The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7,8-dihydroguanine). In bacteria and Saccharomyces cerevisiae, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient E. coli mutM mutY strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen.DNA bases are susceptible to damage in vivo by lipid peroxidation and other endogenous cellular processes that generate reactive oxygen species, as well as by low-level ionizing radiation and oxidative effects of near-ultraviolet (320-380 nm) light. At present there is no direct method to measure the in vivo rate of generation of such endogenous oxidative DNA damage, so its relative importance remains uncertain; recent estimates for mammalian cells have varied from a few hundred to 4 ϫ 10 5 oxidatively altered DNA base residues being introduced per day in each cell (1, 2). Major expected variations between different cell types and subcellular compartments further complicate the issue. The existence of specific DNA repair enzymes that remove oxidized bases from DNA strongly indicates that this form of endogenous damage is physiologically relevant. DNA pyrimidine residues can be oxidized to noncoding ring-saturated and ring-fragmented derivatives (e.g., thymine glycol, cytosine glycol, and Nsubstituted urea). A DNA glycosylase with an associated apurinic͞apyrimidinic (AP) lyase activity can initiate DNA repair of such lesions, and an Escherichia coli enzyme of this type, endonuclease III, has been extensively characterized (3). A human homolog of E. coli endonuclease III exists, and the cDNA encoding the enzyme has been cloned and functionally expressed (4). In purines, the imidazole ring is more susceptible to oxidative damage than the pyrimidine ring, so DNA purine residues are converted to the ring-saturated and ringfragmented derivatives, 8-hydroxyguanine and formamidopyrimidines. A distinct DNA glycosylase is present to remove such damage, and the E. coli enzyme excises both types of lesions at similar rates (5). This formamidopyrimidine-DNA glycosylase (Fpg, MutM) was initially discovered by its action on the cytotoxic ring-fragmented purines (6, 7), but the most important substrate for the enzyme appears to be the mutagenic base 8-hydroxyguanine, which usually occurs in its keto form, 8-oxo-7,8-dihydroguanine (8-oxoG) (8).

Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in Escherichia coli that catalyzes the excision of pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic͞apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (TSC2) in chromosome region 16p13.3, we identified a gene, OCTS3, that encodes a 1-kb transcript. Analysis of OCTS3 cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with E. coli endonuclease III and a related enzyme from Schizosaccharomyces pombe, including a conserved active site region and an iron͞sulfur domain. The product of the OCTS3 gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in E. coli and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron͞sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic͞ apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of E. coli endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells.Reactive oxygen species generated either as a by-product of cellular respiration or by ionizing radiation and other oxidizing agents can cause extensive damage to DNA bases and disrupt the phosphodiester backbone. Unrepaired oxygen radicalderived DNA lesions have been implicated as a causative factor in both cancer and aging (1, 2). Repair of most forms of oxidative DNA damage is mediated via the base excisionrepair pathway (3, 4), which has been well characterized in Escherichia coli. One of the key components of the base excision-repair pathway in that organism is endonuclease III, the product of the nth gene (5). The crystal structure of this iron͞sulfur protein has been determined recently at 1.85 Å resolution (6, 7).Although originally isolated as an activity that incised at certain sites of DNA damage generated by ultraviolet (UV) irradiation in a Mg 2ϩ -independent reaction (8), endonuclease III was subsequently shown to act primarily as a DNA glycosylase that hydrolyses the N-glycosyl bond linking damaged pyrimidines to the DNA backbone. Numerous different substrates for endonuclease III have been identified representing saturated, opened, or contracted pyrimidine rings, such as thymine and cytosine glycol, urea, and N-substituted urea, 5-hydroxy-5-methylhydantoin, and 5,6-dihydrothymine (9-11). The originally observed DNA nicking activity associated with endonuclease III was subsequently shown to result not from hydrolytic cleavage of phosphodiester bonds, but from ␤-e...

The Escherichia coli endonuclease III (Nth-Eco) protein is involved in the removal of damaged pyrimidine residues from DNA by base excision repair. It is an iron-sulphur enzyme possessing both DNA glycosylase and apurinic/apyrimidinic lyase activities. A database homology search identified an open reading frame in genomic sequences of Schizosaccharomyces pombe which encodes a protein highly similar to Nth-Eco. The gene has been subcloned in an expression vector and the protein purified to apparent homogeneity. The S.pombe Nth homologue (Nth-Spo) is a 40.2 kDa protein of 355 amino acids. Nth-Spo possesses glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polymers. The eukaryotic protein removes urea more efficiently than the prokaryotic enzyme, whereas its efficiency in excising thymine glycol is lower. A nicking assay was used to show that the enzyme also exhibits an AP lyase activity on UV- and gamma-irradiated DNA substrates. These findings show that Nth protein is structurally and functionally conserved from bacteria to fission yeast.