We previously purified a bovine pyrimidine hydratethymine glycol DNA glycosylase/AP lyase. The amino acid sequence of tryptic bovine peptides was homologous to Escherichia coli endonuclease III, theoretical proteins of Saccharomyces cerevisiae and Caenorhabditis elegans, and the translated sequences of rat and human 3 -expressed sequence tags (3 -ESTs) (Hilbert, T. P., Boorstein, R. J., Kung, H. C., Bolton, P. H., Xing, D., Cunningham, R. P., Teebor, G. W. (1996) Biochemistry 35, 2505-2511). Now the human 3 -EST was used to isolate the cDNA clone encoding the human enzyme, which, when expressed as a GST-fusion protein, demonstrated thymine glycol-DNA glycosylase activity and, after incubation with NaCNBH 3 , became irreversibly cross-linked to a thymine glycol-containing oligodeoxynucleotide, a reaction characteristic of DNA glycosylase/AP lyases. Amino acids within the active site, DNA binding domains, and [4Fe-4S] cluster of endonuclease III are conserved in the human enzyme. The gene for the human enzyme was localized to chromosome 16p13.2-.3. Genomic sequences encoding putative endonuclease III homologues are present in bacteria, archeons, and eukaryotes. The ubiquitous distribution of endonuclease III-like proteins suggests that the 5,6-double bond of pyrimidines is subject to oxidation, reduction, and/or hydration in the DNA of organisms of all biologic domains and that the resulting modified pyrimidines are deleterious to the organism.When a pyrimidine residue in cellular DNA becomes modified by oxidation, reduction, or hydration of its 5,6-double bond, repair is initiated by a DNA-glycosylase activity that cleaves the N-glycosyl bond of the damaged residue, releasing the modified base and creating an abasic (AP) site in the DNA backbone. Such DNA glycosylase activities have been identified in bacteria, yeast, and mammalian species (1-8) The first such enzyme described was Escherichia coli endonuclease III, which was identified not on the basis of its DNA glycosylase activity, but rather because it nicked UV-irradiated DNA (9). For this reason it was termed an endonuclease, because it was thought that nicking resulted from enzyme-catalyzed hydrolysis of internucleotide phosphodiester bonds at sites of DNA damage. It has since been determined that the enzyme nicks DNA not via hydrolysis, but by catalyzing ␤-elimination of the 3Ј-phosphate group at the AP site formed as a result of the enzyme's DNA glycosylase activity (10 -12). The modified base that was enzymatically released from UV-irradiated DNA proved to be cytosine and/or uracil hydrate (8). Enzymes that effect base release together with strand cleavage via ␤-elimination are now termed DNA glycosylase/AP lyases and, in addition to endonuclease III, include the Fpg protein of E. coli (13), the OGG1 protein of Saccharomyces cerevisiae (14, 15), and T4 endonuclease V (16).DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) enzyme-substrate intermediates (17). Such enzyme-substrate intermediates can be chemically reduced to stable seco...

DNA N-glycosylase/AP (apurinic/apyrimidinic) lyase enzymes of the endonuclease III family (nth in Escherichia coli and Nth1 in mammalian organisms) initiate DNA base excision repair of oxidized ring saturated pyrimidine residues. We generated a null mouse (mNth1 ؊/؊ ) by gene targeting. After almost 2 years, such mice exhibited no overt abnormalities. Tissues of mNth1 ؊/؊ mice contained an enzymatic activity which cleaved DNA at sites of oxidized thymine residues (thymine glycol [Tg]). The activity was greater when Tg was paired with G than with A. This is in contrast to Nth1, which is more active against Tg:A pairs than Tg:G pairs. We suggest that there is a back-up mammalian repair activity which attacks Tg:G pairs with much greater efficiency than Tg:A pairs. The significance of this activity may relate to repair of oxidized 5-methyl cytosine residues (5meCyt). It was shown previously (S. Zuo, R. J. Boorstein, and G. W. Teebor, Nucleic Acids Res. 23:3239-3243, 1995) that both ionizing radiation and chemical oxidation yielded Tg from 5meCyt residues in DNA. Thus, this previously undescribed, and hence novel, back-up enzyme activity may function to repair oxidized 5meCyt residues in DNA while also being sufficient to compensate for the loss of Nth1 in the mutant mice, thereby explaining the noninformative phenotype.Escherichia coli endonuclease III and its eukaryotic homologs initiate the process of DNA base excision repair (BER) of C-5, C-6 ring saturated pyrimidines (31). The endonuclease III-like enzymes are bifunctional enzymes which first effect release of the damaged base from the DNA backbone and then effect strand cleavage at the resulting abasic (apurinic/apyrimidinic [AP]) site via ␤-elimination (19,20,32). Among the modified bases recognized by endonuclease III-like enzymes are 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol [Tg]), cytosine glycol, and cytosine hydrate. Tg is an oxidative product formed in DNA in vitro by reaction with chemical oxidizing agents such as KMnO 4 and OsO 4 as well as via the indirect action of ionizing radiation under aerobic conditions. In cellular DNA, Tg is formed as a product of exposure to aerobic ionizing radiation and other oxidative stresses such as H 2 O 2 in the presence of added Fe 2ϩ . Ionizing irradiation under anaerobic conditions yields the reduced derivative 5,6-dihydrothymine (4, 9, 28). Exposure of cytosine to oxidative stress yields cytosine glycol, which is in equilibrium with its dehydrated form, 5-hydroxycytosine (5ohCyt). 5ohCyt is prone to deamination yielding 5ohUra. In addition to inducing formation of dimeric photoproducts in DNA, UV irradiation also induces hydration of the 5,6 double bond of pyrimidines, primarily forming 5,6 dihydro-6-hydroxy cytosine (cytosine hydrate) (2, 3). Like 5ohCyt, cytosine hydrate is prone to deamination, which yields uracil hydrate (26).Our laboratory first documented the existence of a family of endonuclease III homologs in several species (11) and isolated the cDNA of the human homolog of endonuclease III (hNth...

Chinese hamster lung fibroblast V79 cells have been widely used in studies of DNA damage and DNA repair. Since the p53 gene is involved in normal responses to DNA damage, we have analyzed the molecular genetics and functional status of p53 in V79 cells and primary Chinese hamster embryonic fibroblast (CHEF) cells. The coding product of the p53 gene in CHEF cells was 76 and 75% homologous to human and mouse p53 respectively, and was 95% homologous to the Syrian hamster cells. The V79 p53 sequence contained two point mutations located within a presumed DNA binding domain, as compared with the CHEF cells. Additional immunocytochemical and molecular studies confirmed that the p53 protein in V79 cells was mutated and nonfunctional. Our results indicate that caution should be used in interpreting studies of DNA damage, DNA repair and apoptosis in V79 cells.