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

Research output: Contribution to journalArticle

204 Citations (Scopus)

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

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Endonucleases
Escherichia coli
Purification
Enzymes
Oligonucleotides
Catalytic Domain
Proteins
Nucleotides
DNA Glycosylases
Deoxyribose
Amino Acids
Pyrimidines
Lyases
Schiff Bases
Deoxyribonuclease I
DNA
Glutathione Transferase
HeLa Cells
Sequence Analysis
Cytoplasm

ASJC Scopus subject areas

  • Biochemistry

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Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III. / Ikeda, Shogo; Biswas, Tapan; Roy, Rabindra; Izumi, Tadahide; Boldogh, Istvan; Kurosky, Alexander; Sarker, Altaf H.; Seki, Shuji; Mitra, Sankar.

In: Journal of Biological Chemistry, Vol. 273, No. 34, 21.08.1998, p. 21585-21593.

Research output: Contribution to journalArticle

Ikeda, S, Biswas, T, Roy, R, Izumi, T, Boldogh, I, Kurosky, A, Sarker, AH, Seki, S & Mitra, S 1998, 'Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III', Journal of Biological Chemistry, vol. 273, no. 34, pp. 21585-21593. https://doi.org/10.1074/jbc.273.34.21585
Ikeda, Shogo ; Biswas, Tapan ; Roy, Rabindra ; Izumi, Tadahide ; Boldogh, Istvan ; Kurosky, Alexander ; Sarker, Altaf H. ; Seki, Shuji ; Mitra, Sankar. / Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 34. pp. 21585-21593.
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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.",
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