Step-by-step mechanism of DNA damage recognition by human 8-oxoguanine DNA glycosylase

Biochim Biophys Acta. 2014 Jan;1840(1):387-95. doi: 10.1016/j.bbagen.2013.09.035. Epub 2013 Oct 3.

Abstract

Background: Extensive structural studies of human DNA glycosylase hOGG1 have revealed essential conformational changes of the enzyme. However, at present there is little information about the time scale of the rearrangements of the protein structure as well as the dynamic behavior of individual amino acids.

Methods: Using pre-steady-state kinetic analysis with Trp and 2-aminopurine fluorescence detection the conformational dynamics of hOGG1 wild-type (WT) and mutants Y203W, Y203A, H270W, F45W, F319W and K249Q as well as DNA-substrates was examined.

Results: The roles of catalytically important amino acids F45, Y203, K249, H270, and F319 in the hOGG1 enzymatic pathway and their involvement in the step-by-step mechanism of oxidative DNA lesion recognition and catalysis were elucidated.

Conclusions: The results show that Tyr-203 participates in the initial steps of the lesion site recognition. The interaction of the His-270 residue with the oxoG base plays a key role in the insertion of the damaged base into the active site. Lys-249 participates not only in the catalytic stages but also in the processes of local duplex distortion and flipping out of the oxoG residue. Non-damaged DNA does not form a stable complex with hOGG1, although a complex with a flipped out guanine base can be formed transiently.

General significance: The kinetic data obtained in this study significantly improves our understanding of the molecular mechanism of lesion recognition by hOGG1.

Keywords: (3-hydroxytetrahydrofuran-2-yl)methyl phosphate; 2-aPu; 2-aminopurine; 7,8-dihydro-8-oxoguanine; BER; Base excision repair; Conformational dynamics; DNA glycosylase; Enzyme kinetics; Human 8-oxoguanine DNA glycosylase; ODN; THF; base excision repair; oligodeoxyribonucleotide; oxoG.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Catalysis
  • DNA Damage / genetics*
  • DNA Glycosylases / genetics
  • DNA Glycosylases / metabolism*
  • DNA Repair
  • Guanine / analogs & derivatives*
  • Guanine / metabolism
  • Humans
  • Kinetics
  • Molecular Conformation
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism*
  • Mutation / genetics
  • Spectrometry, Fluorescence

Substances

  • Mutant Proteins
  • 8-hydroxyguanine
  • Guanine
  • DNA Glycosylases
  • oxoguanine glycosylase 1, human