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

Abstract

In Saccharomyces cerevisiae, the apurinic/apyrimidinic (AP) endonucleases Apn1 and Apn2 act as alternative pathways for the removal of various 3′-terminal blocking lesions from DNA strand breaks and in the repair of abasic sites, which both result from oxidative DNA damage. Here we demonstrate that Tpp1, a homologue of the 3′ phosphatase domain of polynucleotide kinase, is a third member of this group of redundant 3′ processing enzymes. Unlike Apn1 and Apn2, Tpp1 is specific for the removal of 3′ phosphates at strand breaks and does not possess more general 3′ phosphodiesterase, exonuclease, or AP endonuclease activities. Deletion ofTPP1 in an apn1 apn2 mutant background dramatically increased the sensitivity of the double mutant to DNA damage caused by H₂O₂ and bleomycin but not to damage caused by methyl methanesulfonate. The triple mutant was also deficient in the repair of 3′ phosphate lesions left by Tdp1-mediated cleavage of camptothecin-stabilized Top1-DNA covalent complexes. Finally, the tpp1 apn1 apn2 triple mutation displayed synthetic lethality in combination with rad52, possibly implicating postreplication repair in the removal of unrepaired 3′-terminal lesions resulting from endogenous damage. Taken together, these results demonstrate a clear role for the lesion-specific enzyme, Tpp1, in the repair of a subset of DNA strand breaks.

ACKNOWLEDGMENTS

This work was supported in part by the Pew Scholars Program in the Biomedical Sciences of the Pew Charitable Trusts and Public Health Service grant CA-90911 (T.E.W.) and training grant 5T32HL07157 (J.R.V).

We thank Tadahide Izumi (University of Texas Medical Branch) for technical information regarding the preparation of the 3′-PG-containing oligonucleotide.