Genomic consequences of DNA oxidation by peroxynitrite

Abstract

The radicals nitric oxide and superoxide are produced endogenously by activated macrophages and neutrophils and combine in a diffusion-limited reaction to form peroxynitrite, a powerful oxidizing and nitrating agent capable of damaging a variety of biomolecules, including DNA. Of the four nucleobases of DNA, guanine has the lowest oxidation potential and thus considerable attention has been given to the study of the oxidation of this base by peroxynitrite. A variety of DNA lesions are generated from guanine including guanidinohydantoin, spiroiminodihydantoin, oxaluric acid, urea, 2-aminoimidazolone, and 5-guanidino-4-nitroimidazole. In order to assess the biological significance and consequences of peroxynitrite-damaged DNA, it is essential that these lesions be characterized for their genotoxic and mutagenic potential. This work focuses on the elucidation of those properties. In the first study, the 2'-deoxynucleoside of 5-guanidino-4-nitroimidazole was chemically synthesized and incorporated into an oligonucleotide by the phosphoramidite method. In the second study, the genotoxic and mutational properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole were determined in wild-type uninduced and SOS-induced E. coli.

(cont.) In the third study, oxaluric acid was found to hydrolyze to urea in a reaction catalyzed by magnesium cations and bicarbonate. The genotoxic and mutational properties of oxaluric acid and urea were determined in wildtype uninduced and SOS-induced E. coli. In the fourth study, the genotoxic and mutational properties of guanidinohydantoin and spiroiminodihydantoin were determined in wild-type uninduced E. coli. In the fifth study, the genotoxic and mutational properties of guanidinohydantoin, spiroiminodihydantoin, oxaluric acid, urea, 2-aminoimidazolone, and 5-guanidino-4-nitroimidazole were determined in wild-type, polymerase II deficient, polymerase IV deficient, polymerase V deficient, and polymerase II / polymerase IV / polymerase V deficient E. coli under both uninduced and SOS-induced conditions. All of the lesions studied were potent sources of mutations in vivo. Guanidinohydantoin, spiroiminodihydantoin, urea, and 5-guanidino-4-nitroimidazole were significant blocks to replication and were strongly dependent upon induction of the SOS system. Polymerase V was responsible for the majority of translesion synthesis.