Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III

Shogo Ikeda, Tapan Biswas, Rabindra Roy, Tadahide Izumi, Istvan Boldogh, Alexander Kurosky, Altaf H. Sarker, Shuji Seki, Sankar Mitra

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208 Scopus citations

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 NH2 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 x 104 and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, the K(m) was 47 nm, and k(cat) was ~0.6/min, independent of whether DHU paired with G or A. The enzyme carries out β-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 Gin 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.

Original languageEnglish (US)
Pages (from-to)21585-21593
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number34
DOIs
StatePublished - Aug 21 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Ikeda, S., Biswas, T., Roy, R., Izumi, T., Boldogh, I., Kurosky, A., Sarker, A. H., Seki, S., & Mitra, S. (1998). Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III. Journal of Biological Chemistry, 273(34), 21585-21593. https://doi.org/10.1074/jbc.273.34.21585