Novel Substrates of Escherichia Coli Nth Protein and Its Kinetics for Excision of Modified Bases From DNA Damaged by Free Radicals
M. Dizdaroglu, C Bauche, H Rodriguez, J Laval
Escherichia coli Nth protein (endonuclease III) is a DNA glycosylase with a broad substrate specificity for pyrimidine derivatives. We discovered novel substrates of E. coli Nth protein using gas chromatography/isotope-dilution mass spectrometry and DNA samples, which were damaged by γ-irradiation or by H2O2/Fe(III)-EDTA/ascorbic acid. These were 4,6-diamino-5-formamidopyrimidine, 5,6-dihydroxyuracil and 5,6-dihydroxycytosine. The first compound was recognized for the first time as a purine-derived substrate of the enzyme. We also investigated kinetics of excision of a multitude of modified bases from three oxidatively damaged DNA substrates. Excision of modified bases were determined as a function of enzyme concentration, incubation time, and substrate concentration. Excision followed Michaelis-Menten kinetics. Kinetic parameters were determined for the following modified bases: 4,6-diamino-5-formamidopyrimidine, cis- and trans-thymine glycols, 5-hydroxycytosine, cis- and trans-uracil glycols, 5-hydroxyuracil, 5-hydroxy-5-methylhydantoin, alloxan, 5,6-dihydroxycytosine, 5,6-dihydroxyuracil, 5-hydroxy-6-hydrothymine and 5-hydroxy-6-hydrouracil. The results show that three newly discovered substrates were excised by the enzyme with a preference similar to excision of its known major substrates such as thymine glycol and 5-hydroxycytosine. Excision kinetics significantly depended on the nature of the damaged DNA substrates in agreement with previous results on other DNA glycosylases. Specificity constants (kcat/KM) of E. coli Nth protein were compared to those of its previously investigated functional homologues such as human and Schizosaccharmyces pombe Nth proteins and Saccharomyces cerevisiae Ntgl and Ntg2 proteins. This comparison shows that significant differences exist with respect to substrate specificity and kinetic parameters despite extensive structural conservation among the Nth homologues.
4, 6 - diamino, 5 formamidopyrimidine, 5.6-dihydroxysytosine, base excision repair, DNA damage, DNA glycosylases, oxidative, substrate specificity
, Bauche, C.
, Rodriguez, H.
and Laval, J.
Novel Substrates of Escherichia Coli Nth Protein and Its Kinetics for Excision of Modified Bases From DNA Damaged by Free Radicals, Biochemistry
(Accessed February 23, 2024)