Substrate specificities of FPG protein (also known as formamidopyrimidine DNA glycosylase) and 8-hydroxyguanine endonuclease were compared by using defined duplex oligodeoxynucleotides containing single residues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and 2,6-diamino-4-hydroxy-5-(N-methyl)formamidopyrimidine (Me-Fapy). Duplexes containing 8-oxodG positioned opposite dC, dG, or dT were cleaved, whereas single-stranded DNA and duplexes containing 8-oxodGdA or 8-oxodA positioned opposite any of the four DNA bases were relatively resistant. Both enzymes cut duplexes containing 8-oxoG-dC 3' and 5' to the modified base but failed to cleave duplex DNA containing synthetic abasic sites, mismatches containing dG, or unmodified DNA. 8-Oxoguanine, identified by HPLC-electrochemical detection techniques, was released during the enzymatic reaction. Apparent Km values for FPG protein acting on duplex substrates containing a single Me-Fapy or 8-oxodG residue positioned opposite dC were 41 and 8 nM, respectively, and those for 8-hydroxyguanine endonuclease were 30 and 13 nM, respectively. Comparison of the properties of the two enzyme activities suggest that they are identical. In view of the widespread distribution of 8-oxodG in cellular DNA, the demonstrated miscoding and mutagenic properties of this lesion, and the existence of a bacterial gene coding for FPG protein, we propose that 8-oxodG DNA is the primary physiological substrate for a constituent glycosylase found in bacteria and mammalian cells.Active oxygen species, generated by ionizing radiation and by endogenous oxidation processes, react with deoxyguanosine (dG) residues in DNA to form 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) (reviewed in ref.

The roles of nitric oxide (NO) in numerous disease states have generated considerable discussion over the past several years. NO has been labeled as the causative agent in different pathophysiological mechanisms, yet appears to protect against various chemical species such as those generated under oxidative stress. Similarly, NO appears to exert a dichotomy of effects within the multistage model of cancer. Chronic inflammation can lead to the production of chemical intermediates, among them NO, which in turn can mediate damage to DNA. Yet, NO also appears to be critical for the tumoricidal activity of the immune system. Furthermore, NO can also have a multitude of effects on other aspects of tumor biology, including angiogenesis and metastasis. This report will discuss how the chemistry of NO may impact the initiation and progression stages of cancer.

We have investigated the excision of a variety of modified bases from DNA by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) [Boiteux, S., O'Connor, T. R., Lederer, F., Gouyette, A., & Laval, J. (1990) J. Biol. Chem. 265, 3916-3922]. DNA used as a substrate was modified either by exposure to ionizing radiation or by photosensitization using visible light in the presence of methylene blue (MB). The technique of gas chromatography/mass spectrometry, which can unambiguously identify and quantitate pyrimidine- and purine-derived lesions in DNA, was used for analysis of hydrolyzed and derivatized DNA samples. Thirteen products resulting from pyrimidines and purines were detected in gamma-irradiated DNA, whereas only the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) was observed in visible light/MB-treated DNA. Analysis of gamma-irradiated DNA after incubation with the Fpg protein followed by precipitation revealed that the Fpg protein significantly excised 4,6-diamino-5-formamidopyrimidine (FapyAde), FapyGua, and 8-OH-Gua. The excision of a small but detectable amount of 8-hydroxyadenine was also observed. The detection of these products in the supernatant fractions of the same samples confirmed their excision by the enzyme. Nine pyrimidine-derived lesions were not excised. The Fpg protein also excised FapyGua and 8-OH-Gua from visible light/MB-treated DNA. The presence of these products in the supernatant fractions confirmed their excision.(ABSTRACT TRUNCATED AT 250 WORDS)

Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiationinduced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells. Ionizing radiation may produce cancer, death, and loss of neural function in humans and animals, and it may induce killing, mutation, and chromosomal aberrations in cells (1). Humans are exposed to low doses of radiation-during air travel, from radon in homes, during space travel, or in areas of low-level contamination, including former sites of nuclear weapon production-and can encounter much higher radiation doses in contaminated areas such as Chernobyl or during radiotherapy (1-5). However, ionizing radiation induces a plethora of types of DNA damages (6), and the identity of specific lesions responsible for the biological effects of radiation remains uncertain. Understanding the long-term effects of low and high doses of ionizing radiation on living organisms requires identification of critical radiation-induced DNA lesions, measurement of their repairability, and determination of the consequences of misrepaired or unrepaired persistent lesions.Lethal and mutagenic effects of ionizing radiation result principally from incompletely or incorrectly repaired DNA lesions (7,8). Ionizing radiation induces high levels of isolated DNA lesions, including SSBs, damaged bases, and abasic sites, located at a distance from other damages (6). Such isolated damages are generally repaired efficiently, and their repair may be increased by priming ionizing radiation doses (9).Ionizing radiation also induces closely spaced lesions, including double-strand breaks (DSBs)-two or more SSBs on opposing strands within about 10-20 bp (10, 11)-and has been postulated to produce other c...

The critical regulatory function of nitric oxide (NO) in many physiologic processes is well established. However, in an aerobic aqueous environment NO is known to generate one or more reactive and potentially toxic nitrogen oxide (NOx) metabolites. This has led to the speculation that mechanisms must exist in vivo by which these reactive intermediates are detoxified, although the nature of these mechanisms has yet to be elucidated. This report demonstrates that among the primary bioorganic products of the reaction of cellular constituents with the intermediates of the NO/O2 reaction are S-nitrosothiol (S-NO) adducts. Anaerobic solutions of NO are not capable of nitrosating cysteine or glutathione, while S-NO adducts of these amino acids are readily formed in the presence of O2 and NO. Investigation of the kinetics for the formation of these S-NO adducts has revealed a rate equation of d[RSNO]/dt = kSNO[NO]2[O2], where kSNO = (6 +/- 2) x 10(6) M-2S-1, a value identical to that for the formation of reactive intermediates in the autoxidation of NO. Competition studies performed with a variety of amino acids, glutathione, and azide have shown that cysteine residues have an affinity for the NOx species that is 3 orders of magnitude greater than that of the nonsulfhydryl amino acids, and > 10(6) times greater than that of the exocyclic amino groups of DNA bases. The dipeptide alanyltyrosine reacts with the intermediates of the NO/O2 reaction with an affinity 150 times less than that of the sulfhydryl-containing compounds. Furthermore, Chinese hamster V79 lung fibroblasts depleted of glutathione display enhanced cytotoxicity on exposure to NO.(ABSTRACT TRUNCATED AT 250 WORDS)

The excision of modified bases from DNA by Escherichia coli endonuclease III was investigated. Modified bases were produced in DNA by exposure of dilute buffered solutions of DNA to ionizing radiation under oxic or anoxic conditions. The technique of gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify 16 pyrimidine- and purine-derived DNA lesions. DNA substrates were incubated either with the native enzyme or with the heat-inactivated enzyme. Subsequently, DNA was precipitated. Pellets were analyzed by GC/MS after hydrolysis and derivatization. Supernatant fractions were analyzed after derivatization without hydrolysis. The results provided unequivocal evidence for the excision by E. coli endonuclease III of a number of thymine- and cytosine-derived lesions from DNA. These were 5,6-dihydrothymine, 5-hydroxy-5-methylhydantoin, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydrouracil, alloxan, uracil glycol, and 5-hydroxy-6-hydrouracil. None of the purine-derived lesions was excised by endonuclease III. The present work extends the substrate specificity of E. coli endonuclease III to another thymine-derived and four cytosine-derived lesions. It is the first investigation of the substrate specificity of this repair enzyme in the context of a large number of pyrimidine- and purine-derived lesions in DNA.

Exocyclic DNA adducts are generated in cellular DNA by various industrial pollutants such as the carcinogen vinyl chloride and by endogenous products of lipid peroxidation. The etheno derivatives of purine and pyrimidine bases 3,N 4 -ethenocytosine (C), 1,N 6 -ethenoadenine (A), N 2 ,3-ethenoguanine, and 1,N 2 -ethenoguanine cause mutations. The A residues are excised by the human and the Escherichia coli 3-methyladenine-DNA glycosylases (ANPG and AlkA proteins, respectively), but the enzymes repairing C residues have not yet been described. We have identified two homologous proteins present in human cells and E. coli that remove C residues by a DNA glycosylase activity. The human enzyme is an activity of the mismatch-specific thymine-DNA glycosylase (hTDG). The bacterial enzyme is the double-stranded uracil-DNA glycosylase (dsUDG) that is the homologue of the hTDG. In addition to uracil and C-DNA glycosylase activity, the dsUDG protein repairs thymine in a G͞T mismatch. The fact that C is recognized and efficiently excised by the E. coli dsUDG and hTDG proteins in vitro suggests that these enzymes may be responsible for the repair of this mutagenic lesion in vivo and be important contributors to genetic stability.

The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A-T to G-C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Eschenchia cofi and mammalia cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase H coded for by the alA gene. This conclusion is based on the following facts: (s) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight,(ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coil akA-mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AIkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10-9 M as compared to 5 x 10-9 M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T. G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG proteinthe 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively-were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested.In DNA, hydrolytic deamination of adenine and guanine occurs under physiological conditions, yielding hypoxanthine and xanthine, respectively (1, 2). Hypoxanthine in DNA is potentially mutagenic since it can pair not only with thymine but also with cytosine and therefore would result in A-T to G-C transitions after DNA replication (3, 4). Hypoxanthine (Hyp) DNA glycosylase excises hypoxanthine from DNA containing dIMP residues in mammalian cells as well as in Escherichia coli (5-8).The potential mutagenic properties of dIMP residues have been substantiated by site-specific mutagenesis in vivo. A single inosine residue at a specific locus in a M13mp9 replicative form molecule constructed in vitro exhibits miscoding mutagenesis in E. coli (3). In mammalian cells, a synthetic c-Ha-ras gene containing hypoxanthine resulted in increased focus formation (9).During purification ofthe Hyp DNA glycosylase, we found that in E. coli Hyp DNA glycosylase activity was associated with the 3-methyladenine (3-MeAde) DNA glycosylase (TagII; AlkA protein) coded by alkA (10). The alIkA gene is inducible during the adaptive response (11, 12). Furthermore, we show that the human ANPG protein (13), the rat APDG protein (14), and the yeast MAG protein (15), all having 3-MeAde DNA glycosylase activity, also e...