Cloning and characterization of a mouse homologue (mNthl1) of Escherichia coli endonuclease III

Altaf H. Sarker, Shogo Ikeda, Hironobu Nakano, Hiroaki Terato, Hiroshi Ide, Kotoe Imai, Kosuke Akiyama, Ken Tsutsui, Zhang Bo, Kihei Kubo, Kazuo Yamamoto, Akira Yasui, Michihiro C. Yoshida, Shuji Seki

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

Endonuclease III (endoIII; nth gene product) of Escherichia coli is known to be a DNA repair enzyme having a relatively broad specificity for damaged pyrimidine bases of DNA. Here, we describe the cloning and characterization of the cDNA and the gene for a mouse homologue (mNthl1/mNth1) of endoIII. The cDNA was cloned from a mouse T-cell cDNA library with a probe prepared by PCR using the library and specific PCR primers synthesized based on the reported information of partial amino acid sequences of bovine NTHL1/NTH1 and of EST Data Bases. The cDNA is 1025 nucleotides long and encodes a protein consisting of 300 amino acids with a predicted molecular mass of 33.6 kDa. The amino acid sequence exhibits significant homologies to those of endoIII and its prokaryotic and eukaryotic homologues. The recombinant mNthl1 with a hexahistidine tag was overexpressed in a nth::cm(r) nei::Km(r) double mutant of E. coli, and purified to apparent homogeneity. The enzyme showed thymine glycol DNA glycosylase, urea DNA glycosylase and AP lyase activities. Northern blot analysis indicated that mNthl1 mRNA is about 1 kb and is expressed ubiquitously. A 15 kb DNA fragment containing the mNthl1 gene was cloned from a mouse genomic library and sequenced. The gene consists of six exons and five introns spanning 6.09 kb. The sequenced 5' flanking region lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors such as Ets, Sp1, AP-1 and AP-2. The mNthl1 gene was shown to lie immediately adjacent to the tuberous sclerosis 2 (Tsc2) gene in a 5'-to-5' orientation by sequence analysis and was assigned to chromosome 17A3 by in situ hybridization.

Original languageEnglish
Pages (from-to)761-774
Number of pages14
JournalJournal of Molecular Biology
Volume282
Issue number4
DOIs
Publication statusPublished - Oct 2 1998
Externally publishedYes

Keywords

  • AP lyase
  • DNA glycosylase
  • Mouse Nthl1 gene
  • Thymine glycol DNA glycosylase
  • Urea DNA glycosylase

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Fingerprint Dive into the research topics of 'Cloning and characterization of a mouse homologue (mNthl1) of Escherichia coli endonuclease III'. Together they form a unique fingerprint.

  • Cite this

    Sarker, A. H., Ikeda, S., Nakano, H., Terato, H., Ide, H., Imai, K., Akiyama, K., Tsutsui, K., Bo, Z., Kubo, K., Yamamoto, K., Yasui, A., Yoshida, M. C., & Seki, S. (1998). Cloning and characterization of a mouse homologue (mNthl1) of Escherichia coli endonuclease III. Journal of Molecular Biology, 282(4), 761-774. https://doi.org/10.1006/jmbi.1998.2042