George W. Teebor

The Repairability of Oxidative Free Radical Mediated Damage to DNA: A Review

Many DNA repair enzyme activities are present in both prokaryotic and eukaryotic organisms. Among these are DNA exo- and endonucleases and DNA glycosylases which remove oxidatively damaged portions of the DNA molecule, thereby initiating excision-repair. The existence of these enzymes may be taken as evidence that cellular DNA is continuously subject to endogenous oxidative stress. Many of the lesions introduced by ionizing and ultraviolet radiation are identical to those introduced into DNA by reactive oxygen species generated by activated white cells, and are substrates for the repair enzymes. The chemical nature of the lesions, their biologic effects, and the mechanism of their repairability are described.

Cloning and expression of the cDNA encoding the human homologue of the DNA repair enzyme, Escherichia coli endonuclease III

We previously purified a bovine pyrimidine hydrate-thymine 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 NaCNBH3, 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.

Targeted deletion of mNth1 reveals a novel DNA repair enzyme activity.

ABSTRACT 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.

5-hydroxymethylcytosine DNA glycosylase activity in mammalian tissue

The enzymatic release of 5-hydroxymethylcytosine from T2 bacteriophage DNA was effected by an extract of calf thymus. Like the previously described 5-hydroxymethyluracil DNA glycosylase, 5-hydroxymethylcytosine DNA glycosylase was not detectable in bacterial extracts. The phylogenetic distribution of these activities indicates that their primary function is the maintenance of methylcytosine residues in differentiated tissue.

Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA ☆

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1-/-Neil1-/- mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1-/- and Neil1-/-. The pulmonary tumors contained, exclusively, activating GGT-->GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT-->GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1-/- and Neil1-/-Nth1-/- mice. The high incidence of tumors in our Nth1-/-Neil1-/- mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.

The Initiation of DNA Excision-Repair

Publisher Summary The excision-repair of Deoxyribonucleic acid (DNA) is an enzymatically mediated process by which modified bases or fragments of bases are removed from cellular DNA together with adjoining normal nucleotides. This segment of excised DNA is then resynthesized through the action of a DNA polymerase using the opposite strand as a template. The last phosphodiester bond is sealed through the action of DNA ligase. The importance of this process to human well being through the prevention of cancer was emphasized by the demonstration that in contrast to normal cells, cells of individuals with xeroderma pigmentosum (XP)—a hereditary disease—did not remove ultraviolet (UV) radiation-induced pyrimidine dimers from their nuclear DNA. The importance of the DNA repair system is emphasized by the demonstration that alkylating agents caused the onset of DNA repair synthesis when applied to human (HeLa) cells in culture. Subsequently it became evident that the DNA repair system could affect the removal of many types of modified bases in addition to pyrimidine dimmers and that DNA repair constituted a general defense mechanism against a wide variety of potentially mutagenic and/or carcinogenic DNA modifications.

Inhibition of thymine dimer excision by the phorbol ester, phorbol myristate acetate☆

Abstract The effect of the promoting agent, phorbol myristate acetate, on repair of UV-induced damage in HeLa cells was studied. The agent decreased survival and subsequent colony-forming ability of irradiated cells and inhibited removal of UV-induced thymine-containing dimers from DNA of irradiated cells.

Normal endonuclease activities for damaged DNA during hepatocarcinogenesis.

Two endonuclease activities in rat liver for damaged DNA were assayed. Double-stranded, covalently closed DNA from phage PM2 was damaged by either ultraviolet irradiation or by heating at acid pH, and used as substrate for endonucleases specific for ultraviolet DNA damage and for DNA apurinic sites, respectively. The levels of both enzyme activities in livers of normal rats were compared to levels in livers of rats fed N-2-acetylaminofluorene. At critical stages of the carcinogenic regimen levels of both endonuclease activities were normal. This, together with other data, suggests that depression of excision-repair of DNA damage does not take place during experimental carcinogenesis.

Depression of Tryptophan Pyrrolase Induction in Regenerating Rat Liver.

Summary The induction of tryptophan pyrrolase by hydrocortisone has been studied in the regenerating rat liver. An initial depression of response, presumably related to the operative procedure, was observed in both sham operated and partially hepatectomized animals. In partially hepatectomized animals, a second depression was observed during that period in which DNA synthesis has been shown to occur. It has been suggested that alterations in enzyme control mechanisms, such as the failure of tryptophan pyrrolase induction by cortisone in minimum deviation hepatomas, may be a fundamental biochemical alteration in tumors. This study shows that a depressed response to hormonal enzyme induction is found in non-neoplastic hepatic cells preparing to divide.

Hydrocortisone Induction of Tyrosine Transaminase in Regenerating Rat Liver

THE “minimal deviation” hepatomas—well differentiated rat hepatocellular carcinomas—demonstrate many abnormal enzyme regulatory mechanisms1. It has been suggested that these alterations are critical in neoplastic transformation2. The corticosteroid induction of the hepatic enzymes tryptophan pyrrolase (TP) and tyrosine transaminase (TT) has been extensively studied in these tumours1. Administration of corticosteroid causes a considerable increase in the activities of TP and TT in the normal adult rat liver within 4–5 h (refs. 3 and 4). In hepatomas, however, there is little or no increase in TP activity after administration of corticosteroid, and baseline enzyme activity is usually depressed1.