Purification and Characterization of Human NTH1, a Homolog ofEscherichia coli Endonuclease III

Abstract

The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH 2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 ؋ 10 4 and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, the K m was 47 nM, and k cat was ϳ0.6/min, independent of whether DHU paired with G or A. The enzyme carries out ␤-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptideoligonucleotide adduct. Furthermore, replacing Lys-212 with Gln inactivated the enzyme. However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.Reactive oxygen species are generated as by-products of oxidative phosphorylation or by ionizing radiation and induce extensive base damage that is mainly repaired via the base excision repair (BER) 1 pathway. This repair is initiated by removal of the damaged base, catalyzed by a DNA glycosylase (1-3). There are two classes of DNA glycosylases with distinct substrate specificities: the monofunctional simple glycosylase and the glycosylase with associated AP lyase activity. All oxidized base lesions are removed from DNA by DNA glycosylase/AP lyases, which not only catalyze removal of the base lesion but also cause strand cleavage via ␤-elimination. The Escherichia coli endonuclease III (Nth) recognizes a wide range of oxidized pyrimidine derivatives, including ring-saturated and ring-fragmented derivatives such as thymine glycol (Tg), 5-hydroxycytosine, 5,6-dihydrouracil (DHU), and urea (1, 3-6). This enzyme has been well conserved from E. coli to the humans (7,8). On the other hand, oxidized purine lesions are also repaired by DNA glycosylase/AP lyases, i.e. Mut M (Fpg) of E. coli or OGG of eukaryotes (yeast and mammals), which do not share extensive sequence similarity. Furthermore, unlike these e...

Detection of cancer‐related gene expression profiles in severe cervical neoplasia

“…This enzyme specifically cleaves oxidatively damaged pyrimidines in DNA. 45,46 Lack of this activity in cervical cancer could contribute to further genomic instability. Interestingly, 2 of the cancer-related genes in clusters 1A and 7A are regulated in a way which is not consistent with data from the literature.…”

Effects of base excision repair proteins on mutagenesis by 8-oxo-7,8-dihydroguanine (8-hydroxyguanine) paired with cytosine and adenine

“…It has been reported that the overexpression of OGG1 and MUTYH suppress mutations by 8-oxo-Gua in cultured cells [30,31]. The NTH1 and NEIL1 proteins are homologues of Escherichia coli Nth and Nei, DNA glycosylases that are specific for oxidized pyrimidine bases [32][33][34][35][36][37][38][39]. The observation that NTH1 can be efficiently trapped to an oligonucleotide duplex containing 8-oxo-Gua:C by treatment with NaBH 4 , suggests that NTH1 binds to 8-oxo-Gua:C DNA [40].…”

DNA Recombination-Initiation Plays a Role in the Extremely Biased Inheritance of Yeast [rho−] Mitochondrial DNA That Contains the Replication Origin ori5

“…The Ntg1 protein exists primarily in mitochondria, although some is found in the nucleus as well (55). Ntg1 is required for the repair of oxidative DNA damage in vivo, and it shares amino acid sequence similarity with E. coli endonuclease III and the human Nth1 protein (21). Ntg1 has DNA repair glycosylase and apurinic/apyrimidinic (AP) lyase activities.…”