Robert J. Boorstein

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.

DNA Base Excision Repair Stimulates Poly(ADP-Ribose) Synthesis

3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthesis, is toxic to cells which incorporate and repair 5-hydroxymethyl-2’-deoxyuridine (HmdUrd). To demonstrate that incorporation and repair of HmdUrd stimulates synthesis of poly(ADP-ribose) from intracellular NAD, V79 hamster cells were treated with HmdUrd and intracellular NAD levels were measured. HmdUrd is incorporated into DNA as a thymidine analogue resulting in extensive substitution of thymine residues with 5hydroxymethyluracil (HmUra) residues. These HmUra residues are then subject to excision by action of HmUra-DNA glycosylase. Following HmdUrd treatment, NAD levels fell markedly (80-90%) within four hours and remained low for at least 10 hours before partially recovering by 24 hours. The degree of NAD lowering was dose dependent and paralleled net HmdUrd incorporation. The NAD lowering was largely prevented by concurrent treatment with 4 mM 3AB. No effects on NAD levels were seen following treatment with dThd or Brdurd, indicating that the effects on NAD result from incorporation of a nucleoside which puts large amounts of a repairable modification into DNA. To confirm that both incorporation and repair are necessary for the effects of HmdUrd on intracellular NAD, mutant cell strains derived from V79 cells deficient in either the ability to incorporate or to repair HmdUrd were examined. HmdUrd did not produce NAD lowering in either mutant cell strain. These results demonstrate that poly(ADP-ribose) synthesis can result directly and exclusively from repair of DNA base modifications.

5-Hydroxymethyluracil in Cellular DNA is Repaired and Sensitizes Cells to Inhibitors of Poly(ADP-Ribose) Synthesis

5-Hydroxymethyluracil (HmUra) can be formed in DNA from thymidine by the action of ionizing radiation or by activated leukocytes (1–7). HmUra can be removed from DNA in vitro through the action of HmUra-DNA glycosylase (8,9) The repairability of HmUra suggests that it is deleterious to cells but the nature of its effects on cell function are uncertain. HmUra can also be introduced into cellular DNA as a result of the incorporation of the nucleoside, 5-hydroxymethy1–2’-deoxyuridine (HmdUrd) (10–12). HmUrd has been reported to be toxic to cells in culture and to animals (11,13,14) but the mechanism of this toxicity is unknown.