Saccharomyces cerevisiae Ntg1p and Ntg2p: broad specificity N-glycosylases for the repair of oxidative DNA damage in the nucleus and mitochondria

Biochemistry. 1999 Aug 31;38(35):11298-306. doi: 10.1021/bi991121i.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Cell Nucleus / drug effects
  • Cell Nucleus / enzymology
  • Cell Nucleus / genetics*
  • Cell Nucleus / radiation effects
  • DNA Damage*
  • DNA Repair*
  • DNA, Mitochondrial / drug effects
  • DNA, Mitochondrial / genetics*
  • DNA, Mitochondrial / radiation effects
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • Gamma Rays
  • Green Fluorescent Proteins
  • Hydrogen Peroxide / toxicity
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • N-Glycosyl Hydrolases / biosynthesis
  • N-Glycosyl Hydrolases / genetics
  • N-Glycosyl Hydrolases / metabolism*
  • Oxidation-Reduction
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Fusion Proteins / radiation effects
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / radiation effects
  • Saccharomyces cerevisiae Proteins*
  • Substrate Specificity

Substances

  • DNA, Mitochondrial
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Green Fluorescent Proteins
  • Hydrogen Peroxide
  • N-Glycosyl Hydrolases
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • NTG1 protein, S cerevisiae
  • NTG2 protein, S cerevisiae