Saccharomyces cerevisiae Ntg1p and Ntg2p:  Broad Specificity N-Glycosylases for the Repair of Oxidative DNA Damage in the Nucleus and Mitochondria

Abstract

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.

Spontaneous DNA Damage in Saccharomyces cerevisiae Elicits Phenotypic Properties Similar to Cancer Cells

“…Genomic DNA samples were prepared as follows. 10 g of BamH1-and ApaL1-digested genomic DNA was incubated with 5 g of recombinant glutathione S-transferase-tagged NTG1 protein in a 25-l reaction mixture containing buffer B (15 mM KH 2 PO 4 , pH 6.8, 10 mM EDTA, 10 mM ␤-mercaptoethanol, and 40 mM KCl) (6). All sample volumes were equalized by adding TE and then incubated for 30 min at 37°C.…”

“…Prior to loading, denaturing dye (50 mM NaOH, 1 mM EDTA, pH 8) was added to each sample. The recombinant Ntg1p used in these experiments is N-terminally tagged with glutathione S-transferase and expressed in Escherichia coli as described previously (6). The protein was purified by glutathione-agarose chromatography followed by Mono-S fast-performance liquid chromatography (18).…”

“…It is believed that repair of oxidative damage is mediated primarily by this pathway (6). In BER, the DNA damage is recognized by a specific DNA glycosylase that catalyzes the release of the damaged base and creates an abasic site.…”

DNA Recombination-Initiation Plays a Role in the Extremely Biased Inheritance of Yeast [rho−] Mitochondrial DNA That Contains the Replication Origin ori5

“…Thus, it is very unlikely that RPO41, OGG1, or NUC1 is involved in double-stranded cleavage in the ori5 region. The Ntg1 protein exists primarily in mitochondria, although some is found in the nucleus as well (55). Ntg1 is required for the repair of oxidative DNA damage in vivo, and it shares amino acid sequence similarity with E. coli endonuclease III and the human Nth1 protein (21).…”

“…Ntg1 has DNA repair glycosylase and apurinic/apyrimidinic (AP) lyase activities. It recognizes various oxidatively modified bases and causes singlestranded breakage (nicking) in double-stranded DNA (15,45,55). Thus, the effect of NTG1 disruption on ori5-specific double-stranded cleavage is not readily explainable by the known biochemical activities of the Ntg1 protein.…”