title: Adenine Glycosylase MutY of Corynebacterium pseudotuberculosis presents the antimutator phenotype and evidences of glycosylase/AP lyase activity in vitro.
abstract: Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis, a disease that predominantly affects small ruminants, causing significant economic losses worldwide. As a facultative intracellular pathogen, this bacterium is exposed to an environment rich in reactive oxygen species (ROS) within macrophages. To ensure its genetic stability, C. pseudotuberculosis relies on efficient DNA repair pathways for excision of oxidative damage such as 8-oxoguanine, a highly mutagenic lesion. MutY is an adenine glycosylase involved in adenine excision from 8-oxoG:A mismatches avoiding genome mutation incorporation. The purpose of this study was to characterize MutY protein from C. pseudotuberculosis and determine its involvement with DNA repair. In vivo functional complementation assay employing mutY gene deficient Escherichia coli transformed with CpmutY showed a 13.5-fold reduction in the rate of spontaneous mutation, compared to cells transformed with empty vector. Also, under oxidative stress conditions, CpMutY protein favored the growth of mutY deficient E. coli, relative to the same strain in the absence of CpMutY. To demonstrate the involvement of this enzyme in recognition and excision of 8-oxoguanine lesion, an in vitro assay was performed. CpMutY protein was capable of recognizing and excising 8-oxoG:A but not 8-oxoG:C presenting evidences of glycosylase/AP lyase activity in vitro. In silico structural characterization revealed the presence of preserved motifs related to the MutY activity on DNA repair, such as catalytic residues involved in glycosylase/AP lyase activity and structural DNA-binding elements, such as the HhH motif and the [4Fe-4S] cluster. The three-dimensional structure of CpMutY, generated by comparative modeling, exhibits a catalytic domain very similar to that of E. coli MutY. Taken together, these results indicate that the CpmutY encodes a functional protein homologous to MutY from E. coli and is involved in the prevention of mutations and the repair of oxidative DNA lesions.
authors.affiliation: Laboratory of Molecular Biology, Federal University of São João Del-Rei (CCO), Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: fael.faria09@yahoo.com.br.
authors.forename: Rafael Cançado
authors.initials: RC
authors.lastname: de Faria
authors.affiliation: Laboratory of Molecular Biology, Federal University of São João Del-Rei (CCO), Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: lgvilanova@yhaoo.com.br.
authors.forename: Liliane Gonçalves
authors.initials: LG
authors.lastname: Vila-Nova
authors.affiliation: Laboratory of Genetics and Biochemistry, Department of Biochemistry, ICB, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil. Electronic address: duonlumo@gmail.com.
authors.forename: Mainá
authors.initials: M
authors.lastname: Bitar
authors.affiliation: Laboratory of Molecular Biology, Federal University of São João Del-Rei (CCO), Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: bruno_carvalho_resende@hotmail.com.
authors.forename: Bruno Carvalho
authors.initials: BC
authors.lastname: Resende
authors.affiliation: Laboratory of Molecular Biology, Federal University of São João Del-Rei (CCO), Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: larissarantes1@hotmail.com.
authors.forename: Larissa Sousa
authors.initials: LS
authors.lastname: Arantes
authors.affiliation: Laboratory of Molecular Biology, Federal University of São João Del-Rei (CCO), Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: arnaldo.br@me.com.
authors.forename: Arnaldo Basso
authors.initials: AB
authors.lastname: Rebelato
authors.affiliation: Laboratory of Cell and Molecular Genetics, Department of General Biology, ICB, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil. Electronic address: vascoariston@gmail.com.
authors.forename: Vasco Ariston Carvalho
authors.initials: VAC
authors.lastname: Azevedo
authors.affiliation: Laboratory of Genetics and Biochemistry, Department of Biochemistry, ICB, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil. Electronic address: gfranco@icb.ufmg.br.
authors.forename: Glória Regina
authors.initials: GR
authors.lastname: Franco
authors.affiliation: Laboratory of Genetics and Biochemistry, Department of Biochemistry, ICB, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil. Electronic address: crmachad@icb.ufmg.br.
authors.forename: Carlos Renato
authors.initials: CR
authors.lastname: Machado
authors.affiliation: Laboratory of Molecular Biology, Federal University of São João Del-Rei (CCO), Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: llaramg@hotmail.com.
authors.forename: Luciana Lara Dos
authors.initials: LLD
authors.lastname: Santos
authors.affiliation: Laboratory of Molecular Biology, Federal University of São João Del-Rei (CCO), Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: debora@ufsj.edu.br.
authors.forename: Débora
authors.initials: D
authors.lastname: de Oliveira Lopes
mesh_terms: D000595:Amino Acid Sequence; D016925:Corynebacterium pseudotuberculosis / Q000235:genetics* / Q000378:metabolism*; D045647:DNA Glycosylases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D018384:Oxidative Stress; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins
chemical_list: D011994:Recombinant Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C437225:adenine glycosylase; C061738:mutY adenine glycosylase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase
keywords: 8-oxoguanine; Corynebacterium pseudotuberculosis; DNA repair; Functional assays; GO system; MutY