title: The role of yeast DNA 3'-phosphatase Tpp1 and rad1/Rad10 endonuclease in processing spontaneous and induced base lesions.
abstract: Tpp1 is a DNA 3'-phosphatase in Saccharomyces cerevisiae that is believed to act during strand break repair. It is homologous to one domain of mammalian polynucleotide kinase/3'-phosphatase. Unlike in yeast, we found that Tpp1 could confer resistance to methylmethane sulfonate when expressed in bacteria that lack abasic endonuclease/3'-phosphodiesterase function. This species difference was due to the absence of delta-lyase activity in S. cerevisiae, since expression of bacterial Fpg conferred Tpp1-dependent resistance to methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and Apn2. In contrast, beta-only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3' alpha,beta-unsaturated aldehydes. In parallel experiments, mutations of TPP1 and RAD1, encoding part of the Rad1/Rad10 3'-flap endonuclease, caused synthetic growth defects in yeast strains lacking Apn1. In contrast, Fpg expression led to a partial rescue of apn1 apn2 rad1 synthetic lethality by converting lesions into Tpp1-cleavable 3'-phosphates. The collected experiments reveal a profound toxicity of strand breaks with irreparable 3' blocking lesions, and extend the function of the Rad1/Rad10 salvage pathway to 3'-phosphates. They further demonstrate a role for Tpp1 in repairing endogenously created 3'-phosphates. The source of these phosphates remains enigmatic, however, because apn1 tpp1 rad1 slow growth could be correlated with neither the presence of a yeast delta-lyase, the activity of the 3'-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.
authors.affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602, USA.
authors.forename: Anandi S
authors.initials: AS
authors.lastname: Karumbati
authors.forename: Rajashree A
authors.initials: RA
authors.lastname: Deshpande
authors.forename: Arshad
authors.initials: A
authors.lastname: Jilani
authors.forename: John R
authors.initials: JR
authors.lastname: Vance
authors.forename: Dindial
authors.initials: D
authors.lastname: Ramotar
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wilson
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics; D004249:DNA Damage / Q000502:physiology; D004260:DNA Repair / Q000502:physiology*; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins; D043382:DNA-Formamidopyrimidine Glycosylase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases
chemical_list: D000447:Aldehydes; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D009153:Mutagens; D016877:Oxidants; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D008741:Methyl Methanesulfonate; D006861:Hydrogen Peroxide; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; C083274:Apn1 protein, S cerevisiae; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D009708:Nucleotidases; C039170:deoxynucleotide 3'-phosphatase; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D045643:DNA Repair Enzymes