RESUMEN
Double-stranded RNA (dsRNA) is a biologically active molecule that plays important roles in normal cell growth and function. Accordingly, the cell uses multiple mechanisms to control its level. The tumor suppressor protein p53 possesses intrinsic 3'â5' exonuclease activity. The aim of the present study was to elucidate the degradation of dsRNA by the exonuclease activity of p53. The results show that recombinant, purified wtp53 and endogenous protein in cytoplasmic fractions of cells remove nucleotides from 3'-ends of dsRNA. Several lines of evidence support a connection between p53 and dsRNase activity in cytoplasm: (1) this activity parallels the status of endogenous cytoplasmic p53; (2) the endogenous exonuclease displays a similar dsRNA excision profile characteristic for purified wtp53; (3) cytoplasmic fractions of HCT116(p53+/+) cells exert higher levels of exonuclease activity compared to those of HCT116(p53-/-) cells; (4) transfection of the wtp53, but not exonuclease-deficient mutant p53-R175H, into HCT116 (p53-/-) cells induced high levels of dsRNase activity in cytoplasm; (5) the accumulation of p53 in cytoplasm following the γ-irradiation stress stimuli correlates with the increase in the excision of dsRNA and (6) the dsRNA forms a complex with a protein that can be disrupted by an anti-p53 antibody. Our data suggest that the degradation of dsRNA by p53 protein may direct either the complete degradation of and decrease in the level of dsRNA or incomplete degradation and the generation of short dsRNA products. The possible roles of p53 dsRNase activity in cytoplasm in the inhibition of translation and induction of cell apoptosis, is discussed.
Asunto(s)
Citoplasma/enzimología , Exonucleasas/metabolismo , ARN Bicatenario/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Anticuerpos Monoclonales , Apoptosis , Western Blotting , Línea Celular Tumoral , Daño del ADN , Ensayo de Cambio de Movilidad Electroforética , Humanos , ARN Bicatenario/genética , Proteínas Recombinantes/metabolismo , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
OBJECTIVE: Nucleoside analogs, used against HIV, can be incorporated into a mitochondrial DNA by DNA polymerase gamma. Both the decrease in mitochondrial DNA and increased mutations of mitochondrial DNA may lead to mitochondrial diseases. The tumor suppressor protein p53 exhibits 3' --> 5' exonuclease activity and can provide a proofreading function for DNA polymerases. In the present study, we investigated the ability of p53 to excise incorporated nucleoside analogs from DNA in mitochondria. DESIGN: The functional interaction of p53 and DNA polymerase gamma during the incorporation of nucleoside analog was examined in mitochondrial fractions of p53-null H1299 cells, as the source of DNA polymerase gamma. METHODS: Primer extension reactions were carried out to elucidate the incorporation and removal of nucleoside analogs. RESULTS: The results demonstrate that the excision of incorporated nucleoside analogs in mitochondrial fractions of H1299 cells increased in the presence of purified recombinant p53, or cytoplasmic extracts of large cell carcinoma 2 cells expressing endogenous wild-type p53 (but not specifically predepleted extracts) or cytoplasmic extracts of H1299 cells overexpressing wild-type p53, but not exonuclease-deficient mutant p53-R175H. The amount of nucleoside analogs incorporated into the elongated DNA with mitochondrial fractions of human colon carcinoma 116 (HCT116)(p53+/+) cells was lower than that of HCT116(p53-/-) cells. Furthermore, mitochondrion-localized elevation of p53 in HCT116(p53+/+) cells, following the irradiation-stress stimuli, correlates with the reduction in incorporation of nucleoside analogs and wrong nucleotides. CONCLUSION: p53 in mitochondria may functionally interact with DNA polymerase gamma, thus providing a proofreading function during mitochondrial DNA replication for excision of nucleoside analogs and polymerization errors.
Asunto(s)
Daño del ADN/genética , ADN Mitocondrial/genética , Infecciones por VIH/genética , VIH-1 , Nucleósidos/genética , Proteína p53 Supresora de Tumor/genética , Western Blotting , Carcinoma de Células Grandes/genética , Línea Celular Tumoral , Células Cultivadas , ADN Polimerasa gamma , Cartilla de ADN , ADN Mitocondrial/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN , Endopeptidasa K , Infecciones por VIH/metabolismo , Humanos , Nucleósidos/metabolismoRESUMEN
p53 in cytoplasm displays an intrinsic 3'-->5' exonuclease activity. To understand the significance of p53 exonuclease activity in cytoplasm, cytoplasmic extracts of various cell lines were examined for exonuclease activity with different single-stranded RNA (ssRNA) substrates. Using an in vitro RNA degradation assay, we observed in cytoplasmic extracts of LCC2 cells, expressing high levels of endogenous wtp53, an efficient 3'-->5' exonuclease activity with RNA substrates, removing the 3'-terminal nucleotides. Interestingly, RNA containing AU-rich sequences (ARE) is the permissive substrate for exonucleolytic degradation. Evidence that exonuclease function with RNA detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: (1) this activity closely parallels with status and levels of endogenous cytoplasmic p53; (2) the endogenous exonuclease exerts identical RNA substrate specificity and excision profile characteristic for purified baculovirus-or bacterially-expressed wtp53s; (3) the exonuclease activity with ARE RNA is competed out by the presence of ss or double-stranded DNA substrate utilized by p53 protein in cytoplasm; (4) immunoprecipitation by specific anti-p53 antibodies markedly reduced the exonuclease activity with both RNA and DNA substrates; and (5) transfection of the wtp53, but not exonuclease-deficient mutant p53-R175H, into p53-null H1299 or HCT116 cells induced high levels of exonuclease activity with ARE RNA substrate in cytoplasm with characteristic excision profile. The efficient ARE RNA degradation correlates with the efficient binding of p53 to ARE RNA in cytoplasm. The possible role of p53 exonuclease activity in ARE-mRNA destabilization in cytoplasm, which may be important for expression of proteins that control cell growth and/or apoptosis is discussed.