RESUMEN
Electron paramagnetic resonance (EPR) signals of peroxyl radicals of poly(acrylic acid), PAA, and of single- and double-stranded DNA were generated at 293 K and pH 6.6 by in situ photolysis of O2-saturated aqueous solutions containing the macromolecules and H2O2. From time-resolved EPR measurements upper limits for the rate constants of reaction of glutathione (GSH) with the peroxyl radicals of PAA [kb < or = (0.8 +/- 0.2) x 10(2) dm3 mol-1S-1] and of single- and double-stranded DNA from calf thymus [kb < or = (2 +/- 2) x 10(2) dm3 mol-1S-1] were determined. The low value of kb for reaction of GSH with DNA peroxyl radicals is in agreement with the observed lack of protective effect of the thiol in radiation-induced DNA strand break reactions (Liphard et al. 1990).
Asunto(s)
Resinas Acrílicas , ADN de Cadena Simple/química , ADN/química , Glutatión/química , Peróxidos , Animales , Bovinos , ADN/efectos de la radiación , Daño del ADN , Ácido Ditionitrobenzoico , Espectroscopía de Resonancia por Spin del Electrón/métodos , Glutatión/análogos & derivados , Disulfuro de Glutatión , Peróxido de Hidrógeno , Cinética , Oxidación-Reducción , Timo , Factores de TiempoRESUMEN
The photoreaction of 2-methyl-1,4-naphthoquinone (MQ, menadione) with DNA and polynucleotides in argon-saturated aqueous solution (pH 7) was studied. Results from laser flash photolysis experiments indicate that triplet quinone reacts with DNA and polyA but not detectably with polyU by one-electron oxidation of the bases of the nucleic acid with formation of the radical anion of the quinone. Irradiation of argon-saturated solutions containing MQ and DNA or polynucleotides (polyU, polyA, polyG or polyC) with 334 nm light leads to an increase in molecular weight for single-stranded DNA, polyA and to a much less extent for polyU. This finding indicates crosslink formation with quantum yields in the range of 10(-5)-10(-3).
Asunto(s)
ADN/efectos de los fármacos , Polinucleótidos/metabolismo , Vitamina K/farmacología , Animales , Bovinos , ADN/metabolismo , ADN/efectos de la radiación , ADN de Cadena Simple/efectos de los fármacos , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/efectos de la radiación , Oxidación-Reducción , Fotoquímica , Fotólisis , Polinucleótidos/efectos de la radiación , Vitamina K/química , Vitamina K/metabolismoRESUMEN
DNA of plasmid pBR322 irradiated with laser at a wavelength of 193 mm was treated with an extract containing proteins from E.coli K12 AB1157 (wild-type). The enzymes were found to produce single- and double-strand DNA breaks, which was interpreted as a transformation of a portion of cyclobutane pyrimidine dimers and (6-4) photoproducts into nonrepairable single-strand DNA breaks. The products resulted from ionization of DNA, in particular, single-strand breaks, transform to double-strand breaks. A comparison of these data with the data on survival of plasmid upon transformation of E.coli K12 AB1157 enables one to assess the biological significance of single- and double-strand breaks. The inactivation of the plasmid (in AB1157) is mainly determined by the number of directly formed laser-induced single-strand breaks, whereas the contribution of enzymatically produced single- and double-strand breaks is insignificant.
Asunto(s)
Daño del ADN , ADN Bacteriano/genética , Escherichia coli/genética , Plásmidos/genética , Rayos LáserRESUMEN
Reactions of OH radicals with poly(U) (polyuridylic acid) in the presence of Ru(bpy)32+ or Ru(phen)32+ in aqueous solutions were studied. OH radicals were produced by pulse radiolysis and their reactions with ruthenium complexes were measured spectrophotometrically under conditions were the complexes are attached to the polynucleotide. The OH radical adds to either the uracil moiety or the ruthenium complexes. The ratio of the radicals produced depends only on the ratio of their rate constants and the concentrations of poly(U) and ruthenium complexes. Similar results were obtained with uridine-5'-monosphosphate, where the ruthenium complexes are not attached to the nucleotide. Surprisingly, the yield of single-strand break formation from the OH adducts of uracil in poly(U) is much smaller than that expected on the basis of the yield measured in the absence of ruthenium complexes. Possible reasons for this behaviour are discussed.
Asunto(s)
2,2'-Dipiridil/análogos & derivados , Daño del ADN , Depuradores de Radicales Libres/farmacología , Radical Hidroxilo , Compuestos Organometálicos/farmacología , Fenantrolinas/farmacología , Fármacos Fotosensibilizantes/farmacología , Poli U/efectos de la radiación , 2,2'-Dipiridil/farmacología , Complejos de CoordinaciónRESUMEN
Escherichia coli K12 bacteria strains AB1157 (repair-deficient wild-type, uvrA+ recA+), AB1886 (urvA-), AB2463 (recA-) and AB2480 (urvA- recA-) were exposed to 254 nm germicidal UV and 216 or 193 nm laser radiation. The mean lethal incident dose (D37) for AB1157 does not change significantly with wavelength, whereas it increases for the other strains on going from lambda irr = 254 to 193 nm, e.g. eightfold for AB2480. Quantum yields for inactivation, due to light absorbed by the chromosomal DNA, have been estimated from the D37 values. The large differences in D37 between uvrA+ and uvrA- strains indicate a significant contribution of pyrimidine dimers and (6-4) photoproducts to photodamage of DNA in the cells. The formation of dimers even with lambda irr = 193 nm is supported by the result that the photoreactivable sector is larger than 63%. Inactivation of E. coli upon irradiation at 193 and 216 nm is therefore due to damage to intracellular DNA rather than to membrane or protein damage as is the case for mammalian cells. The ratio of the lethal doses for AB1157 vs AB2480 is approximately 900 with lambda irr = 254 nm, but only 160 with lambda irr = 193 nm. We propose that this is due to the better repair of photodimers and (6-4) photoproducts than of damage induced by photoionization via upper excited states. Irradiation at 193 nm inactivates AB1157 mainly by damage due to photoionization and AB2480 by damage due to photodimers in the chromosomal DNA.
Asunto(s)
Reparación del ADN , Proteínas de Escherichia coli , Escherichia coli/efectos de la radiación , Rayos Ultravioleta , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Dosis de Radiación , Rec A Recombinasas/metabolismo , Dedos de ZincRESUMEN
Some of the recent progress made in the understanding of the quantitative aspects of the oxygen effect in radiation biology by several groups is summarized. Examples are: the importance of unrepairable damage for the quantitative description of the oxygen effect; proof that protein thiols hardly contribute to protection in cells in the absence of oxygen; the proposal that protection by thiols in concentration ranges where all DNA radicals react with oxygen is due to the formation of hydroperoxides which can be repaired enzymatically by glutathione peroxydase; the finding that unscavengeable damage in plasmid DNA is mainly due to spur-induced clustered damages, but that the precursors of the scavengeable and the unscavengeable damage are comparably well repaired by thiols; the result that E. coli repair wild type strains are better protected by addition of thiols than strains with deficiencies in enzymatic repair capacities.
Asunto(s)
Daño del ADN , ADN Bacteriano/efectos de la radiación , Ditiotreitol/farmacología , Depuradores de Radicales Libres/farmacología , Oxígeno , Plásmidos/efectos de la radiación , Reactivos de Sulfhidrilo/farmacología , Animales , Bacteriófago M13/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Escherichia coli/efectos de la radiación , Rayos gamma , Glutatión/farmacología , Glicerol/farmacología , Radical Hidroxilo/farmacología , Metanol/farmacología , Tolerancia a Radiación , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
The influence of three different kinds of single-strand breaks (ssb) on the biological activity of plasmid DNA (pBR322) was studied. The single-strand breaks were produced either by gamma-irradiation (together with base and sugar damage) or by DNase I digestion which introduced ligatable ssb. Non-ligatable ssb--single-strand gaps of three nucleotides in length--were generated in the nicked DNA by exonuclease III treatment. The biological activity (N/N(o)) of this damaged DNA was assessed in vivo by transformation of E. coli (CMK) repair wild-type cells. The activity of the enzymes of E. coli was studied in vitro by incubation in a protein extract of E. coli making use of an in vitro assay introduced earlier, which makes it possible to distinguish between enzymatic degradation (dsb formation) and repair of damaged plasmid DNA. The biological activity (D37) of DNA with non-ligatable ssb, as determined by electrotransformation, was about 56% lower than that of DNA with ligatable ssb. The biological activity of enzymatically damaged DNA is greater in calcium-treated cells than in electroporated cells. It is proposed that this is due to a calcium-dependent inhibition of nucleases. In contrast to the enzymatically damaged DNA, with gamma-radiation-damaged DNA a calcium-dependent increase in survival was not observed. Therefore, calcium-dependent nucleases do not play a role in the repair of damage produced by gamma-irradiation. The enzyme activity data show that the single-strand damages are either converted into dsb or repaired. A comparison of the efficiency of dsb formation in the extract for two of the single-strand damages is presented. The efficiency depends on the kind of damage and on the presence of cofactors, especially ATP and dNTPs.
Asunto(s)
Daño del ADN , ADN Recombinante/metabolismo , ADN Recombinante/efectos de la radiación , Desoxirribonucleasa I/metabolismo , Plásmidos , Animales , Bovinos , Escherichia coli/genéticaRESUMEN
pBR322 plasmid DNA was exposed to 254 nm UV radiation and examined for enzymatically produced single-strand break (sbb) and double-strand break (dsb) formation by treatment with an extract containing the proteins of Escherichia coli (AB1157 (uvrA+ recA+) and AB2480 (uvrA- recA-)). Enzymatic conversion of the 254 nm-induced lesions into ssbs on treatment with an extract from AB1157 was observed, but not conversion into dsbs. The rate of enzymatic ssb formation in the AB1157 extract is initially higher than in the AB2480 extract, the sbb formation levels off leading to plateau values with increasing incubation time. The rate of ssb formation in the AB2480 extract is initially lower, but does not level off, and the ssb yield becomes larger at longer incubation times than that with the AB1157 extract. The biological inactivation of the plasmids was measured as a function of 254 nm fluence by transformation of E. coli AB1157 and AB2480. Inactivation with AB2480 is mainly due to a single photoproduct, a cyclobutane-type pyrimidine dimer, per DNA molecule. Inactivation with AB1157 occurs with a quantum yield which is virtually identical with that of the plateau values of enzymatic ssb formation, as measured by incubation in the AB1157 extract. A possible interpretation is that the formation of irreparable ssbs is the lethal step in the sequence of events leading to inactivation of plasmid DNA in the repair wild-type strain. The quantum yield of inactivation is 10-20 times smaller for transformation of AB1157 than for AB2480, indicating that enzymatic repair of photolesions of the plasmid occurs in AB1157.
Asunto(s)
Cromosomas Bacterianos/efectos de la radiación , Daño del ADN , ADN Bacteriano/efectos de la radiación , ADN de Cadena Simple/aislamiento & purificación , Escherichia coli/metabolismo , Plásmidos/efectos de la radiación , Rayos Ultravioleta , Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/efectos de la radiación , Electroforesis en Gel de Agar , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Cinética , Plásmidos/aislamiento & purificación , Factores de TiempoRESUMEN
The survival of E. coli K12 strain AB1157 and the isogenic repair-deficient mutant E. coli AB2480 (recA13, uvrA6) was measured after gamma-irradiation in the presence of various alcohols as well as after incubation and subsequent removal of the alcohols before irradiation. Irradiation in the presence of alcohols leads to the already known protection effect, which has been attributed to OH radical scavenging. However, it was not possible to explain the protection solely in terms of the reactivity of the OH radical with the various alcohols, because addition of some of the alcohols did not yield the expected survival values. It was found that incubation with and subsequent removal of various alcohols before irradiation led to radiosensitization. The degree of radiosensitization increases with the hydrophobicity of the alcohol. In the case of glycerol no radiosensitization was observed. We can conclude that alcohols protect predominantly by OH radical scavenging. The comparatively small protection of cell survival by the more hydrophobic alcohols can be attributed to the sensitizing effect of these alcohols.
Asunto(s)
Alcoholes/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Protectores contra Radiación/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , 1-Propanol/farmacología , 1-Propanol/toxicidad , Alcoholes/toxicidad , Butanoles/farmacología , Butanoles/toxicidad , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Depuradores de Radicales Libres , Glicerol/farmacología , Glicerol/toxicidad , PropanolesRESUMEN
We have measured the yields of strand break formation and biological inactivation as a function of OH scavenger concentration for 60Co gamma-irradiated pBR322 plasmid and M13mp9 RF phage DNA. The yields of single-strand breaks (ssbs), double-strand breaks formed proportionally to dose (alpha dsbs), and lethal damage (LD) decrease with increasing scavenging capacity sigma, their ratios remaining approximately constant up to sigma approximately 10(8) s-1. On a double-logarithmic plot the yields decrease linearly with sigma in parallel lines. At higher scavenging capacities, the yields, while still decreasing, level off to a different extent. Our results for the yields of ssbs and alpha dsbs confirm those of Krisch et al. (1991) using SV40 DNA. The data were analysed assuming that DNA damage is brought about by OH radicals, and a non-scavengeable portion arising from the direct radiation effect. Using a model based on non-homogeneous scavenging kinetics, the dependence on scavenging capacity of the ssb yield could be quantitatively accounted for. From the scavenging dependence of the yield of dsbs which are formed quadratically with dose (beta dsbs) and which are the result of two independent ssbs within a critical distance h, a value of about 13 basepairs was obtained for h. The parallel decrease in the yield of ssbs and alpha dsbs with scavenging capacity was rationalized in terms of the Siddiqi-Bothe mechanism (Siddiqi and Bothe 1987). The efficiency of this mechanism was found to be approximately 0.01. From the analysis of the LD yields it was shown that up to sigma approximately 10(8) s-1, inactivation is predominantly due to single OH radicals which lead to LD with an efficiency of 0.12 per OH-induced ssb. At higher scavenging capacities, a non-scavengeable spur effect similar to the locally multiply damaged sites mechanism of Ward (1988) mainly contributes to LD.
Asunto(s)
Bacteriófagos/genética , Daño del ADN , ADN de Cadena Simple/efectos de la radiación , ADN/efectos de la radiación , Plásmidos/genética , Alcoholes , Radioisótopos de Cobalto , Genética de Radiación , AguaRESUMEN
Plasmid pTZ18R and calf thymus DNA in aerated neutral aqueous solution were irradiated by continuous 254 nm light. The quantum yields are phi ssb = 4.0 x 10(-5) and phi dsb = 1.4 x 10(-6) for single- and double-strand break formation, respectively, phi br = 2.3 x 10(-5) for base release, phi dn = 2.1 x 10(-3) for destruction of nucleotides, and phi icl approximately phi lds approximately 1 x 10(-6) for interstrand cross-links and locally denatured sites, respectively. The presence of Tris-HCl/ethylenediaminetetraacetic acid (10:1, pH 7.5) buffer strongly reduces phi ssb. The corresponding phi values, obtained on employing pulsed 193 nm laser irradiation, are much larger than those using lambda irr = 254 nm. This is ascribed to a contribution of chemical reactions induced by photoionization, which is absent for 254 nm irradiation. The quantum yields of inactivation of plasmid DNA (lambda irr = 254 nm) were measured by transformation of the Escherichia coli strains AB1157 (wild type), phi ina (1157) = 1.6 x 10(-4), AB1886 (uvr-), phi ina (1886) = 4.2 x 10(-4), AB2463 (rec-), phi ina (2463) = 4.1 x 10(-4) and AB2480 (uvr- rec-), phi ina (2480) = 3.1 x 10(-3). The quantum yields of inactivation of plasmid DNA are compared with those of the four E. coli strains (denoted as chromosomal DNA inactivation) obtained from the literature. The results for E. coli strain AB2480 show that the chromosomal DNA and the plasmid DNA are both inactivated by a single pyrimidine photodimer per genome.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
ADN/efectos de la radiación , Rayos Láser , Plásmidos/efectos de la radiación , Rayos Ultravioleta , ADN Bacteriano/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , FotólisisRESUMEN
In the 400-MHz 1H NMR spectra of gamma-irradiated, N2O-saturated aqueous solutions of polyuridylic acid (polyU), polycytidylic acid (polyC), polyadenylic acid (polyA), and single-stranded DNA from calf thymus, well-resolved signals of the undamaged free bases were identified. Upon incubation of the solutions at 95 degrees C for 2 h after irradiation, the amount of release of undamaged bases increased and in the case of polyA and DNA additional signals in the region 7.7-8.3 ppm due to 4,6-diamino-5-(formylamino)-pyrimidine and 8-hydroxyadenine (8-OH-adenine) were identified. Probably because of their low solubility guanine and modified guanine bases were not detected, whereas the lack of signals of low molecular weight pyrimidine derivatives is explained by the stability of the corresponding glycosidic bonds toward hydrolysis under our conditions. From the spectral intensities G values for release of unaltered and modified nucleic bases were estimated for radiation doses corresponding to approximately 2.5% conversion for polyU and polyC, to approximately 6% conversion for polyA and to approximately 30-60% conversion for DNA. The NMR data were in agreement with G values reported for release of undamaged bases from irradiated polyU and for double-strand DNA obtained on the basis of chromatographic procedures in combination with UV absorption (D.J. Deeble, D. Schulz, and C. von Sonntag, Int. J. Radiat. Biol. 49, 915-926, 1986; J.F. Ward and I. Kuo, Radiat. Res. 66, 485-498, 1976). Broadening of the NMR peaks of the polymers at high radiation doses (> 10 kGy) was dependent on the magnetic field strength. Therefore, it is ascribed to chemical shift heterogeneity caused by a variety of irradiation products with strongly overlapping signals rather than to a decrease in internal mobility caused by crosslinking of the polymer chains.
Asunto(s)
ADN de Cadena Simple/efectos de la radiación , Polirribonucleótidos/efectos de la radiación , Adenina , Radioisótopos de Cobalto , Citosina , Daño del ADN , ADN de Cadena Simple/metabolismo , Rayos gamma , Espectroscopía de Resonancia Magnética , Óxido Nitroso , Soluciones , Timina , Uracilo , AguaRESUMEN
Double-strand breaks (dsbs) have been produced in plasmid DNA by various restriction endonucleases and the survival and the deletion mutation incidence have been measured in E. coli. The deletion formation is known to depend upon the occurrence of short direct repeats within the DNA molecule. In order to study the role of these repeats we constructed plasmid molecules with repeats of various lengths or with a 10-base pair repeat at different distances from each other. Furthermore the influence of the location and the structure of the dsb was studied. Repair and deletion frequencies of the linearized plasmids were measured after transformation of E. coli. The yield of the specific deletion mutation (the one which occurs between the introduced repeats) increases nearly linearly with the square of the length of the repeat, while the yield of the correctly repaired DNA and the yield of all other deletion mutants remained constant. The slope of the linear increase of the yield of the specific deletion depends on the location and the structure of the dsb. The yield of the specific deletion mutation decreases with increasing distance between the repeats. A proposal for the rate-determining step of the deletion formation is made.
Asunto(s)
Daño del ADN , Enzimas de Restricción del ADN/metabolismo , ADN Bacteriano/genética , Escherichia coli/genética , Mutagénesis , Plásmidos , Eliminación de Secuencia , Composición de Base , Secuencia de Bases , Reparación del ADN , Modelos Genéticos , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The survival of M13 DNA was studied after partial replacement of thymine by uracil in the bacteriophage. Uracils carry the same genetic information as the thymines. Nevertheless in Escherichia coli wild-type cells, uracils in DNA are replaced by thymines by excision repair initiated by uracil-DNA glycosylase (UDG). Thus inactivation of uracil-containing phage DNA is solely due to repair initiated by UDG. Incorporation of uracils was achieved in one or in both strands, either randomly or site-specifically using differently uracylated oligonucleotides. The results show that up to 580 uracils can be repaired without a significant decrease in survival if the uracils are localized in the (-) strand only. Incorporation of 246 uracils into the (+) strand leads to approximately 30% or approximately 10% survival when expressed in Escherichia coli strains CMK and JM103, respectively. However, when uracils are distributed over both strands a sharp decrease in survival occurs. This shows that the repair of two uracils localized in close proximity on opposite strands of the DNA by the excision repair mechanism is difficult, whereas uracils occurring in one strand are repaired more efficiently, irrespective of their number.
Asunto(s)
Colifagos/genética , Reparación del ADN/genética , ADN Viral/genética , Nucleótidos de Uracilo/genética , Secuencia de Bases , Daño del ADN/genética , ADN de Cadena Simple/genética , Electroforesis , Escherichia coli/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/genética , Transformación Bacteriana/genéticaRESUMEN
The yields of single-strand breakage (ssb) in single-stranded calf thymus DNA (ssDNA) have been determined after 60Co gamma-irradiation of aqueous anoxic solutions in the presence of different concentrations of dithiothreitol (DTT), ascorbate or trans-4,5-dihydroxy-1,2-dithiane, using low-angle laser light scattering. The influence of DTT on the kinetics of ssb formation has been determined by conductivity measurements in pulse radiolysis. The results suggest that strand breakage in ssDNA proceeds via two modes of about equal contribution and with half-lives of about 7 ms and 0.8s, respectively. Both modes reflect reactions of at least two DNA radicals, which react with DTT by hydrogen-atom transfer reactions with similar rate constants of about 5-9 x 10(5) dm3 mol-1 s-1. These hydrogen-atom transfer reactions inhibit strand break formation. The slow mode is shown to represent the decay of base-radicals to generate sugar radicals. The involvement of the oxidizing .OH adduct radical of guanine in the formation of strand breaks can be ruled out and there is no evidence for a contribution from the anion or radical anion of DTT to the inhibition of strand breaks via electron transfer reactions to DNA radicals.
Asunto(s)
Acetofenonas , Daño del ADN , ADN de Cadena Simple/efectos de la radiación , Ditiotreitol , Fármacos Sensibilizantes a Radiaciones , Animales , Hidróxidos , Radical Hidroxilo , Técnicas In Vitro , Oxígeno , Soluciones , AguaRESUMEN
Single- and double-stranded calf thymus DNA and two polynucleotides (0.4 mM) were studied in aqueous solution at pH approximately 7 using pulsed, 20 ns laser excitation at 193 nm. Monophotonic ionization of the nucleic acids is suggested from the linear dependences of the concentration of ejected electrons and the number of single- and double-strand breaks (ssb, dsb, respectively) on laser intensity (IL) in the range (0.2-3) x 10(6) W cm-2. The quantum yields of formation of hydrated electrons (phi e-) and ssb and dsb (phi ssb and phi dsb) are therefore independent of IL. In contrast, under 248 nm excitation these quantum yields increase linearly with IL under otherwise comparable conditions. Nevertheless, several effects and mechanistic implications are analogous using lambda exc = 193 and 248 nm. For polycytidylic acid, poly(C), in Ar-saturated solution for example, the efficiency of ssb per radical cation (eta RC = phi ssb/phi e-) is similar to the efficiency of ssb per OH radical (eta OH). For polyadenylic acid, poly(A), and single- and double-stranded DNA eta RC (lambda exc = 193 nm) is significantly smaller than eta OH. The ratio phi ssb (N2O)/phi ssb (Ar) is approximately 2 for poly(C), approximately 4 for poly(A) approximately 10 for DNA; the conversion of hydrated electrons into OH radicals in N2O-saturated solution and smaller eta RC than eta OH values in the case of DNA account for these results. For double-stranded DNA phi dsb does not depend on IL but increases linearly with the dose, indicating an accumulative effect of two ssb to generate one dsb. The critical distance for this event is 60-85 phosphoric acid diester bonds.
Asunto(s)
ADN/efectos de la radiación , Animales , Bovinos , ADN/química , Daño del ADN , ADN de Cadena Simple/efectos de la radiación , Técnicas In Vitro , Rayos Láser , Fotoquímica , Poli A/efectos de la radiación , Poli C/efectos de la radiaciónRESUMEN
The absorption spectra of polyadenylic acid (polyA) radicals in N2O saturated aqueous solution have been measured as a function of time (up to 15 s) following an 0.4 microsecond electron pulse. The spectra and their changes were analysed by comparison with those from monomeric adenine derivatives (nucleosides and nucleotides) which had been studied by Steenken. The reaction of OH. radicals with the adenine moiety in polyA results in the formation of two hydroxyl adducts at the positions C-4 [polyA4OH.] and C-8 [polyA8OH.]. Each OH-adduct undergoes a unimolecular transformation reaction before any bimolecular or other unimolecular decay occurs. These reactions are characterized by different rate constants and pH dependencies. The polyA4OH. adduct undergoes a dehydration reaction to yield a neutral N6 centered radical (rate constant kdeh = 1.4 x 10(4)s-1 at pH 7.3). This reaction is strongly inhibited by H+. In comparison with the analogous reactions in adenosine phosphates, the kinetic pK value for its inhibition is two pH units higher. This shift is the result of the counter ion condensation or double-strand formation. The polyA8OH. adduct undergoes an imidazole ring opening reaction to yield an enol type of formamidopyrimidine radical with the resulting base damage (kr.o. = 3.5 x 10(4)s-1 at pH 7.3). This reaction in contrast is strongly catalysed by H+ and OH-, similar as for adenosine but different compared to the nucleotides.
Asunto(s)
Hidróxidos/química , Poli A/química , Radiólisis de Impulso , Fenómenos Químicos , Química Física , Radicales Libres , Concentración de Iones de Hidrógeno , Radical Hidroxilo , IsomerismoAsunto(s)
Enzimas de Restricción del ADN/metabolismo , ADN/química , ADN/efectos de la radiación , Desoxirribonucleasa I/metabolismo , Radiación Ionizante , Animales , Muerte Celular/efectos de la radiación , Cromatina/ultraestructura , Aberraciones Cromosómicas/genética , ADN/metabolismo , Humanos , Mutación/efectos de la radiación , Transformación Genética/efectos de la radiaciónRESUMEN
Gaps of various lengths were generated in duplex M13mp18DNA by exonuclease III digestion of nicked DNA. The length of the gap increased essentially linearly with time of digestion. Survival in E. coli, however, was not a linear function of gap length. Similar results were obtained when gaps were produced by stopping the polymerization reaction. The survival (N/No) of the gapped DNA in SOS-induced E. coli cells transformed by electroporation and uninduced cells transformed by the calcium chloride method can be quantitatively accounted for by a kinetic model assuming a single-strand endonucleolytic activity (Pd) in the cell which increases linearly with gap length (L) and a repair activity by a polymerase (Pr) which is independent of gap length (formula 1). With uninduced cells transformed by electroporation the results can be mathematically described if assumptions are made concerning the protection of single-stranded parts of the DNA by single-strand affinic proteins.