The role of yeast DNA 3'-phosphatase Tpp1 and rad1/Rad10 endonuclease in processing spontaneous and induced base lesions.
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.
Pubmed ID: 12783866 RIS Download
Aldehydes | Aspartic Acid | DNA Damage | DNA Repair | DNA Repair Enzymes | DNA-Binding Proteins | DNA-Formamidopyrimidine Glycosylase | Deoxyribonuclease (Pyrimidine Dimer) | Endodeoxyribonucleases | Endonucleases | Escherichia coli Proteins | Fungal Proteins | Hydrogen Peroxide | Methyl Methanesulfonate | Mutagens | Mutation | N-Glycosyl Hydrolases | Nucleotidases | Oxidants | Phenotype | Saccharomyces cerevisiae | Saccharomyces cerevisiae Proteins | Single-Strand Specific DNA and RNA Endonucleases