Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases

Abstract

Formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) share an overall common three-dimensional structure and primary amino acid sequence in conserved structural motifs but have different substrate specificities, with bacterial Fpg proteins recognizing formamidopyrimidines, 8-oxoguanine (8-oxoG) and its oxidation products guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) and bacterial Nei proteins recognizing primarily damaged pyrimidines. In addition to bacteria, Fpg has also been found in plants, while Nei is sparsely distributed among the prokaryotes and eukaryotes. Phylogenetic analysis of Fpg and Nei DNA glycosylases demonstrated, with 95% bootstrap support, a clade containing exclusively sequences from plants and fungi. Members of this clade exhibit sequence features closer to bacterial Fpg proteins than to any protein designated as Nei based on biochemical studies. The Candida albicans (Cal) Fpg DNA glycosylase and a previously studied Arabidopsis thaliana (Ath) Fpg DNA glycosylase were expressed, purified and characterized. In oligodeoxynucleotides, the preferred glycosylase substrates for both enzymes were Gh and Sp, the oxidation products of 8-oxoG, with the best substrate being a site of base loss. GC/MS analysis of bases released from γ-irradiated DNA show FapyAde and FapyGua to be excellent substrates as well. Studies carried out with oligodeoxynucleotide substrates demonstrate that both enzymes discriminated against A opposite the base lesion, characteristic of Fpg glycosylases. Single turnover kinetics with oligodeoxynucleotides showed that the plant and fungal glycosylases were most active on Gh and Sp, less active on oxidized pyrimidines and exhibited very little or no activity on 8-oxoG. Surprisingly, the activity of AthFpg1 on an AP site opposite a G was extremely robust with a k obs of over 2500 min −1 .

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo

“…We therefore used a "pooled assay" in which gas chromatography/ mass spectrometry (GC/MS) was used to detect the lesions released by a glycosylase from γ-irradiated calf thymus DNA containing multiple lesions. This method allows the measurement of substrate specificity under conditions where the enzyme concentration is far less than the substrate concentration and the enzyme encounters numerous lesions on the same substrate (21). In addition, by normalizing the signals for all substrates recognized by a given glycosylase, we can compare the substrate recognition patterns of different glycosylases (21).…”

“…This method allows the measurement of substrate specificity under conditions where the enzyme concentration is far less than the substrate concentration and the enzyme encounters numerous lesions on the same substrate (21). In addition, by normalizing the signals for all substrates recognized by a given glycosylase, we can compare the substrate recognition patterns of different glycosylases (21). Interestingly, MmuNeil3Δ324 exhibits a broad lesion recognition pattern and prefers FapyA and FapyG followed by 5-OHU, 5-OHC, and 5OHMH, then by Tg and 8-oxoA.…”

“…To create base lesions in vitro, calf thymus DNA was γ-irradiated with 40 Gray in N 2 O saturated phosphate buffer solution as previously described (21,44). Then 50 μg DNA was incubated with 1 μg glycosylase in 50 mM phosphate buffer (pH 7.4) containing 100 mM KCl, 1 mM EDTA and 0.1 mM DTT at 37°C for 1 h. The subsequent GC/MS analysis was performed as described (21,44) and the data were collected from three independent experiments using three independently prepared DNA substrates. Normalization of the data is described in SI Materials and Methods.…”

The Fpg/Nei Family of DNA Glycosylases

“…Phylogenetic analysis and functional studies of the Fpg/Nei family indicate that in Actinobacteria alone, six gene clades occur, two within the Nei proteins and four within the Fpg clade (52). The plant and fungi clade is clearly part of the Fpg family while within metazoans, Neil2 and Neil3 form their own clade separate from Neil1.…”

Structural Characterization of Viral Ortholog of Human DNA Glycosylase NEIL1 Bound to Thymine Glycol or 5-Hydroxyuracil-containing DNA

“…When performing pre-steady state kinetics on DNA glycosylases, either a single-or double-exponential model can be used to fit the data depending on the substrates tested. The Tg:A data were fit to a single-exponential model, as observed for Candida albicans Fpg with a double-stranded Tg substrate (48). We note that another group reported that kinetics data obtained with human NEIL1 and Tg-containing DNA were fit to a doubleexponential model, presumably because of the interconversion of the cis-and trans-Tg epimers (49).…”