title: Modulation of the turnover of formamidopyrimidine DNA glycosylase.
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.
authors.affiliation: Center for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, L606, Portland, Oregon 97239-3098, USA.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Harbut
authors.forename: Michael
authors.initials: M
authors.lastname: Meador
authors.forename: M L
authors.initials: ML
authors.lastname: Dodson
authors.forename: R S
authors.initials: RS
authors.lastname: Lloyd
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D043382:DNA-Formamidopyrimidine Glycosylase