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Arabidopsis Thaliana Ogg1 Protein Excises 8-Hydroxyguanine and 2,6- Diamino-4-hydroxy-5-formamidopyrimidine from Oxidative Damaged DNA Containing Multiple Lesions

Published

Author(s)

T Morales-Ruiz, M Birincioglu, Pawel Jaruga, H Rodriguez, T. Roldan-Arjona, Miral M. Dizdar

Abstract

A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thaliana has recently been cloned, isolated, and characterized [Garcia-Ortiz, M.V., Ariza, R.R., and Roldan-Arjona, T. (2001) Plant Mol. Biol. 47, 795-804]. This enzyme (AtOggl) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster and human Oggl proteins. We investigated the substrate specificity and kinetics of AtOggl for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the seventeen modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins. Excision depended on incubation time, enzyme concentration and substrate concentration, and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligonucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua;C pair with some or very low activity on other pairs in agreement with activity of other Oggl proteins. The results unequivocally show that AtOggl possesses common substrates with other eukaryotic Oggl proteins albeit significant differences between their excision kinetics.
Citation
Biochemistry
Volume
42
Issue
10

Keywords

8-hroxyguanine, DNA repair, eukaryotic dnzymes, Formamidopyrimidines, Hydroxyl radical, Oxidative DNA damage

Citation

Morales-Ruiz, T. , Birincioglu, M. , Jaruga, P. , Rodriguez, H. , Roldan-Arjona, T. and Dizdar, M. (2003), Arabidopsis Thaliana Ogg1 Protein Excises 8-Hydroxyguanine and 2,6- Diamino-4-hydroxy-5-formamidopyrimidine from Oxidative Damaged DNA Containing Multiple Lesions, Biochemistry (Accessed March 28, 2024)
Created March 17, 2003, Updated October 12, 2021