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...