RESUMEN
Activity of microsomal enzymes was studied using an indirect method for estimation of the rate of antipyrine secretion with saliva in 70 healthy persons at the age from 16 to 86 years. Distinct individual differences were observed in the activity of the enzymes: time of the antipyrine half-secretion varied from 6.4 hrs to 50.1 hrs. The time of antipyrine half-secretion was significantly higher in the older (60-86 years) than in younger persons (16-30 years), suggesting decreased activity of the microsomal enzymes in older people. The microsomal enzymes activity might be increased distinctly under effect of several drugs, especially of hypotensive drugs--reserpine and dibasole. The data obtained suggest that for effective medicinal therapy it is essential to control the initial activity of microsomal enzymes as well as its alterations in the course of medicamentous treatment; the activity of microsomal enzymes in a given person must be taken into consideration for correct individual selection of doses of the drugs.
Asunto(s)
Biotransformación , Microsomas/enzimología , Adolescente , Adulto , Anciano , Antipirina/análisis , Bencimidazoles/farmacología , Femenino , Humanos , Masculino , Métodos , Persona de Mediana Edad , Parasimpatolíticos/farmacología , Reserpina/farmacología , Saliva/análisisRESUMEN
To understand the mechanism involved in the coordination of the sequential repair reactions that lead to long-patch BER, we have investigated interactions between proteins involved in this pathway. We find that human AP endonuclease 1 (APE1) physically interacts with flap endonuclease 1 (FEN1) and with proliferating cell nuclear antigen. An oligonucleotide substrate containing a reduced abasic site, which was pre-incised with APE1, was employed to reconstitute the excision step of long-patch BER with purified human DNA polymerase beta and FEN1. We demonstrate that addition of APE1 to the excision reaction mixture slightly (1.5-2-fold) stimulates the removal of the displaced flap by FEN1. These results suggest the possibility that long-patch BER is coordinated and directed by protein-protein interactions.
Asunto(s)
Liasas de Carbono-Oxígeno/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Liasas de Carbono-Oxígeno/inmunología , Liasas de Carbono-Oxígeno/aislamiento & purificación , Células Cultivadas , ADN Polimerasa beta/metabolismo , Replicación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/inmunología , Endonucleasas de ADN Solapado , Humanos , Pruebas de PrecipitinaRESUMEN
The repair of oxidative base lesions in DNA is a coordinated chain of reactions that includes removal of the damaged base, incision of the phosphodiester backbone at the abasic sugar residue, incorporation of an undamaged nucleotide and sealing of the DNA strand break. Although removal of a damaged base in mammalian cells is initiated primarily by a damage-specific DNA glycosylase, several lyases and DNA polymerases may contribute to the later stages of repair. DNA polymerase beta (Pol beta) was implicated recently as the major polymerase involved in repair of oxidative base lesions; however, the identity of the lyase participating in the repair of oxidative lesions is unclear. We studied the mechanism by which mammalian cell extracts process DNA substrates containing a single 8-oxoguanine or 5,6-dihydrouracil at a defined position. We find that, when repair synthesis proceeds through a Pol beta-dependent single nucleotide replacement mechanism, the 5'-deoxyribosephosphate lyase activity of Pol beta is essential for repair of both lesions.
Asunto(s)
ADN Polimerasa beta/metabolismo , ADN Polimerasa beta/fisiología , Guanina/análogos & derivados , Liasas/metabolismo , Oxígeno/metabolismo , Uracilo/análogos & derivados , Animales , Secuencia de Bases , Liasas de Carbono-Oxígeno/metabolismo , Línea Celular , ADN/metabolismo , Daño del ADN , ADN Polimerasa beta/genética , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Fibroblastos/metabolismo , Guanina/metabolismo , Humanos , Liasas/química , Ratones , Ratones Noqueados , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Uracilo/metabolismoRESUMEN
It was found that the pineal gland of active ground squirrels (Citellus erythrogenys) differed from this gland in rats by a more pronounced differentiation into cortical and medullary zones, by larger pineocytes with polymorphous nuclei and numerous pseudokariosomes. Hybernation was accompanied by reduced concentration of serotonin in the pineal gland, by the disappearance of differences between the cortical and the medullary zones and by a number of morphological signs of functional depression. The appearing changes were similar to those occurring in denervation of the pineal gland.
Asunto(s)
Hibernación , Glándula Pineal/fisiología , Roedores/fisiología , Sciuridae/fisiología , Animales , Núcleo Celular , Glándula Pineal/citología , Glándula Pineal/metabolismo , Ratas , Serotonina/metabolismoRESUMEN
The repair pathways involved in the removal of thymine glycol (TG) from DNA by human cell extracts have been examined. Closed circular DNA constructs containing a single TG at a defined site were used as substrates to determine the patch size generated after in vitro repair by cell extracts. Restriction analysis of the repair incorporation in the vicinity of the lesion indicated that the majority of TG was repaired through the base excision repair (BER) pathways. Repair incorporation 5' to the lesion, characteristic for the nucleotide excision repair pathway, was not found. More than 80% of the TG repair was accomplished by the single-nucleotide repair mechanism, and the remaining TGs were removed by the long patch BER pathway. We also analyzed the role of the xeroderma pigmentosum, complementation group G (XPG) protein in the excision step of BER. Cell extracts deficient in XPG protein had an average 25% reduction in TG incision. These data show that BER is the primary pathway for repair of TG in DNA and that XPG protein may be involved in repair of TG as an accessory factor.
Asunto(s)
Reparación del ADN , ADN/metabolismo , Timina/análogos & derivados , Secuencia de Bases , Línea Celular , Fibroblastos/metabolismo , Humanos , Datos de Secuencia Molecular , Mutagénesis , Mapeo Restrictivo , Timina/metabolismo , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/metabolismoRESUMEN
Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.
Asunto(s)
ADN Polimerasa beta/metabolismo , Reparación del ADN , Afidicolina/farmacología , ADN/biosíntesis , ADN/química , ADN Polimerasa III/metabolismo , Reparación del ADN/efectos de los fármacos , Didesoxinucleósidos/farmacología , Humanos , Modelos Genéticos , Especificidad por SustratoRESUMEN
In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3'-OH and 5'-sugar phosphate ends. In mammalian cells, the 5'-sugar phosphate is removed by the AP lyase activity of DNA polymerase beta (Pol beta). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol beta-null mouse cells and show that the reduced repair in Pol beta-null extracts can be complemented by addition of purified Pol beta. Using this complementation assay, we demonstrate that mutated Pol beta without dRPase activity is able to stimulate long patch BER. Mutant Pol beta deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol beta-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol beta alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol beta are coupled and that both of these Pol beta functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.