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 δ-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, β-only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3′ α,β-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 δ-lyase, the activity of the 3′-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.