RESUMEN
Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos/metabolismo , Elementos Aisladores , Factores de Transcripción NFI/metabolismo , Animales , Sitios de Unión , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Factor de Unión a CCCTC , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Virus de la Leucemia Murina de Friend/genética , Virus de la Leucemia Murina de Friend/metabolismo , Silenciador del Gen , Vectores Genéticos/genética , Células HeLa , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFI/genética , Plásmidos/genética , Plásmidos/metabolismo , Proto-Oncogenes/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección , Transgenes , Integración ViralRESUMEN
Escherichia coli Nth protein (endonuclease III) is a DNA glycosylase with a broad substrate specificity for pyrimidine derivatives. We discovered novel substrates of E. coli Nth protein using gas chromatography/isotope-dilution mass spectrometry and DNA samples, which were damaged by gamma-irradiation or by H(2)O(2)/Fe(III)-EDTA/ascorbic acid. These were 4, 6-diamino-5-formamidopyrimidine, 5,6-dihydroxyuracil, and 5, 6-dihydroxycytosine. The first compound was recognized for the first time as a purine-derived substrate of the enzyme. We also investigated kinetics of excision of a multitude of modified bases from three damaged DNA substrates. Excision of modified bases was determined as a function of enzyme concentration, incubation time, and substrate concentration. Excision followed Michaelis-Menten kinetics. Kinetic parameters were determined for the following modified bases: 4,6-diamino-5-formamidopyrimidine, cis- and trans-thymine glycols, 5-hydroxycytosine, cis- and trans-uracil glycols, 5-hydroxyuracil, 5-hydroxy-5-methylhydantoin, alloxan, 5, 6-dihydroxycytosine, 5,6-dihydroxyuracil, 5-hydroxy-6-hydrothymine, and 5-hydroxy-6-hydrouracil. The results show that three newly discovered substrates were excised by the enzyme with a preference similar to excision of its known major substrates such as thymine glycol and 5-hydroxycytosine. Excision kinetics significantly depended on the nature of the damaged DNA substrates in agreement with previous results on other DNA glycosylases. Specificity constants (k(cat)/K(M)) of E. coli Nth protein were compared to those of its previously investigated functional homologues such as human and Schizosaccharomyces pombe Nth proteins and Saccharomyces cerevisiae Ntg1 and Ntg2 proteins. This comparison shows that significant differences exist with respect to substrate specificity and kinetic parameters despite extensive structural conservation among the Nth homologues.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina) , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Proteínas Bacterianas/metabolismo , Barbitúricos/metabolismo , Radicales Libres/farmacología , Cromatografía de Gases y Espectrometría de Masas , Cinética , Estructura Molecular , N-Glicosil Hidrolasas , Pirimidinas/metabolismo , Saccharomyces cerevisiae/enzimología , Especificidad por Sustrato , Uracilo/análogos & derivados , Uracilo/metabolismoRESUMEN
A DNA repair enzyme has recently been isolated from the ionizing radiation-resistant bacterium Deinococcus radiodurans [Bauche, C., and Laval, J. (1999) J. Bacteriol. 181, 262-269]. This enzyme is a homologue of the Fpg protein of Escherichia coli. We investigated the substrate specificity of this enzyme for products of oxidative DNA base damage using gas chromatography/isotope-dilution mass spectrometry and DNA substrates, which were either gamma-irradiated or treated with H(2)O(2)/Fe(III)-EDTA/ascorbic acid. Excision of purine lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 8-hydroxyguanine (8-OH-Gua) was observed among 17 lesions detected in damaged DNA substrates. The extent of excision was determined as a function of enzyme concentration, time, and substrate concentration. FapyGua and FapyAde were excised with similar specificities from three DNA substrates, whereas 8-OH-Gua was the least preferred lesion. The results show that D. radiodurans Fpg protein and its homologue E. coli Fpg protein excise the same modified DNA bases, but the excision rates of these enzymes are significantly different. Formamidopyrimidines are preferred substrates of D. radiodurans Fpg protein over 8-OH-Gua, whereas E. coli Fpg protein excises these three lesions with similar efficiencies from various DNA substrates. Substrate specificities of these enzymes were also compared with that of Saccharomyces cerevisiae Ogg1 protein, which excises FapyGua and 8-OH-Gua, but not FapyAde.
Asunto(s)
Reparación del ADN , Proteínas de Escherichia coli , Cocos Grampositivos/enzimología , N-Glicosil Hidrolasas/metabolismo , ADN/metabolismo , ADN/efectos de la radiación , Daño del ADN , ADN-Formamidopirimidina Glicosilasa , Rayos gamma , Guanina/análogos & derivados , Guanina/metabolismo , Cinética , Óxido Nitroso , Pirimidinas/metabolismo , Especificidad por SustratoRESUMEN
Deinococcus radiodurans is able to resist and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. It is believed that it possesses highly efficient DNA repair mechanisms. To characterize the repair pathway of oxidized purines in this bacteria, we have purified, from crude extracts, proteins that recognize these oxidized bases. We report here that D. radiodurans possesses two proteins excising the oxidized purines (formamidopyrimidine and 8-oxoguanine) by a DNA glycosylase-a purinic/apyrimidine lyase mechanism. Moreover, one of those proteins is endowed with a thymine glycol DNA glycosylase activity. One of these proteins could be the homolog of the Escherichia coli Fpg enzyme, which confirms the existence of a base excision repair system in this bacteria.