Substrate specificities of the Ntg1 and Ntg2 proteins of Saccharomyces cerevisiae for oxidized DNA bases are not identical

Two genes of Saccharomyces cerevisiae, NTG1 and NTG2, encode proteins with a significant sequence homology to the endonuclease III of Escherichia coli . The Ntg1 and Ntg2 proteins were overexpressed in E. coli and purified to apparent homogeneity, The substrate specificity of Ntg1. and Ntg2 proteins for modified bases in oxidatively damaged DNA was investigated using gas ch romatography/isotope-dilution mass spectrometry. The substrate used was cal f-thymus DNA exposed to gamma-radiation in N2O-saturated aqueous solution. The results reveal excision by Ntg1 and Ntg2 proteins of six pyrimidine-der ived lesions, 5-hydroxy-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5-hydroxy- 5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine and thymine glycol, a nd two purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from gamma-irradiated DNA. In contras t, Ntg1 and Ntg2 proteins do not release 8-hydroxyguanine or 8-hydroxyadeni ne from gamma-irradiated DNA. The Ntg1 and Ntg2 proteins also release 2,6-d iamino-4-hydroxy-5-N-methylformamido-pyrimidine from dam aged poly(dG-dC).p oly(dG-dC). Excision was measured as a function of enzyme concentration and time. Furthermore, kinetic parameters were determined for each lesion. The results show that kinetic constants varied among the different lesions for the same enzyme. We also investigated the capacity of the Ntg1 and Ntg2 pr oteins to cleave 34mer DNA duplexes containing a single 8-OH-Gua residue mi spaired with each of the four DNA bases. The results show that the Ntg1 pro tein preferentially cleaves a DNA duplex containing 8-OH-Gua mispaired with a guanine. Moreover, the Ntg1 protein releases free 8-OH-Gua from 8-OH-Gua /Gua duplex but not from duplexes containing 8-OH-Gua mispaired with adenin e, thymine or cytosine. in contrast, the Ntg2 protein does not incise duple xes containing 8-OH-Gua mispaired with any of the four DNA bases. These res ults demonstrate that substrate specificities of the Ntg1 and Ntg2 proteins are similar but not identical and clearly different from that of the endon uclease III of E. coli and its homologues in Schizosaccharomyces pombe or h uman cells.