Modulation of the turnover of formamidopyrimidine DNA glycosylase

Michael B. Harbut, Michael Meador, M. L. Dodson, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

In recent years, significant progress has been made in determining the catalytic mechanisms by which base excision repair (BER) DNA glycosylases and glycosylase-abasic site (AP) lyases cleave the glycosyl bond. While these investigations have identified active site residues and active site architectures, few investigations have analyzed postincision turnover events. Previously, we identified a critical residue (His16) in the T4-pyrimidine dimer glycosylase (T4-Pdg) that, when mutated, interferes with enzyme turnover [Meador et al. (2004) J. Biol. Chem. 279, 3348-3353]. To test whether comparable residues and mechanisms might be operative for other BER glycosylase:AP-lyases, molecular modeling studies were conducted comparing the active site regions of T4-Pdg and the Escherichia coli formamidopyrimidine DNA glycosylase (Fpg). These analyses revealed that His71 in Fpg might perform a similar function to His16 in T4-Pdg. Site-directed mutagenesis of the Fpg gene and analyses of the reaction mechanism of the mutant enzyme revealed that the H71A enzyme retained activity on a DNA substrate containing an 8-oxo-7,8-dihydroguanine (8-oxoG) opposite cytosine and DNA containing an AP site. The H71A Fpg mutant was severely compromised in enzyme turnover on the 8-oxoG-C substrate but had turnover rates comparable to that of wild-type Fpg on AP-containing DNA. The similar mutant phenotypes for these two enzymes, despite a complete lack of structural or sequence homology between them, suggest a common mechanism for the rate-limiting step catalyzed by BER glycosylase:AP-lyases.

Original languageEnglish (US)
Pages (from-to)7341-7346
Number of pages6
JournalBiochemistry
Volume45
Issue number23
DOIs
StatePublished - Jun 13 2006

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DNA-Formamidopyrimidine Glycosylase
DNA-(Apurinic or Apyrimidinic Site) Lyase
Pyrimidine Dimers
Modulation
DNA Repair
Enzymes
Catalytic Domain
Repair
DNA
DNA Glycosylases
Mutagenesis
Molecular modeling
Cytosine
Enzyme activity
Substrates
Sequence Homology
Site-Directed Mutagenesis
Escherichia coli
Genes
Phenotype

ASJC Scopus subject areas

  • Biochemistry

Cite this

Modulation of the turnover of formamidopyrimidine DNA glycosylase. / Harbut, Michael B.; Meador, Michael; Dodson, M. L.; Lloyd, Robert (Stephen).

In: Biochemistry, Vol. 45, No. 23, 13.06.2006, p. 7341-7346.

Research output: Contribution to journalArticle

Harbut, Michael B. ; Meador, Michael ; Dodson, M. L. ; Lloyd, Robert (Stephen). / Modulation of the turnover of formamidopyrimidine DNA glycosylase. In: Biochemistry. 2006 ; Vol. 45, No. 23. pp. 7341-7346.
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