The Corynebacterium pseudotuberculosis genome contains two formamidopyrimidine-DNA glycosylase enzymes, only one of which recognizes and excises 8-oxoguanine lesion.
The GO-system is a DNA repair mechanism that prevents and corrects oxidative DNA damage. Formamidopyrimidine-DNA glycosylase (FPG/MutM) participates in this system, avoiding the mutagenic effects of 8-oxoguanine lesion into DNA. Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis, is a facultative intracellular microorganism vulnerable to oxidative DNA damage. Since inefficiencies in the DNA damage repair system can lead to death, the characterization of repair genes may provide valuable molecular targets for caseous lymphadenitis therapy. The purposes of this study were to functionally characterize MutM1 and MutM2 proteins from C. pseudotuberculosis in silico, in vivo, and in vitro and to examine their role in the repair of 8-oxoguanine damage. In silico investigation revealed that both proteins have conserved domains typical of DNA glycosylases, such as DNA binding domains and DNA glycosylase/AP lyase catalytic domain. In comparison with the MutM protein of Escherichia coli, however, CpMutM2 was found to lack residues that are essential for recognizing and excising 8-oxoguanine damage. Molecular docking calculations have shown a native-like orientation of 8-oxoguanine at the CpMutM1 active site, while the same is not observed for CpMutM2, which seems to poorly interact with DNA. Surface charge analyses have corroborated this finding. Overexpression of CpMutM1 or CpMutM2 has toxic effects on E. coli strain BH20 (mutM-), as shown by growth curves obtained in the presence of hydrogen peroxide and cell viability assays. This cytotoxicity can be attributed to an imbalance in the repair pathway, resulting from hyperactivity of DNA glycosylases, leading to formation of AP sites and DNA strand breakage at levels that exceed the processing capacity of other enzymes in the BER pathway. In order to demonstrate the involvement of these enzymes in the recognition and excision of 8-oxoguanine lesion, glycosylase activity was evaluated in vitro. Only the CpMutM1 protein was proven to be capable of recognizing and excising 8-oxoguanine. Taken together, these results suggest that although the formamidopyrimidine-DNA glycosylase domain is conserved in both proteins, only one proved to be functional in recognizing and excising 8-oxoguanine lesion.
Laboratory of Molecular Biology, Federal University of São João Del-Rei, Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: larissarantes1@hotmail.com.
Larissa Souza
LS
Arantes
Laboratory of Molecular Biology, Federal University of São João Del-Rei, Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: lgvilanova@yhaoo.com.br.
Liliane Gonçalves Vila
LG
Nova
Laboratory of Molecular Biology, Federal University of São João Del-Rei, Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil.
Bruno Carvalho
BC
Resende
Laboratory of Genetics and Biochemistry, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil. Electronic address: mbitarufmg@gmail.com.
Mainá
M
Bitar
Laboratory of Molecular Biology, Federal University of São João Del-Rei, Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: ivanglista2009@yahoo.com.br.
Ivan Evangelista Vale
IE
Coelho
Laboratory of Molecular and Cellular Genetics, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil. Electronic address: miyoshi@icb.ufmg.br.
Anderson
A
Miyoshi
Laboratory of Molecular and Cellular Genetics, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil. Electronic address: vascoariston@gmail.com.
Vasco Ariston
VA
Azevedo
Laboratory of Molecular Biology, Federal University of São João Del-Rei, Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: llaramg@hotmail.com.
Luciana
L
Lara Dos Santos
Laboratory of Genetics and Biochemistry, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil. Electronic address: crmachad@icb.ufmg.br.
Carlos Renato
CR
Machado
Laboratory of Molecular Biology, Federal University of São João Del-Rei, Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address: debora@ufsj.edu.br.
Débora
D
de Oliveira Lopes
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016925:Corynebacterium pseudotuberculosis / Q000201:enzymology / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D043382:DNA-Formamidopyrimidine Glycosylase / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C024829:8-hydroxyguanine; D006147:Guanine; D043382:DNA-Formamidopyrimidine Glycosylase
8-Oxoguanine; Corynebacterium pseudotuberculosis; DNA repair; GO system; MutM; Oxidative damage