Excision by the human methylpurine DNA N-glycosylase of cyanuric acid, a stable and mutagenic oxidation product of 8-oxo-7,8-dihydroguanine

PURPOSE: 1-(2-Deoxy-beta-D-erythro-pentofuranosyl)-cyanuric acid (cyanuric acid nucleoside or dCa) has been shown to be formed upon exposure of 8-oxo-7,8-dihydroguanine- (8-oxoG) containing oligodeoxyribonucleotides (ODN) to oxidizing agents. When present in DNA, cyanuric acid (Ca) is readily bypassed by Escherichia coli DNA polymerases, which preferentially incorporate 2'-deoxyadenosine-5'-monophosphate (dAMP) opposite to the lesion. Therefore, Ca could be a mutagenic DNA lesion yielding G.C to T.A transversions like 8-oxoG. These results call attention to the potential importance of secondary oxidation products of 8-oxoG. The present study investigates the capability of several DNA N-glycosylases to remove the Ca lesion in DNA. MATERIALS AND METHODS: A site-specifically modified 22-mer ODN containing a single Ca residue was hybridized with complementary sequences yielding four DNA duplexes harbouring Ca opposite each of the regular DNA bases. The four Ca.N duplexes were used as substrates for nine DNA N-glycosylases from bacterial, yeast or human origin. RESULTS: The results show that the human methylpurine DNA N-glycosylase (Mpg) can remove Ca from DNA duplexes. Interestingly, oxidized base-specific DNA N-glycosylases, Fpg, Nth, Ntg1, Ntg2, Ogg1, hNth1 and hOgg1, cannot repair Ca in DNA. Furthermore, the removal of Ca by Mpg varied markedly depending on the opposite DNA base, the rank being Ca.C=Ca.T>Ca.G=Ca.A. CONCLUSIONS: 8-OxoG-derived lesions in DNA such as spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), oxaluric acid (Oa), oxazolone (Oz) and Ca are substrates of base excision repair DNA N-glycosylases. Most of them, Sp, Gh, Oa and Oz, are substrates of the oxidized bases-specific enzymes such as Nth or Fpg. In contrast, Ca is substrate of the human methylpurine DNA N-glycosylase (Mpg).     

Increased mutability and decreased repairability of a three-lesion clustered DNA-damaged site comprised of an AP site and bi-stranded 8-oxoG lesions

Abstract

Purpose: Ionizing radiation induces DNA damage, some of which are present in clusters, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. These clusters are thought to contribute to the detrimental effects of radiation, in part due to the compromised repair of clustered DNA damaged sites.

Materials and methods: The repair of three-lesion cluster present in oligonucleotides were determined in vitro using the hamster cell line CHO-K1 nuclear extract or purified proteins involved in base excision repair. The mutagenic potential of these clusters present in a plasmid was determined using an Escherichia coli reporter assay.

Results: We have shown that the repair of an abasic (AP) site within a three-lesion cluster, comprised of an AP site and bi-stranded 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, is retarded compared to that of an isolated AP site in an in vitro base excision repair (BER) assay. Further, the mutation frequency of the clustered damaged site is up to three times greater than that of an isolated 8-oxoG lesion.

Conclusions: As a consequence of enhanced mutagenic potential of clusters, non-double-strand break (DSB) DNA damage may contribute to the detrimental effects of radiation, in addition to the effects of DSB.     

Efficiency of radiation-induced base lesion excision and the order of enzymatic treatment

Purpose: To clarify whether initial base excision repair processes at clustered DNA damage sites comprising multiple base lesions affect subsequent excision processes via the formation of additional strand breaks by glycosylase and apurinic/apyrimidinic (AP) endonuclease base excision enzymes.

Materials and methods: Plasmid DNA (pUC18) as a model DNA molecule was exposed to high-linear-energy-transfer (LET) ionizing radiation (He^2+?or C^6+?ions) or low-LET ionizing radiation (X-rays) under various conditions to produce varied radical-scavenging effects. pUC18 was then treated sequentially or simultaneously with two bacterial base excision enzymes (glycosylases), namely, endonuclease III and formamidopyrimidine-DNA glycosylase, which convert pyrimidine (or abasic [AP] site) and purine (or AP site) lesions to single-strand breaks (SSB), respectively. Yields of additional SSB or double-strand breaks (DSB) as digestion products were examined after changing the order of enzymatic treatment.

Results: There were few differences among the enzymatic treatments, indicating that treatment order did not affect the final yields of additional SSB or DSB formed by glycosylase activity. This suggests that of the total damage, the fraction of clustered damage sites with a persistent base lesion dependent on the order of glycosylase treatment was insignificant if present.

Conclusion: Base lesion clusters induced by high- or low-LET radiation appear three or more base pairs apart, and are promptly converted to a DSB by glycosylase, regardless of the order of enzymatic treatment.     

Increased sensitivity to sparsely ionizing radiation due to excessive base excision in clustered DNA damage sites in Escherichia coli

PURPOSE: In order to clarify the cellular processing and repair mechanisms for radiation-induced clustered DNA damage, we examined the correlation between the levels of DNA glycosylases and the sensitivity to ionizing radiation in Escherichia coli. MATERIALS AND METHODS: The lethal effects of gamma-rays, X-rays, alpha-particles and H2O2 were determined in E. coli with different levels of DNA glycosylases. The formation of double-strand breaks by post-irradiation treatment with DNA glycosylase was assayed with gamma-irradiated plasmid DNA in vitro. RESULTS: An E. coli mutM nth nei triple mutant was less sensitive to the lethal effect of sparsely ionizing radiation (gamma-rays and X-rays) than the wild-type strain. Overproduction of MutM (8-oxoguanine-DNA glycosylase), Nth (endonuclease III) and Nei (endonulease VIII) increased the sensitivity to gamma-rays, whereas it did not affect the sensitivity to alpha-particles. Increased sensitivity to gamma-rays also occurred in E. coli overproducing human 8-oxoguanine-DNA glycosylase (hOgg1). Treatment of gamma-irradiated plasmid DNA with purified MutM converted the covalently closed circular to the linear form of the DNA. On the other hand, overproduction of MutM conferred resistance to H2O2 on the E. coli mutM nth nei mutant. CONCLUSIONS: The levels of DNA glycosylases affect the sensitivity of E. coli to gamma-rays and X-rays. Excessive excision by DNA glycosylases converts nearly opposite base damage in clustered DNA damage to double-strand breaks, which are potentially lethal.     

Cationic peptides containing tyrosine protect against radiation-induced oxidative DNA damage

PURPOSE: To examine the effect of the amino acid tyrosine on oxidatively or direct-type damaged DNA damage when it is present in a DNA binding ligand. MATERIALS AND METHODS: We made use of tetralysine ligands to ensure binding to DNA and to condense the DNA, and simulated direct-type damage by using gamma irradiation in the presence of thiocyanate ions. These ligands contained an additional C terminal amino acid. Phenylalanine was used as a control for tyrosine. These ligands were used in conjuction with a plasmid substrate to quantify strand break yields. Base damage yields were estimated by measuring the strand break yield after incubation of the plasmid with the bacterial base excision repair enzyme formamidopyrimidine-DNA N-glycosylase (FPG). RESULTS: When the condensing ligand contains an additional tyrosine or tryptophan residue, the plasmid is protected against the effects of a single electron oxidation, as assayed by sensitivity to a base excision repair enzyme. This protection is significantly greater in condensed plasmid where the amino acid residues are in close proximity to the DNA, and can be observed even when only a small fraction of the ligand contains tyrosine. CONCLUSIONS: Bound tyrosine residues located in close proximity to DNA are capable of reversing oxidative DNA damage far more efficiently than when present unbound in the bulk solution. This suggests that tyrosine residues in DNA binding proteins may participate in the repair of DNA that has been oxidatively damaged by ionizing radiation.     

Direct detection and quantification of abasic sites for in vivo studies of DNA damage and repair

Use of chemotherapeutic agents to induce cytotoxic DNA damage and programmed cell death is a key strategy in cancer treatments. However, the efficacy of DNA-targeted agents such as temozolomide is often compromised by intrinsic cellular responses such as DNA base excision repair (BER). Previous studies have shown that BER pathway resulted in formation of abasic or apurinic/apyrimidinic (AP) sites, and blockage of AP sites led to a significant enhancement of drug sensitivity due to reduction of DNA base excision repair. Since a number of chemotherapeutic agents also induce formation of AP sites, monitoring of these sites as a clinical correlate of drug effect will provide a useful tool in the development of DNA-targeted chemotherapies aimed at blocking abasic sites from repair. Here we report an imaging technique based on positron emission tomography (PET) that allows for direct quantification of AP sites in vivo. For this purpose, positron-emitting carbon-11 has been incorporated into methoxyamine ([¹¹C]MX) that binds covalently to AP sites with high specificity. The binding specificity of [¹¹C]MX for AP sites was demonstrated by in vivo blocking experiments. Using [¹¹C]MX as a radiotracer, animal PET studies have been conducted in melanoma and glioma xenografts for quantification of AP sites. Following induction of AP sites by temozolomide, both tumor models showed significant increase of [¹¹C]MX uptake in tumor regions in terms of radioactivity concentration as a function of time, which correlates well with conventional aldehyde reactive probe (ARP)-based bioassays for AP sites.     

DNA damage formation, DNA repair, and survival after exposure of DNA repair-proficient and nucleotide excision repair-deficient human lymphoblasts to UVA1 and UVB

PURPOSE: The comet assay has been used to visualize DNA damage in single cells after exposure to UV light. These comets are commonly thought to reflect transient, repair-induced DNA breaks. The goal of the work presented here was to further characterize the nature of UV-induced comets and to further elucidate DNA damage formation by different wavelengths of ultraviolet light. MATERIALS AND METHODS: Detailed dose-response and time-course experiments with comet formation were carried out with normal and nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) lymphoblasts. Irradiation was carried out with low, intermediate, or high doses of UVA1 or UVB, comet formation was observed, cell survival and viability were determined, and UV-induced apoptosis was measured. RESULTS: All responses were dose-dependent. With the intermediate dose of UVA1, a pronounced comet formation was observed without subsequent growth inhibition. Raising levels of porphyrins, which act as photosensitizers, by preincubation with 5-amino-levulinic acid increased comet formation with UVA1, but not with UVB. UVA1-sensitivity and comet formation in XP cells was not significantly different from the normal cells. With UVB no comet formation was seen without subsequent apoptotic cell death. XP cells exhibited the known UVB-hypersensitivity, but their comet formation was not significantly different from that of normal cells. CONCLUSIONS: The findings are compatible with the hypothesis that UV-induced comets represent transient repair-induced DNA breaks. Both, the NER of dimers and the base excision repair of oxidative DNA modifications are thought to contribute to comet formation.     

LET dependence of the yield of single-, double-strand breaks and base lesions in fully hydrated plasmid DNA films by 4He(2+) ion irradiation

PURPOSE: To characterize the complexity of DNA damage through determination of the yields of single (SSB) and double strand breaks (DSB), base lesions and clustered damage sites induced in fully hydrated plasmid DNA by direct radiation effects as a function of the ionizing density of the radiation using 4He(2+) ion irradiation with linear energy-transfer (LET) values in the range 19 to 148 keV/microm. MATERIALS AND METHODS: Hydrated plasmid DNA (pUC18) containing 34.5 water molecules/nucleotide was irradiated with He(2+) ions with LET values of 19, 63, 95, 121 and 148 keV/microm. From quantification of the conformational changes of the irradiated samples (closed circular, open or linear forms) analyzed by agarose gel electrophoresis, the yields of SSB and DSB were obtained. Base lesions were visualized as additional strand breaks by treatment with base excision repair enzymes (endonuclease III (Nth) and formamidpyrimidine DNA glycosylase (Fpg)). RESULTS: The yield of prompt SSB does not depend significantly on LET of the 4He(2+) ions, whereas the yield of prompt DSB increases with increasing LET. The yields of isolated base lesions, revealed by Nth and Fpg as additional SSB, decrease drastically with increasing LET. The sum of the yields of DSB and additional DSB revealed by Nth and Fpg increase with increasing LET of the 4He(2+) ions except at the highest LET investigated. CONCLUSION: The yields of clustered damage, revealed as DSB and non-DSB clustered damage sites, but not isolated lesions, namely SSB, increase with increasing ionization density of the 4He(2+) ions except at the highest LET investigated.     

Reaction of guanyl radicals in plasmid DNA with biological reductants: chemical repair of DNA damage produced by the direct effect of ionizing radiation

Purpose : It has been previously argued that the use of the one-electron oxidants (SCN) 2 ?- and Br 2 ?- with plasmid DNA leads to the formation of DNA guanyl radicals. These guanyl radical species are intermediates in the DNA damage produced by processes such as photo-ionization and ionizing irradiation. The present paper evaluates the use of thallium(II) ions (Tl II OH +) as the one-electron oxidant, and also determines rate constants for the reduction (repair) of guanyl radicals in plasmid DNA by a variety of reducing agents including the biologically important compounds ascorbate and glutathione. Materials and methods : Aqueous solutions of plasmid DNA containing 10 -3 mol dm -3 thiocyanate or thallous ions and a reducing agent (azide, nitrite, ferrocyanide, hexachloroiridate(III), iodide, ascorbate, glutathione, glutathione disulphide, methionine, tyrosine, 5-hydroxyindole-3-acetic acid, 10 -7 -10 -4 mol dm -3) were irradiated with 137 Cs γ-rays (662 keV). After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). Strand break yields after incubation were quantified by means of agarose gel electrophoresis. Results : High yields of FPG-sensitive sites produced by the oxidants (SCN)2 ?- and Tl II OH + were strongly attenuated by the presence of the reducing agents. Conclusions : From the results, it is possible to arrive at estimates of the rate constants for the reduction of the DNA guanyl radical by the reducing agents. Values lie in the range 10 4 -10 7 dm 3 mol -1 s -1. Using the values for ascorbate and glutathione, it is possible to estimate an upper limit on the order of milliseconds for the lifetime of DNA guanyl radicals under cellular conditions. The implication is that there may well be a significant chemical repair of DNA base damage by the direct effect of ionizing radiation.     

Synergistic effect of aphidicolin and 1-beta-D-arabinofuranosylcytosine on the repair of gamma-ray-induced DNA damage in normal human fibroblasts.

The effects on enzymatic DNA repair of aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC), two potent inhibitors of long-patch excision repair, were investigated in cultured human cells exposed to 60Co gamma-radiation. Using alkaline-sucrose velocity sedimentation analysis, both drugs were shown to inhibit markedly the repair of radioproducts in cultures exposed to greater than or equal to 150 Gy, indicating that a significant component of gamma-ray-induced DNA damage is operated on by a long-patch excision pathway. Moreover, while the extent of repair inhibited by aphidicolin was comparable to that suppressed by araC, combined exposure of irradiated cultures to the two drugs elicited a synergistic response. Specifically, in all three normal fibroblast strains examined, the yield of aphidicolin- or araC-detectable sites (lesions whose repair could be blocked by each drug alone) observed during the first 2 h after irradiation with 150 Gy ranged from 0.8 to 1.2 per 10(8) daltons genomic DNA, whereas the incidence of sites detected by combined exposure to the inhibitors was increased 4-fold (i.e. 3.8 per 10(8) daltons). This difference in site yield leads us to propose that simultaneous administration of aphidicolin and araC serves to block, in addition to long-patch repair, a second mode of excision repair which is refractory to each drug alone.     

神经特异性转录因子DAT1绿荧光蛋白表达载体的构建及其在C8细胞中的表达定位

目的：构建神经特异性转录因子DAT1与绿荧光蛋白融合表达的表达载体，并检测其在C8星形胶质细胞中的表达。方法：采用基因重组技术将DAT1基因和绿色荧光蛋白基因融合，构建绿荧光蛋白表达载体pEGFP—C1-DAT1，测序验证序列无误后。用脂质体法将重组质粒转入C8细胞，荧光显微镜观察DAT1的表达及定位。结果：测序验证结果表明，重组质粒含有DAT1全长编码序列，转染实验表明EGFP—DAT1能在C8细胞中正确表达，且：DAT1蛋白主要定位于细胞核中。结论：DAT1全长cDNA基因绿色荧光蛋白表达载体构建成功，在C8细胞中可以正确表达，为进一步研究DAT1的功能奠定了基础。     

Reaction of guanyl radicals in plasmid DNA with biological reductants: chemical repair of DNA damage produced by the direct effect of ionizing radiation.

PURPOSE: It has been previously argued that the use of the one-electron oxidants (SCN)2(*-) and Br2(*-) with plasmid DNA leads to the formation of DNA guanyl radicals. These guanyl radical species are intermediates in the DNA damage produced by processes such as photo-ionization and ionizing irradiation. The present paper evaluates the use of thallium(II) ions (Tl(II)OH(+)) as the one-electron oxidant, and also determines rate constants for the reduction (repair) of guanyl radicals in plasmid DNA by a variety of reducing agents including the biologically important compounds ascorbate and glutathione. MATERIALS AND METHODS: Aqueous solutions of plasmid DNA containing 10(-3) mol dm(-3) thiocyanate or thallous ions and a reducing agent (azide, nitrite, ferrocyanide, hexachloroiridate(III), iodide, ascorbate, glutathione, glutathione disulphide, methionine, tyrosine, 5-hydroxyindole-3-acetic acid, 10(-7)-10(-4) mol dm(-3)) were irradiated with 137Cs gamma-rays (662 keV). After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). Strand break yields after incubation were quantified by means of agarose gel electrophoresis. RESULTS: High yields of FPG-sensitive sites produced by the oxidants (SCN)2(*-) and Tl(II)OH(+) were strongly attenuated by the presence of the reducing agents. CONCLUSIONS: From the results, it is possible to arrive at estimates of the rate constants for the reduction of the DNA guanyl radical by the reducing agents. Values lie in the range 10(4)-10(7) dm(3) mol(-1) s(-1). Using the values for ascorbate and glutathione, it is possible to estimate an upper limit on the order of milliseconds for the lifetime of DNA guanyl radicals under cellular conditions. The implication is that there may well be a significant chemical repair of DNA base damage by the direct effect of ionizing radiation.     

The molecular genetics of the incision step in the DNA excision repair process

This review describes the evolution of research into the genetic basis of how different organisms use the process of excision repair to recognize and remove lesions from their cellular DNA. One particular aspect of excision repair, DNA incision, and how it is controlled at the genetic level in bacteriophage, bacteria, S. cerevisae, D. melanogaster, rodent cells and humans is examined. In phage T4, DNA is incised by a DNA glycosylase-AP endonuclease that is coded for by the denV gene. In E. coli, the products of three genes, uvrA, uvrB and uvrC, are required to form the UVRABC excinuclease that cleaves DNA and releases a fragment 12-13 nucleotides long containing the site of damage. In S. cerevisiae, genes complementing five mutants of the RAD3 epistasis group, rad1, rad2, rad3, rad4 and rad10 have been cloned and analyzed. Rodent cells sensitive to a variety of mutagenic agents and deficient in excision repair are being used in molecular studies to identify and clone human repair genes (e.g. ERCC1) capable of complementing mammalian repair defects. Most studies of the human system, however, have been done with cells isolated from patients suffering from the repair defective, cancer-prone disorder, xeroderma pigmentosum, and these cells are now beginning to be characterized at the molecular level. Studies such as these that provide a greater understanding of the genetic basis of DNA repair should also offer new insights into other cellular processes, including genetic recombination, differentiation, mutagenesis, carcinogenesis and aging.     

DNA base and strand damage in X-irradiated monkey CV-1 cells: influence of pretreatment using small doses of radiation

Base damage in alpha DNA from irradiated monkey CV-1 cells was determined by measuring release of 5'-32P-end labelled DNA fragments after digestion with endonuclease III of E. coli. The frequency and base sequence locations of the enzyme-sensitive sites were determined. Fragments were released from irradiated DNA at sequence sites of pyrimidines and guanines. The time for repair of half the single strand breaks was approximately 1.5 h. Repair of base damage as judged from loss of enzyme-sensitive sites in DNA was slower, with more than half of the damaged bases still detectable after 4 h of repair. Two important changes in the pattern of fragment release from DNA were produced when small radiation doses preceded the large ones needed to produce measurable DNA strand breaks and base damage. 5 Gy to cells incubated several hours before 320 Gy increased by five-fold the abundance of small DNA fragments with 3'-phosphoryl termini detected in high-resolution denaturing gels. These increases were detectable with doses as small as 0.2 Gy and were accompanied by the appearance of new species of DNA fragments of intermediate mobility at specific locations in the base sequence. The patterns resemble those produced by digesting DNA from heavily irradiated cells with endonuclease III.     

Redox equilibrium between guanyl radicals and thiocyanate influences base damage yields in gamma irradiated plasmid DNA. Estimation of the reduction potential of guanyl radicals in plasmid DNA in aqueous solution at physiological ionic strength

Purpose : Gamma irradiation of an aqueous solution containing thiocyanate ions produces the strongly oxidizing intermediate (SCN) 2 £ -. Reaction of this species with plasmid DNA produces damage that is revealed as strand breaks after incubation with the Escherichia coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). It has been previously reported that the yield of damage is highly sensitive to the experimental conditions, leading to the suspicion that electron transfer between DNA and (SCN) 2 £ - is reversible. In principle this makes it possible to determine the oxidation potential for plasmid DNA (more formally the reduction potential of oneelectron oxidized plasmid DNA), a fundamental parameter describing the reactivity of DNA towards electron transfer reactions. Materials and methods : Aqueous solutions of plasmid DNA and thiocyanate ions were subjected to 137 Cs n -irradiation. After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). The yield of this damage was quantified by using agarose gel electrophoresis to identify the fraction of the plasmid population that contains strand breaks. Results : The yield of FPG-sensitive sites decreases with increasing thiocyanate concentration, decreasing DNA concentration, and increasing dose rate. By making some simple assumptions about the chemical reactions that produce DNA damage, it is possible to derive a quantitative mathematical model for the yield of FPG-sensitive sites. A good agreement was found between this model and the experimental observations over a wide range of conditions (thiocyanate concentrations, DNA concentrations, and dose rates that vary by 20-, 40-, and 150-fold respectively). Conclusions : It was possible to assign a value to the equilibrium constant for the one electron transfer reaction between the two radical species (SCN) 2 £ - and DNA-G £ +. This leads to an estimate of the reduction potential at pH 7 for the couple DNA G £ + /DNA of E 7 = +1.39 - 0.01V.     

Redox equilibrium between guanyl radicals and thiocyanate influences base damage yields in gamma irradiated plasmid DNA. Estimation of the reduction potential of guanyl radicals in plasmid DNA in aqueous solution at physiological ionic strength.

PURPOSE: Gamma irradiation of an aqueous solution containing thiocyanate ions produces the strongly oxidizing intermediate (SCN)2*-. Reaction of this species with plasmid DNA produces damage that is revealed as strand breaks after incubation with the Escherichia coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). It has been previously reported that the yield of damage is highly sensitive to the experimental conditions, leading to the suspicion that electron transfer between DNA and (SCN)2*- is reversible. In principle this makes it possible to determine the oxidation potential for plasmid DNA (more formally the reduction potential of one-electron oxidized plasmid DNA), a fundamental parameter describing the reactivity of DNA towards electron transfer reactions. MATERIALS AND METHODS: Aqueous solutions of plasmid DNA and thiocyanate ions were subjected to 137Cs gamma-irradiation. After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). The yield of this damage was quantified by using agarose gel electrophoresis to identify the fraction of the plasmid population that contains strand breaks. RESULTS: The yield of FPG-sensitive sites decreases with increasing thiocyanate concentration, decreasing DNA concentration, and increasing dose rate. By making some simple assumptions about the chemical reactions that produce DNA damage, it is possible to derive a quantitative mathematical model for the yield of FPG-sensitive sites. A good agreement was found between this model and the experimental observations over a wide range of conditions (thiocyanate concentrations, DNA concentrations, and dose rates that vary by 20-, 40-, and 150-fold respectively). CONCLUSIONS: It was possible to assign a value to the equilibrium constant for the one electron transfer reaction between the two radical species (SCN)2*- and DNA-G*+. This leads to an estimate of the reduction potential at pH 7 for the couple DNA G*+/DNA of E7 = +1.39+/-0.01V.     

Hyperthermic effects on DNA repair of UV-irradiated Dictyostelium discoideum

DNA repair of a lower eukaryote, Dictyostelium discoideum, has been investigated through the analysis of heat effects on cell mortality and DNA repair of UV-irradiated amoeboid cells. In a wild-type strain (NC4), an increase in temperature immediately after UV irradiation resulted in an increase in cell mortality, though similar heat treatment before UV irradiation had no such effect. Similar results were obtained in another wild-type strain, HPS83. In NC4, heat treatment after UV irradiation did not inhibit the nicking of DNA strands during excision repair processes, but did inhibit the rejoining of the DNA strand breaks. Removal of thymine-containing pyrimidine dimers from DNA molecules was also depressed by heat treatment after UV irradiation. In contrast, heat treatment before UV irradiation had no effect on any stage of the nicking process, the excision of the dimers or the rejoining. On the other hand, a radiation-sensitive mutant (TW8) defective in an incision step of the excision repair process did not show an increase in cell mortality in response to heat treatment administered either before or after UV irradiation. Though the optimum temperature for cell growth of the amoebae was 23 degrees C, the critical temperature for effective enhancement of cell killing was ca. 30 degrees C. Hence we assume that the excision repair of UV-damaged DNA is selectively sensitive to heat treatment.     

Kinetics of Double Strand Break Repair in the DNA of X-irradiated Synchronized Mammalian Cells

Base damages, sugar damages, and single-strand breaks produced by free radicals are the preponderant lesions produced in DNA by ionizing radiation. These lesions have been individually introduced into substrate, template, and biologically active DNA molecules and enzymatic processing and biological consequences determined. Free radical-induced DNA lesions are processed by base excision repair and many are potentially lethal in simple viral systems. Furthermore, a number of free radical modifications of purine and pyrimidine bases are premutagenic lesions. The results of the enzymatic and biological processing of a number of the more well-studied and stable lesions are summarized.