Novel substrates of Escherichia coli Nth protein and its kinetics for excision of modified bases from DNA damaged by free radicals

Escherichia coli Nth protein (endonuclease III) is a DNA glycosylase with a broad substrate specificity for pyrimidine derivatives. We discovered nove l substrates of E. coli Nth protein using gas chromatography/isotope-diluti on mass spectrometry and DNA samples, which were damaged by gamma-irradiati on or by H2O2/Fe(III)-EDTA/ascorbic acid. These were 4,6-diamiao-5-formamid opyrimidine, 5,6-dihydroxyuracil, and 5,6-dihydroxycytosine. The first comp ound was recognized for the first time as a purine-derived substrate of the enzyme. We also investigated kinetics of excision of a multitude of modifi ed bases from three damaged DNA substrates. Excision of modified bases was determined as a function of enzyme concentration, incubation time, and subs trate concentration. Excision followed Michaelis-Menten kinetics. Kinetic p arameters were determined for the following modified bases: 4,6-diamino-5-f ormamidopyrimidine, cis- and trans-thymine glycols, 5-hydroxycytosine, cis- and trans-uracil glycols, 5-hydroxyuracil, 5-hydroxy-5-methylhydantoin, al loxan, 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 excisio n of its known major substrates such as thymine glycol and 5-hydroxycytosin e. Excision kinetics significantly depended on the nature of the damaged DN A substrates in agreement with previous results on other DNA glycosylases. Specificity constants (k(cat)/K-M) of E. coli Nth protein were compared to those of its previously investigated functional homologues such as human an d Schizosaccharomyces pombe Nth proteins and Saccharomyces cerevisiae Ntg1 and Ntg2 proteins. This comparison shows that significant differences exist with respect to substrate specificity and kinetic parameters despite exten sive structural conservation among the Nth homologues.