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Effect of Single Mutations on the Specificity of Esherichia Coli FPG Protein for Excision of Purine Lesions From DNA Damaged By Free Radicals

Published

Author(s)

O. Sidorkina, M. Dizdaroglu, J. Laval

Abstract

The Formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys57->Gly (FpgK57G), Lys57->Arg (FpgK57R), Lys155->Ala (FpgK155A), Pro2->Gly (FpgP2G) and Pro2->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA
Citation
Free Radical Biology and Medicine
Volume
31
Issue
No. 6

Keywords

2, 6-Diamino-4-hydroxy-t-formamidopyrimid, 8-Hydroxyguanine, DNA repair, Fpg protein, single mutations, substrate specificity

Citation

Sidorkina, O. , Dizdaroglu, M. and Laval, J. (2001), Effect of Single Mutations on the Specificity of Esherichia Coli FPG Protein for Excision of Purine Lesions From DNA Damaged By Free Radicals, Free Radical Biology and Medicine (Accessed June 16, 2024)

Issues

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Created September 1, 2001, Updated February 19, 2017