Characterization of Escherichia coli endonuclease VIII

Dongyan Jiang, Zafer Hatahet, Robert J. Melamede, Yoke Wah Kow, Susan S. Wallace

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Abstract

Escherichia coli endonuclease VIII (endo VIII) was identified as an enzyme that, like endonuclease III (endo III), removes radiolysis products of thymine including thymine glycol, dihydrothymine, β-ureidoisobutyric acid, and urea from double-stranded plasmid or phage DNA and cleaves the DNA strand at abasic (AP) sites (Melamede, R. J., Hatahet, Z., Kow, Y. W., Ide., H., and Wallace, S. S. (1994) Biochemistry 33, 1255-1264). Using apparently homogeneous endo VIII protein, we now show that endo VIII removes from double-stranded oligodeoxyribonucleotides the stable oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil. Endo VIII cleaved the damage-containing DNA strand by β,δ-elimination as does formamidopyrimidine DNA glycosylase (Fpg). Like Fpg, endo VIII also excised the 5'-terminal deoxyribose phosphate from an endonuclease IV (endo IV) pre-incised AP site. Thus, in addition to amine acid sequence homology (Jiang, D., Hatahet, Z., Blaisdell, J., Melamede, R. J., and Wallace, S.S. (1997) J. Bacteriol. 179, 3773-3782), endo VIII shares a number of catalytic properties with Fpg. In addition, endo VIII specifically bound to oligodeoxynucleotides containing a reduced AP site with a stoichiometry of 1:1 for protein to DNA with an apparent equilibrium dissociation constant of 3.9 nM. Like Fpg and endo III, the DNase I footprint was small with contact sites primarily on the damage- containing strand; unlike Fpg and endo III, the DNA binding of endo VIII to DNA was asymmetriC, 3' to the reduced AP site.

Original languageEnglish (US)
Pages (from-to)32230-32239
Number of pages10
JournalJournal of Biological Chemistry
Volume272
Issue number51
DOIs
StatePublished - Dec 19 1997

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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