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1.
Int J Radiat Biol ; 54(5): 709-22, 1988 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-2902166

RESUMEN

Unlabelled and radiolabelled methoxyamine have been used to characterize DNA damage caused by gamma-rays or by the chemical reagent osmium tetroxide (OsO4). Both treatments introduce in DNA a number of methoxyamine-binding sites proportional to the dose. Whereas the number of these sites remains constant after the OsO4 treatment it increases during postirradiation incubation; the postirradiation appearance of methoxyamine-binding sites is enhanced by the presence of methoxyamine. OsO4 treatment and gamma-irradiation also induce the formation of alkali-labile sites in DNA. Whereas the number of these sites remains constant after OsO4 treatment, it increases during postirradiation incubation and an alkaline medium accelerates their formation. A fraction of the alkali-labile sites found in gamma-irradiated DNA is methoxyamine-labile; by contrast, the OsO4-treated DNA is stable in the presence of methoxyamine.


Asunto(s)
Daño del ADN , ADN Bacteriano/efectos de la radiación , Tetróxido de Osmio/farmacología , Osmio/farmacología , ADN Bacteriano/efectos de los fármacos , Escherichia coli/genética , Rayos gamma , Hidroxilaminas , Indicadores y Reactivos
2.
Mutat Res ; 181(1): 45-56, 1987 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-2444877

RESUMEN

The paper is an outline review of the main aspects concerning the formation and repair of AP (apurinic/apyrimidinic) sites in DNA as well as some of the chemical properties allowing their quantitative determination. A new method for the measurement of AP sites based on their reaction with [14C]methoxyamine is described. It has been applied to the measurement of AP sites produced in DNA either by physical (gamma-rays) or chemical (methyl methanesulphonate, osmium tetroxide) agents. The method has also been used to quantify the excision of abnormal bases from DNA under the action of specific DNA glycosylases and to prevent the chemical or enzymatic degradation of DNA containing AP sites. The paper contains data about the purification and characterization of uracil-DNA glycosylase and AP endodeoxyribonuclease from carrot cells, two enzymes involved in the first steps of base excision repair through AP site intermediates. The biological effects of unrepaired AP sites are also discussed.


Asunto(s)
Daño del ADN , ADN Glicosilasas , Reparación del ADN , ADN/análisis , ADN/genética , ADN/efectos de la radiación , ADN Ligasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/aislamiento & purificación , Rayos gamma , Hidroxilaminas , Metilmetanosulfonato/farmacología , N-Glicosil Hidrolasas/aislamiento & purificación , N-Glicosil Hidrolasas/metabolismo , Tetróxido de Osmio/farmacología , Proteínas de Plantas/aislamiento & purificación , Uracil-ADN Glicosidasa
3.
Biochim Biophys Acta ; 867(1-2): 24-30, 1986 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-3011095

RESUMEN

Using [32P]DNA alkylated with [3H]methyl methanesulfonate, depurinated by heating at 50 degrees C for various periods, then treated with sodium hydroxide, a table was constructed giving the DNA fraction soluble in 5% perchloric acid at 0 degree C as a function of the frequency of strand breaks. The alkaline treatment placed a break near each apurinic site; the apurinic sites were counted in two ways which gave consonant results: by the loss of [3H]methyl groups and by reaction with [14C]methoxyamine. The 32P label of DNA was used to measure the acid-solubility.


Asunto(s)
ADN Bacteriano/metabolismo , Purinas , Escherichia coli/análisis , Calor , Concentración de Iones de Hidrógeno , Hidroxilaminas/metabolismo , Metilmetanosulfonato , Metilación , Percloratos , Hidróxido de Sodio , Solubilidad
4.
J Biol Chem ; 260(9): 5252-8, 1985 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-2580833

RESUMEN

This paper describes the use of methoxyamine to study the enzymatic reactions catalyzed by uracil-DNA glycosylase and by AP (apurinic/apyrimidinic) endodeoxyribonuclease isolated from mammalian cells. [14C]Methoxyamine permits one to follow the formation of AP sites in a uracil-containing polydeoxyribonucleotide incubated with calf thymus uracil-DNA glycosylase. The number of methoxyamine-reacted AP sites is equal to that of uracil released. Methoxyamine has no effect on the uracil-DNA glycosylase activity and may be added together with the enzyme in order to block the AP sites and prevent the degradation of the polynucleotide by the AP endonucleases that may be present in a crude preparation. Addition of methoxyamine to AP sites prevents not only the enzymatic hydrolysis of the adjacent phosphodiester bond but also the degradation of the polynucleotide by NaOH. This protective effect disappears after methoxyamine is removed by acetaldehyde.


Asunto(s)
ADN Glicosilasas , Reparación del ADN , Hidroxilaminas/metabolismo , Animales , Bovinos , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/metabolismo , N-Glicosil Hidrolasas/metabolismo , Ratas , Uracilo/metabolismo , Uracil-ADN Glicosidasa
5.
Biochim Biophys Acta ; 740(4): 410-6, 1983 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-6349690

RESUMEN

A simple and rapid method is described for the determination of AP (apurinic/apyrimidinic) sites in DNA. The method involves the reaction of [14C]methoxyamine with the aldehyde group present in the deoxyribose moiety after a base loss. Studies with alkylated-depurinated DNA and with uracil-containing polydeoxyribonucleotides depyrimidinated by uracil-DNA glycosylase show that methoxyamine reacts with both apurinic and apyrimidinic sites in a rapid and exhaustive way. Under standard conditions (30-min incubation with 5 mM methoxyamine at 37 degrees C, pH 7.2) untreated DNA is almost unreactive and the [14C]methoxyamine incorporation in DNA is proportional to the number of AP sites. Since the methoxyamine reaction is free from any degradative effect on DNA, AP sites may be estimated from a simple determination of the acid-insoluble radioactivity.


Asunto(s)
Ácido Apurínico/metabolismo , ADN Bacteriano/metabolismo , Hidroxilaminas/metabolismo , Polinucleótidos/metabolismo , Alquilación , Radioisótopos de Carbono , Escherichia coli , Concentración de Iones de Hidrógeno , Cinética , Polidesoxirribonucleótidos
6.
Eur J Biochem ; 124(3): 435-40, 1982 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-6286302

RESUMEN

Uracil-DNA glycosylase and apurinic/apyrimidinic (AP) endodeoxyribonuclease have been purified from cultured carrot cells. The two enzymes, separated by affinity chromatography on Sepharose-poly(rU), were found to have properties similar to those of the homologous bacterial and mammalian enzymes. The action of AP endodeoxyribonuclease on (dA)230 . (dT, dU)230 partially depyrimidinated by uracil-DNA glycosylase suggests that these two enzymes might act successively to initiate the repair of uracil-containing DNA.


Asunto(s)
ADN Glicosilasas , Reparación del ADN , Desoxirribonucleasas/aislamiento & purificación , Endonucleasas/aislamiento & purificación , Proteínas de Escherichia coli , N-Glicosil Hidrolasas/aislamiento & purificación , Plantas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Desoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Escherichia coli/enzimología , Cinética , N-Glicosil Hidrolasas/metabolismo , Factores de Tiempo , Uracil-ADN Glicosidasa
7.
J Biol Chem ; 257(3): 1208-14, 1982 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-7056715

RESUMEN

The bovine uracil-DNA glycosylase previously isolated from thymocyte nuclei was further purified by 1 order of magnitude with the aid of affinity chromatography. The final preparation was totally devoid of DNase and apurinic or apyrimidinic (AP) endonuclease activities, and it corresponded to purifications of 457-fold over the nuclear extract and of about 2000-fold over the crude tissue homogenate. Most of the general enzyme properties already described were confirmed. Furthermore, this mammalian uracil-DNA glycosylase was shown to bind specifically with polymerized and not with monomeric nucleotide compounds, while having a preference for double-stranded forms. It cleaved N-glycosyl linkages only at the deoxyuridyl units located in internal positions of polynucleotide chains. The enzyme also used RNA-DNA hybrids as functional substrates and was practically ineffective on deoxyuridyl residues at the 3'-ends of nucleic acids. The activity of the glycosylase was greatly impaired in assays with DNA substrates that contained amounts of AP sites exceeding 5 microM. The inhibitory concentrations of AP residues were about 100 times lower than those found equally effective for the other reaction product, i.e. free uracil, and were almost comparable to the Km values for deoxyuridyl nucleotides in the DNA substrates. This all appears as a modulation of the glycosylase catalysis by the relative amounts of its substrate and product structures in DNA. The data lead us to surmise that the removal of uracil from cellular DNA is functionally coupled to the expected elimination of the formed AP sites by specific endonucleases. Base-exchange and base-insertion experiments with the purified enzyme yielded negative results under various conditions. The glycosylase behaved essentially as a hydrolase which has no associated base-insertase properties and irreversibly excises uracil from DNA by a mechanism for channeling the process to the next steps of the repair pathway.


Asunto(s)
ADN Glicosilasas , Reparación del ADN , N-Glicosil Hidrolasas/metabolismo , Timo/enzimología , Animales , Radioisótopos de Carbono , Bovinos , Cinética , N-Glicosil Hidrolasas/aislamiento & purificación , Especificidad por Sustrato , Tritio , Uracilo/farmacología , Uracil-ADN Glicosidasa
9.
Biochim Biophys Acta ; 518(1): 138-49, 1978 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-629972

RESUMEN

Selected samples of heterogeneous DNA from calf thymus with similar number-average molecular weight, Mn, and a low incidence of single-strand breaks were exposed in aqueous solutions to a mild X-ray dose of 1500 rads. The irradiation produced on the average about 0.2 bihelical and 2.2 monohelical scissions per DNA molecule of 1708 000 Mn. The percent distribution of the chemical termini released at the radiation nicks of DNA was as follows: 64.0 OH, 9.0 PO4 and 27.0 unknowns at the 3' ends: 3.8 OH, 68.2 PO4 and 28.0 unknowns at the 5' ends. A nuclease-free polynucleotide ligase I purified about 3000-fold over the crude homogenate from calf thymus succeeded in rejoining 50% of the breaks in the X-irradiated DNA. The ability of the enzyme to close radiation nicks in DNA directly was confirmed also by experiments on synthetic poly(dA).poly([3H]dT),poly(dT)-cellulose substrates with an irradiated dT chain at either the 3' or the 5' side of the functional break. The poor discrimination of mammalian ligase versus nicked DNA containing radiation damage is of practical relevance. While rejoining altered nucleotide chains in the helices of DNA, the enzyme might contribute to the fixation of premutational lesions in the genetic material.


Asunto(s)
ADN Ligasas/metabolismo , ADN/efectos de la radiación , Polinucleótido Ligasas/metabolismo , Animales , Bovinos , Cinética , Relación Estructura-Actividad , Timo/enzimología
10.
J Antibiot (Tokyo) ; 28(8): 580-9, 1975 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-1158783

RESUMEN

The chemical dimers of rifamycin SV resembled the corresponding monomeric analogs with respect to the inhibitory properties versus the nucleic acid polymerases. At low doses, such compounds blocked the initiation step of the DNA transcription catalyzed by the bacterial RNA polymerase, as observed for the parental antibiotic and its derivative rifampicin which are largely used in therapy. At concentrations one to two orders of magnitude higher, the chemically modified rifamycins inhibited also other nucleotidyltransferases. The widespread toxicity of the dimeric and monomeric semisynthetic rifamycins versus these enzymes was not causally related with an enhancement of their lipophily. The observed effects might be due to a loss of selectivity in the inhibition mechanism which was originally specific for the RNA polymerase from E. coli at the beginning of its catalysis. The rifamycin derivatives might then react with the catalytic portion of other nucleotidyltransferases interfering adversely with the enzyme activity in a number of ways and/or at different levels.


Asunto(s)
ADN Nucleotidiltransferasas/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Rifamicinas/toxicidad , Animales , Bovinos , Fenómenos Químicos , Química , ADN/biosíntesis , ADN Bacteriano/biosíntesis , ARN/metabolismo , ARN Bacteriano/metabolismo , Rifampin/toxicidad
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