title: Roles of base excision repair enzymes Nth1p and Apn2p from Schizosaccharomyces pombe in processing alkylation and oxidative DNA damage.
abstract: Schizosaccharomyces pombe Nthpl, an ortholog of the endonuclease III family, is the sole bifunctional DNA glycosylase encoded in its genome. The enzyme removes oxidative pyrimidine and incises 3' to the apurinic/apyrimidinic (AP) site, leaving 3'-alpha,beta-unsaturated aldehyde. Analysis of nth1 cDNA revealed an intronless structure including 5'- and 3'-untranslated regions. An Nth1p-green fluorescent fusion protein was predominantly localized in the nuclei of yeast cells, indicating a nuclear function. Deletion of nth1 confirmed that Nth1p is responsible for the majority of activity for thymine glycol and AP site incision in the absence of metal ions, while nth1 mutants exhibit hypersensitivity to methylmethanesulfonate (MMS). Complementation of sensitivity by heterologous expression of various DNA glycosylases showed that the methyl-formamidopyrimidine (me-fapy) and/or AP sites are plausible substrates for Nth1p in repairing MMS damage. Apn2p, the major AP endonuclease in S. pombe, also greatly contributes to the repair of MMS damage. Deletion of nth1 from an apn2 mutant resulted in tolerance to MMS damage, indicating that Nth1p-induced 3'-blocks are responsible for MMS sensitivity in apn2 mutants. Overexpression of Apn2p in nth1 mutants failed to suppress MMS sensitivity. These results indicate that Nth1p, not Apn2p, primarily incises AP sites and that the resultant 3'-blocks are removed by the 3'-phosphodiesterase activity of Apn2p. Nth1p is dispensable for cell survival against low levels of oxidative stress, but wild-type yeast became more sensitive than the nth1 mutant at high levels. Overexpression of Nth1p in heavily damaged cells probably induced cell death via the formation of 3'-blocked single-strand breaks.
authors.affiliation: Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridaicho, Okayama 700-0005, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Sugimoto
authors.forename: Emi
authors.initials: E
authors.lastname: Igawa
authors.forename: Haruna
authors.initials: H
authors.lastname: Tanihigashi
authors.forename: Mayumi
authors.initials: M
authors.lastname: Matsubara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ide
authors.forename: Shogo
authors.initials: S
authors.lastname: Ikeda
mesh_terms: D000478:Alkylation / Q000187:drug effects; D004249:DNA Damage / Q000502:physiology; D045647:DNA Glycosylases / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D008741:Methyl Methanesulfonate / Q000633:toxicity; D009097:Multienzyme Complexes / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics*; D012568:Schizosaccharomyces / Q000187:drug effects / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D009097:Multienzyme Complexes; D009153:Mutagens; C492078:Nth1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D008741:Methyl Methanesulfonate; D045647:DNA Glycosylases; C480729:Apn2 protein, S pombe; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase