Cells exposed to DNA damaging agents in their natural environment do not undergo continuous cycles of replication but are more frequently engaged in gene transcription. Luciferase gene expression analysis with DNA templates containing uracil or 8-oxoguanine, placed at a defined position, indicated that in nondividing Escherichia coli cells, efficient mutagenic lesion bypass does occur in vivo during transcription. Sequence analyses of the transcript population revealed that RNA polymerase inserts adenine opposite to uracil, and adenine or cytosine opposite to 8-oxoguanine. Surprisingly, deletions were also detected for 8-oxoguanine-containing templates, indicating RNA polymerase slippage over this lesion. Genetic analyses showed that, in E. coli, 8-oxoguanine is subject to transcription-coupled repair. Consequently, DNA damages alter transcription fidelity in vivo, which may lead to the production of mutant proteins that have the potential to change the phenotype of nondividing cells.

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 chromatography/isotope-dilution mass spectrometry. The substrate used was calf-thymus DNA exposed to gamma-radiation in N2O-saturated aqueous solution. The results reveal excision by Ntg1 and Ntg2 proteins of six pyrimidine-derived lesions, 5-hydroxy-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5-hydroxy-5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine and thymine glycol, and two purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from gamma-irradiated DNA. In contrast, Ntg1 and Ntg2 proteins do not release 8-hydroxyguanine or 8-hydroxyadenine from gamma-irradiated DNA. The Ntg1 and Ntg2 proteins also release 2, 6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine from damaged poly(dG-dC).poly(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 proteins to cleave 34mer DNA duplexes containing a single 8-OH-Gua residue mispaired with each of the four DNA bases. The results show that the Ntg1 protein 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 adenine, thymine or cytosine. In contrast, the Ntg2 protein does not incise duplexes containing 8-OH-Gua mispaired with any of the four DNA bases. These results demonstrate that substrate specificities of the Ntg1 and Ntg2 proteins are similar but not identical and clearly different from that of the endonuclease III of E.coli and its homologues in Schizosaccharomyces pombe or human cells.

Saccharomyces cerevisiae possesses two functional homologues (Ntg1p and Ntg2p) of the Escherichia coli endonuclease III protein, a DNA base excision repair N-glycosylase with a broad substrate specificity directed primarily against oxidatively damaged pyrimidines. The substrate specificities of Ntg1p and Ntg2p are similar but not identical, and differences in their amino acid sequences as well as inducibility by DNA damaging agents suggest that the two proteins may have different biological roles and subcellular locations. Experiments performed on oligonucleotides containing a variety of oxidative base damages indicated that dihydrothymine, urea, and uracil glycol are substrates for Ntg1p and Ntg2p, although dihydrothymine was a poor substrate for Ntg2p. Vectors encoding Ntg1p-green fluorescent protein (GFP) and Ntg2p-GFP fusions under the control of their respective endogenous promoters were utilized to observe the subcellular targeting of Ntg1p and Ntg2p in S. cerevisiae. Fluorescence microscopy of pNTG1-GFP and pNTG2-GFP transformants revealed that Ntg1p localizes primarily to the mitochondria with some nuclear localization, whereas Ntg2p localizes exclusively to the nucleus. In addition, the subcellular location of Ntg1p and Ntg2p confers differential sensitivities to the alkylating agent MMS. These results expand the known substrate specificities of Ntg1p and Ntg2p, indicating that their base damage recognition ranges show distinct differences and that these proteins mediate different roles in the repair of DNA base damage in the nucleus and mitochondria of yeast.