MISC

1998年8月

Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III - Direct identification of Lys-212 as the active nucleophilic residue

JOURNAL OF BIOLOGICAL CHEMISTRY
  • S Ikeda
  • ,
  • T Biswas
  • ,
  • R Roy
  • ,
  • T Izumi
  • ,
  • Boldogh, I
  • ,
  • A Kurosky
  • ,
  • AH Sarker
  • ,
  • S Seki
  • ,
  • S Mitra

273
34
開始ページ
21585
終了ページ
21593
記述言語
英語
掲載種別
DOI
10.1074/jbc.273.34.21585
出版者・発行元
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

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, 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 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 similar to 0.6/min, independent of whether DHU paired with G or A. The enzyme carries out p-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 peptide-oligonucleotide 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.

Web of Science ® 被引用回数 : 201

リンク情報
DOI
https://doi.org/10.1074/jbc.273.34.21585
CiNii Articles
http://ci.nii.ac.jp/naid/80010507956
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/9705289
Web of Science
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000075492600023&DestApp=WOS_CPL

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