue RecA. We used GST-fusion and his-tagged protein systems to further investigate the physical interaction between p53 and hRad51, homologue of the yeast Rad51 protein that is involved in recombination and DNA double strand repair. The hRad51 binds to wild-type p53 and to a lesser extent, point mutants 135Y, 249S and 273H. This binding is not mediated by a DNA or RNA intermediate. Mapping studies using a panel of p53 deletion mutants indicate that hRad51 could bind to two regions of p53; one between amino acids 94 and 160 and a second between 264 and 315. Addition of anti-p53 antibody PAb421 (epitope 372-381 amino acids) inhibited the interaction with hRad51. In contrast, p53 interacts with the region between aa 125 and 220 of hRad51, which is highly conserved among Rad51 related proteins from bacteria to human. In Escherichia coli ecA protein, this region is required for homo-oligomerization, suggesting that p53 might disrupt the interaction between RecA and Rad51 subunits, thus inhibiting biochemical functions of Rad51 like proteins. These data are consistent with the hypothesis that p53 interaction with hRAD51 may influence DNA recombination and repair and that additional modifications of p53 by mutation and protein binding may affect this interaction.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Sitios de Unión , Secuencia Conservada , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Modelos Moleculares , Mapeo Peptídico , Mutación Puntual , Unión Proteica , Conformación Proteica , Recombinasa Rad51 , Rec A Recombinasas/química , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Eliminación de Secuencia , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Monoclonal antibodies were produced against recombinant human RAD51 recombination protein. The antibodies of IgG subclasses were isolated from serum-free cell culture medium and purified by affinity chromatography on protein A-Sepharose. The antibodies can be used to detect specifically RAD51 protein on immunoblots of total cell lysates. Native RAD51 protein is specifically precipitated from lysates of human cells. In addition, these antibodies readily detect RAD51 in the cell nucleus by immunofluorescence staining. Epitope mapping on overlapping peptides spanning the complete primary amino acid sequence of human RAD51 revealed that three monoclonals recognize an epitope on RAD51 very close to the N-terminus of the protein (amino acids 16 to 20); the other three monoclonals interact with amino acids 85 to 95 of human RAD51.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Proteínas de Unión al ADN/inmunología , Secuencia de Aminoácidos , Animales , Células COS , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Conformación Proteica , Recombinasa Rad51 , Proteínas Recombinantes/inmunologíaRESUMEN
The tumour suppressor p53 prevents tumour formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage-induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinatorial events. Here we report that p53 physically interacts with key factors of homologous recombination: the human RAD51 protein and its prokaryotic homologue RecA. In vitro, wild-type p53 inhibits defined biochemical activities of RecA protein, such as three-way DNA strand exchange and single strand DNA-dependent ATPase activity. In vivo, temperature-sensitive p53 forms complexes with RAD51 only in wild-type but not in mutant conformation. These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and control the extent and timing of the production of genetic variation via homologous recombination. Gene amplification an other types of chromosome rearrangements involved in tumour progression might occur not only as result of inappropriate cell proliferation but as a direct consequence of a defect in p53-mediated control of homologous recombination processes due to mutations in the p53 gene.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Rec A Recombinasas/metabolismo , Recombinación Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Reparación del ADN/genética , ADN Complementario/genética , ADN Complementario/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Hidrólisis , Técnicas In Vitro , Mutación Puntual , Recombinasa Rad51 , Ratas , Rec A Recombinasas/genéticaRESUMEN
Injection into mice of chimeric proteins consisting of a portion of either the simian virus 40 large tumor antigen or nonstructural protein 1 of influenza A virus or of the murine tumor suppressor p53 on one hand and T-cell epitopes of lymphocytic choriomeningitis virus on the other resulted in antiviral protective immunity, which was independent of the epitopes' position in the protein and the same whether the latter was immunologically nonself or self. Mice of different haplotypes were protected when the corresponding class I molecule-restricted epitopes had been inserted close to each other in one carrier protein.