title: 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 gamma-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 2500min(-1).
authors.affiliation: Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Burlington, VT 05405-0068, United States.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Kathe
authors.forename: Ramiro
authors.initials: R
authors.lastname: Barrantes-Reynolds
authors.forename: Pawel
authors.initials: P
authors.lastname: Jaruga
authors.forename: Michael R
authors.initials: MR
authors.lastname: Newton
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Burrows
authors.forename: Viswanath
authors.initials: V
authors.lastname: Bandaru
authors.forename: Miral
authors.initials: M
authors.lastname: Dizdaroglu
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Bond
authors.forename: Susan S
authors.initials: SS
authors.lastname: Wallace
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002176:Candida albicans / Q000201:enzymology*; D045647:DNA Glycosylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D043382:DNA-Formamidopyrimidine Glycosylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000235:genetics / Q000378:metabolism; D005720:Gamma Rays; D008401:Gas Chromatography-Mass Spectrometry; D006146:Guanidines / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006827:Hydantoins / Q000378:metabolism*; D007700:Kinetics; D011743:Pyrimidines / Q000737:chemistry; D013141:Spiro Compounds / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D018744:DNA, Plant; D006146:Guanidines; D006827:Hydantoins; D011743:Pyrimidines; D013141:Spiro Compounds; C467338:guanidinohydantoin; C408822:spiroiminodihydantoin; D006151:Guanosine; C024829:8-hydroxyguanine; D006147:Guanine; D043224:Deoxyribonuclease (Pyrimidine Dimer); D045647:DNA Glycosylases; C500428:OGG1 protein, Arabidopsis; D043382:DNA-Formamidopyrimidine Glycosylase