Saccharomyces cerevisiae Ntg1p and Ntg2p: Broad specificity N-glycosylasesfor the repair of oxidative DNA damage in the nucleus and mitochondria

Saccharomyces cerevisiae possesses two functional homologues (Ntg1p and Ntg 2p) of the Escherichia coli endonuclease III protein, a DNA base excision r epair N-glycosylase with a broad substrate specificity directed primarily a gainst oxidatively damaged pyrimidines. The substrate specificites of Ntg1p and Ntg2p are similar but not identical, and differences in their amino ac id sequences as well as inducibility by DNA damaging agents suggest that th e two proteins may have different biological roles and subcellular location s. Experiments performed on oligonucleotides containing a variety of oxidat ive base damages indicated that dihydrothymine, urea, and uracil glycol are substrates for Ntg1p and Ntg2p, although dihydrothymine was a poor substra te for Ntg2p. Vectors encoding Ntg1p-green fluorescent protein (GFP) and Nt g2p-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 transforman ts revealed that Ntg1p localizes primarily to the mitochondria with some nu clear 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 s ubstrate specifities of Ntg1p and Ntg2p, indicating that their base damage recognition ranges show distinct differences and that these proteins mediat e different roles in the repair of DNA base damage in the nucleus and mitoc hondria of yeast.