Formation of 8-oxoguanine in cellular DNA of Escherichia coli strains defective in different antioxidant defences |
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Authors: | Alhama, Jose Ruiz-Laguna, Julia Rodriguez-Ariza, Antonio Toribio, Fermin Lopez-Barea, Juan Pueyo, Carmen |
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Affiliation: | Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Avenida de Medina Azahara s/n 14071-Córdoba, España |
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Abstract: | This paper examines the relationship in Escherichia coli betweenthe in vivo content of 8-oxoguanine (8-oxoG) in chromosomalDNA and deficiencies of various key antioxidant defences. Thestructural genes for catalases (katG and katE), cytosolic superoxidedismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase(fpg) were inactivated to obtain bacterial strains lacking thescavenger enzymes for H2O2 or O2· or the DNA repairprotein for 8-oxoG. Wild-type bacteria showed 5-fold increasedsensitivity to both lethality and mutagenesis by H2O2 in K medium(1 % casamino acids and 1 % glucose), as compared with nutrientbroth. This higher sensitivity was associated with increasedchromosomal oxidative damage, estimated as the 8-oxodG content,and with a marked decrease in both catalase and SOD activities.Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayedincreased 8-oxodG content in chromosomal DNA (2.8-fold thatof the wild-type) when grown under standard aerated conditions.Comparatively, no significant difference in 8-oxodG contentwas observed in cells grown without aeration. Bacteria totallydevoid of catalase activity (katG katE mutant) showed wild-typecontents of 8-oxodG in chromosomal DNA when grown under aeratedconditions. Nevertheless, the protective role of catalase inpreventing formation of 8-oxodG in chromosomal DNA became evidentunder oxidative stress conditions: growth under hyperoxygenationand, particularly, following H2O2 exposure. Catalase deficiencyresulted in a dramatic decrease in viability after H2O2 exposure.A deficiency of Fpg protein also sensitized E.coli to H2O2 lethality,though to lesser extent than a deficiency of catalase activity.However, the scavenger enzyme and the DNA repair protein protectedequally against 8-oxoG formed in vivo upon H2O2 treatment. 1To whom correspondence should be addressed. Tel: 57 218695; Fax: 57 218688; Email: bblpucuc{at}uco.es |
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