Repair of DNA Strand Breaks by the Overlapping Functions of Lesion-Specific and Non-Lesion-Specific DNA 3′ Phosphatases

Reactive oxygen species (ROS) generated by the mitochondria during aerobic metabolism can induce several types of DNA damage, including abasic (apurinic/apyrimidinic [AP]) sites, base modifications, and DNA strand breaks. Strand breaks caused by ROS, as well as from exposure to ionizing radiation or treatment with the anticancer agent bleomycin, often contain unconventional terminal groups, such as 3′ phosphoglycolates (PGs) and 3′ phosphates, resulting from fragmentation of deoxyribose sugars (7). These blocking lesions must be removed to allow repair of the breaks by polymerization and ligation. AP endonucleases, which function during base excision repair (BER) of abasic sites, also exhibit 3′ phosphodiesterase activity and provide a major pathway for removal of 3′-terminal lesions (2, 12, 13, 23). Two AP endonucleases, Apn1 and Apn2, have been identified in Saccharomyces cerevisiae. Apn1 is a homologue of Escherichia coli endonuclease IV and represents the major AP endonuclease in yeast, constituting >90% of the activity in cellular extracts (12). Apn2 (also called Eth1) shares homology with E. coli exonuclease III and human Hap1/Ape1 and is induced after exposure to DNA-damaging agents such as the alkylating agent methyl methanesulfonate (MMS) (2, 13). Like Apn1, Apn2 possesses both AP endonuclease and 3′ phosphodiesterase activities; however, only Apn2 has been shown to have an associated 3′-5′ exonuclease activity (23). In S. cerevisiae, apn1 apn2 double mutant strains display a synergistic increase in sensitivity to MMS and H2O2, indicating that these enzymes perform overlapping roles in the repair of abasic sites and in the repair of strand breaks with 3′-terminal lesions (2, 13). We recently identified a DNA 3′ phosphatase in S. cerevisiae, TPP1, based on its homology to the bifunctional human enzyme polynucleotide kinase-3′ phosphatase (hPNKP) (25). hPNKP interacts with XRCC1 and is thought to function during BER–single-strand break repair by restoring normal termini to strand breaks containing 3′ phosphates and 5′ hydroxyls (26), although in vivo evidence to support this is lacking. Interestingly, Tpp1 shares significant homology with only the 3′ phosphatase domain of hPNKP. A corresponding 5′ kinase domain is entirely absent from the genome. Consistent with these observations, biochemical characterization of Tpp1 revealed that the enzyme possesses a robust DNA 3′ phosphatase activity, but an associated 5′ kinase activity was not detected. Tpp1 and Apn1 were identified as the primary constitutive 3′ phosphatase activities in whole-cell extracts, suggesting that these two enzymes, along with Apn2, may represent three alternative pathways for the repair of 3′ phosphate blocking lesions. In addition to oxidative damage, strand breaks bearing 3′ phosphates are thought to arise in cells as one result of the action of DNA topoisomerases. DNA topoisomerase I (Top1 in yeast) forms a covalent intermediate between the active site tyrosine and the 3′ end of the phosphodiester backbone of DNA. Recently, Nash and colleagues identified a tyrosyl-DNA phosphodiesterase, Tdp1, which cleaves the covalent Top1-DNA phosphotyrosine bond to leave a phosphate at the 3′ terminus (18, 28). It is likely that Tdp1 acts to remove Top1 when transcription or replication complexes cause premature dissociation of the 5′ strand. Consistent with this, tdp1 mutant yeast is sensitive to the drug camptothecin, which stabilizes the Top1-DNA covalent intermediate (18). This sensitivity is far less than that observed with recombination mutants (16), however, and so alternative pathways for Top1 removal must exist. In the subset of Top1 lesions that are cleaved by Tdp1, 3′ phosphatases must presumably act downstream. To investigate these possibilities, we examined 3′ phosphate removal by using purified enzymes and by evaluating the sensitivity of mutant strains to oxidative and camptothecin-induced DNA damage. We show that Tpp1 is specific and highly efficient in removing phosphates from 3′ termini, in contrast to Apn1. Surprisingly, these studies also revealed that Apn1, but not Tpp1, acts to remove a single nucleotide at a nick in DNA, regardless of the presence of a 3′-terminal lesion. Tpp1 functions synergistically with Apn1 and Apn2 in the repair of lesions induced by treatment with H2O2 and bleomycin but not in the repair of lesions induced by treatment with MMS. Furthermore, the loss of Tpp1, Apn1, and Apn2 is lethal in the absence of Rad52-dependent recombinational repair and also sensitizes cells to camptothecin in a TDP1-dependent fashion. These findings are discussed in the context of overlapping pathways for the repair of 3′ phosphate blocking lesions.