A new Schizosaccharomyces pombe base excision repair mutant, nth1, reveals overlapping pathways for repair of DNA base damage.
Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or gamma-irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3-methyladenine and 7-methylguanine whereas methyl-formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1-like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears to be their lack of AP lyase activity. The nth1 mutant also exhibited elevated frequencies of spontaneous mitotic intrachromosomal recombination, which is a phenotype shared by the MMS-hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.
Osman
Fekret
F
Department of Biochemistry and Molecular Biology, University College London, Gower St., London WC1E 6BT, UK.
Bjørås
Magnar
M
Alseth
Ingrun
I
Morland
Ingrid
I
McCready
S
S
Seeberg
Erling
E
Tsaneva
Irina
I
D002470:Cell Survival; D004249:DNA Damage; D045647:DNA Glycosylases / Q000378:metabolism; D004260:DNA Repair; D004271:DNA, Fungal / Q000187:drug effects / Q000528:radiation effects; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D011743:Pyrimidines / Q000378:metabolism; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D009153:Mutagens; D011743:Pyrimidines; D008741:Methyl Methanesulfonate; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases