Endogenous DNA abasic sites cause cell death in the absence of Apn1, Apn2 and Rad1/Rad10 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, mutations in APN1, APN2 and either RAD1 or RAD10 genes are synthetic lethal. In fact, apn1 apn2 rad1 triple mutants can form microcolonies of approximately 300 cells. Expression of Nfo, the bacterial homologue of Apn1, suppresses the lethality. Turning off the expression of Nfo induces G(2)/M cell cycle arrest in an apn1 apn2 rad1 triple mutant. The activation of this checkpoint is RAD9 dependent and allows residual DNA repair. The Mus81/Mms4 complex was identified as one of these back-up repair activities. Furthermore, inactivation of Ntg1, Ntg2 and Ogg1 DNA N-glycosylase/AP lyases in the apn1 apn2 rad1 background delayed lethality, allowing the formation of minicolonies of approximately 10(5) cells. These results demonstrate that, under physiological conditions, endogenous DNA damage causes death in cells deficient in Apn1, Apn2 and Rad1/Rad10 proteins. We propose a model in which endogenous DNA abasic sites are converted into 3'-blocked single-strand breaks (SSBs) by DNA N-glycosylases/AP lyases. Therefore, we suggest that the essential and overlapping function of Apn1, Apn2, Rad1/Rad10 and Mus81/Mms4 is to repair 3'-blocked SSBs using their 3'-phosphodiesterase activity or their 3'-flap endonuclease activity, respectively.     

Overlapping specificities of base excision repair, nucleotide excision repair, recombination, and translesion synthesis pathways for DNA base damage in Saccharomyces cerevisiae

The removal of oxidative damage from Saccharomyces cerevisiae DNA is thought to be conducted primarily through the base excision repair pathway. The Escherichia coli endonuclease III homologs Ntg1p and Ntg2p are S. cerevisiae N-glycosylase-associated apurinic/apyrimidinic (AP) lyases that recognize a wide variety of damaged pyrimidines (H. J. You, R. L. Swanson, and P. W. Doetsch, Biochemistry 37:6033-6040, 1998). The biological relevance of the N-glycosylase-associated AP lyase activity in the repair of abasic sites is not well understood, and the majority of AP sites in vivo are thought to be processed by Apn1p, the major AP endonuclease in yeast. We have found that yeast cells simultaneously lacking Ntg1p, Ntg2p, and Apn1p are hyperrecombinogenic (hyper-rec) and exhibit a mutator phenotype but are not sensitive to the oxidizing agents H2O2 and menadione. The additional disruption of the RAD52 gene in the ntg1 ntg2 apn1 triple mutant confers a high degree of sensitivity to these agents. The hyper-rec and mutator phenotypes of the ntg1 ntg2 apn1 triple mutant are further enhanced by the elimination of the nucleotide excision repair pathway. In addition, removal of either the lesion bypass (Rev3p-dependent) or recombination (Rad52p-dependent) pathway specifically enhances the hyper-rec or mutator phenotype, respectively. These data suggest that multiple pathways with overlapping specificities are involved in the removal of, or tolerance to, spontaneous DNA damage in S. cerevisiae. In addition, the fact that these responses to induced and spontaneous damage depend upon the simultaneous loss of Ntg1p, Ntg2p, and Apn1p suggests a physiological role for the AP lyase activity of Ntg1p and Ntg2p in vivo.     

Synergism between base excision repair,mediated by the DNA glycosylases Ntg1 and Ntg2, and nucleotide excision repair in the removal of oxidatively damaged DNA bases in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, inactivation of the two DNA N-glycosylases Ntg1p and Ntg2p does not result in a spontaneous mutator phenotype, whereas simultaneous inactivation of Ntglp, Ntg2p and Radlp or Rad14p, both of which are involved in nucleotide excision repair (NER), does. The triple mutants rad1 ntg1 ntg2 and rad14 ntg1 ntg2 show 15- and 22-fold increases, respectively, in spontaneous forward mutation to canavanine resistance (CanR) relative to the wild-type strain (WT). In contrast, neither of these triple mutants shows an increase in the incidence of Lys+ revertants of the lys1-1 ochre allele. Furthermore, the rad1 ntg1 ntg2 mutant is hypersensitive to the lethal effect of H2O2 relative to WT, rad1 and ntg1 ntg2 mutant strains. Moreover, the rad1 ntg1 ntg2 strain is hypermutable (CanR and Lys+) upon exposure to H2O2, relative to WT, rad1 and ntg1 ntg2 strains. Mutagen sensitivity and enhanced mutagenesis in the rad1 ntg1 ntg2 triple mutant, relative to the other strains tested, were also observed upon exposure to oxidizing agents such as tertbutylhydroperoxide and menadione. In contrast, the sensitivity of the rad1 ntg1 ntg2 triple mutant to gamma-irradiation does not differ from that of the WT. However, the triple mutant shows an increase in the frequency of Lys+ revertants recovered after gamma-irradiation. The results reported in this study demonstrate that base excision repair (BER) mediated by Ntglp and Ntg2p acts synergistically with NER to repair endogenous or induced lethal and mutagenic oxidative DNA damage in yeast. The substrate specificity of Ntg1 p and Ntg2p, and the spectrum of lesions induced by the DNA-damaging agents used, strongly suggest that oxidized DNA bases, presumably oxidized pyrimidines, represent the major targets of this repair pathway.     

Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA

Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.     

Yeast DNA replication protein Dpb11 activates the Mec1/ATR checkpoint kinase

The Saccharomyces cerevisiae Mec1-Ddc2 protein kinase (human ATR-ATRIP) initiates a signal transduction pathway in response to DNA damage and replication stress to mediate cell cycle arrest. The yeast DNA damage checkpoint clamp Ddc1-Mec3-Rad17 (human Rad9-Hus1-Rad1: 9-1-1) is loaded around effector DNA and thereby activates Mec1 kinase. Dpb11 (Schizosaccharomyces pombe Cut5/Rad4 or human TopBP1) is an essential protein required for the initiation of DNA replication and has a role in checkpoint activation. In this study, we demonstrate that Dpb11 directly activates the Mec1 kinase in phosphorylating the downstream effector kinase Rad53 (human Chk1/2) and DNA bound RPA. However, DNA was not required for Dpb11 to function as an activator. Dpb11 and yeast 9-1-1 independently activate Mec1, but substantial synergism in activation was observed when both activators were present. Our studies suggest that Dpb11 and 9-1-1 may partially compensate for each other during yeast checkpoint function.     

Abrogation of the Chk1-Pds1 checkpoint leads to tolerance of persistent single-strand breaks in Saccharomyces cerevisiae

In budding yeast, Apn1, Apn2, Tpp1, and Rad1/Rad10 are important enzymes in the removal of spontaneous DNA lesions. apn1 apn2 rad1 yeast are inviable due to accumulation of abasic sites and strand breaks with 3' blocking lesions. We found that tpp1 apn1 rad1 yeast exhibited slow growth but frequently gave rise to spontaneous slow growth suppressors that segregated as single-gene mutations. Using a candidate gene approach, we identified several tpp1 apn1 rad1 suppressors. Deleting uracil glycosylase suppressed both tpp1 apn1 rad1 and apn1 apn2 rad1 growth defects by reducing the abasic site burden. Mutants affecting the Chk1-Pds1 metaphase-anaphase checkpoint only suppressed tpp1 apn1 rad1 slow growth. In contrast, most S-phase checkpoint mutants were synthetically lethal in a tpp1 apn1 rad1 background. Epistasis analyses showed an additive effect between chk1 and ung1, indicating different mechanisms of suppression. Loss of Chk1 partially restored cell-growth parameters in tpp1 apn1 rad1 yeast, but at the same time exacerbated chromosome instability. We propose a model in which recombinational repair during S phase coupled with failure of the metaphase-anaphase checkpoint allows for tolerance of persistent single-strand breaks at the expense of genome stability.     

Base excision repair activities required for yeast to attain a full chronological life span

The chronological life span of yeast, the survival of stationary (G0) cells over time, provides a model for investigating certain of the factors that may influence the aging of non-dividing cells and tissues in higher organisms. This study measured the effects of defined defects in the base excision repair (BER) system for DNA repair on this life span. Stationary yeast survives longer when it is pre-grown on respiratory, as compared to fermentative (glucose), media. It is also less susceptible to viability loss as the result of defects in DNA glycosylase/AP lyases (Ogg1p, Ntg1p, Ntg2p), apurinic/apyrimidinic (AP) endonucleases (Apn1p, Apn2p) and monofunctional DNA glycosylase (Mag1p). Whereas single BER glycosylase/AP lyase defects exerted little influence over such optimized G0 survival, this survival was severely shortened with the loss of two or more such enzymes. Equally, the apn1delta and apn2delta single gene deletes survived as well as the wild type, whereas a apn1delta apn2delta double mutant totally lacking in any AP endonuclease activity survived poorly. Both this shortened G0 survival and the enhanced mutagenicity of apn1delta apn2delta cells were however rescued by the over-expression of either Apn1p or Apn2p. The results highlight the vital importance of BER in the prevention of mutation accumulation and the attainment of the full yeast chronological life span. They also reveal an appreciable overlap in the G0 maintenance functions of the different BER DNA glycosylases and AP endonucleases.     

Clamping the Mec1/ATR Checkpoint Kinase into Action

The yeast checkpoint protein kinase Mec1, the ortholog of human ATR, is the essential upstream regulator of the cell cycle checkpoint in response to DNA damage and to stalling of DNA replication forks. The activity of Mec1/ATR is not directly regulated by the DNA substrates that signal checkpoint activation. Rather the signal appears to be transduced to Mec1 by factors that interact with the signaling DNA substrates. One of these factors, the DNA damage checkpoint clamp Rad17-Mec3-Ddc1 (human 9-1-1) is loaded onto gapped DNA resulting from the partial repair of DNA damage, and the Ddc1 subunit of this complex activates Mec1. In vertebrate cells, the TopBP1 protein (Cut5 in S. pombe and Dpb11 in S. cervisiae) that is also required for establishment of the replication fork, functions during replication fork dysfunction to activate ATR. Both mechanisms of activation generally upregulate the kinase activity towards all downstream targets.     

Cell-cycle-specific activators of the Mec1/ATR checkpoint kinase

Mec1 [ATR (ataxia telangiectasia mutated- and Rad3-related) in humans] is the principle kinase responsible for checkpoint activation in response to replication stress and DNA damage in Saccharomyces cerevisiae. The heterotrimeric checkpoint clamp, 9-1-1 (checkpoint clamp of Rad9, Rad1 and Hus1 in humans and Ddc1, Rad17 and Mec3 in S. cerevisiae; Ddc1-Mec3-Rad17) and the DNA replication initiation factor Dpb11 (human TopBP1) are the two known activators of Mec1. The 9-1-1 clamp functions in checkpoint activation in G1- and G2-phase, but its employment differs between these two phases of the cell cycle. The Ddc1 (human Rad9) subunit of the clamp directly activates Mec1 in G1-phase, an activity identified only in S. cerevisiae so far. However, in G2-phase, the 9-1-1 clamp activates the checkpoint by two mechanisms. One mechanism includes direct activation of Mec1 by the unstructured C-terminal tail of Ddc1. The second mech-anism involves the recruitment of Dpb11 by the phosphorylated C-terminal tail of Ddc1. The latter mechanism is highly conserved and also functions in response to replication stress in higher eukaryotes. In S. cerevisiae, however, both the 9-1-1 clamp and the Dpb11 are partially redundant for checkpoint activation in response to replication stress, suggesting the existence of additional activators of Mec1.     

Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex

Genotoxic stress activates checkpoint signaling pathways that block cell cycle progression, trigger apoptosis, and regulate DNA repair. Studies in yeast and humans have shown that Rad9, Hus1, Rad1, and Rad17 play key roles in checkpoint activation. Three of these proteins-Rad9, Hus1, and Rad1-interact in a heterotrimeric complex (dubbed the 9-1-1 complex), which resembles a PCNA-like sliding clamp, whereas Rad17 is part of a clamp-loading complex that is related to the PCNA clamp loader, replication factor-C (RFC). In response to genotoxic damage, the 9-1-1 complex is loaded around DNA by the Rad17-containing clamp loader. The DNA-bound 9-1-1 complex then facilitates ATR-mediated phosphorylation and activation of Chk1, a protein kinase that regulates S-phase progression, G2/M arrest, and replication fork stabilization. In addition to its role in checkpoint activation, accumulating evidence suggests that the 9-1-1 complex also participates in DNA repair. Taken together, these findings suggest that the 9-1-1 clamp is a multifunctional complex that is loaded onto DNA at sites of damage, where it coordinates checkpoint activation and DNA repair.     

Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA.

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.     

The role of Schizosaccharomyces pombe DNA repair enzymes Apn1p and Uve1p in the base excision repair of apurinic/apyrimidinic sites

In Schizosaccharomyces pombe the repair of apurinic/apyrimidinic (AP) sites is mainly initiated by AP lyase activity of DNA glycosylase Nth1p. In contrast, the major AP endonuclease Apn2p functions by removing 3'-alpha,beta-unsaturated aldehyde ends induced by Nth1p, rather than by incising the AP sites. S. pombe possesses other minor AP endonuclease activities derived from Apn1p and Uve1p. In this study, we investigated the function of these two enzymes in base excision repair (BER) for methyl methanesulfonate (MMS) damage using the nth1 and apn2 mutants. Deletion of apn1 or uve1 from nth1Delta cells did not affect sensitivity to MMS. Exogenous expression of Apn1p failed to suppress the MMS sensitivity of nth1Delta cells. Although Apn1p and Uve1p incised the oligonucleotide containing an AP site analogue, these enzymes could not initiate repair of the AP sites in vivo. Despite this, expression of Apn1p partially restored the MMS sensitivity of apn2Delta cells, indicating that the enzyme functions as a 3'-phosphodiesterase to remove 3'-blocked ends. Localization of Apn1p in the nucleus and cytoplasm hints at an additional function of the enzyme other than nuclear DNA repair. Heterologous expression of Saccharomyces cerevisiae homologue of Apn1p completely restored the MMS resistance of the nth1Delta and apn2Delta cells. This result confirms a difference in the major pathway for processing the AP site between S. pombe and S. cerevisiae cells.     

The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9

DNA damage activates cell cycle checkpoints that prevent progression through the cell cycle. In yeast, the DNA damage checkpoint response is regulated by a series of genes that have mammalian homologs, including rad1, rad9, hus1, and rad17. On the basis of sequence homology, yeast and human Rad1, Rad9, and Hus1 protein homologs are predicted to structurally resemble the sliding clamp PCNA. Likewise, Rad17 homologs have extensive homology with replication factor C (RFC) subunits (p36, p37, p38, p40, and p140), which form a clamp loader for PCNA. These observations predict that Rad1, Hus1, and Rad9 might interact with Rad17 as a clamp-clamp loader pair during the DNA damage response. In this report, we demonstrate that endogenous human Rad17 (hRad17) interacts with the PCNA-related checkpoint proteins hRad1, hRad9, and hHus1. Mutational analysis of hRad1 and hRad17 demonstrates that this interaction has properties similar to the interaction between RFC and PCNA, a well characterized clamp-clamp loader pair. Moreover, we show that DNA damage affects the association of hRad17 with the clamp-like checkpoint proteins. Collectively, these data provide the first experimental evidence that hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9 in manner similar to the interaction between RFC and PCNA.     

Interaction of apurinic/apyrimidinic endonuclease 2 (Apn2) with Myh1 DNA glycosylase in fission yeast

Oxidative DNA damage is repaired primarily by the base excision repair (BER) pathway in a process initiated by removal of base lesions or mismatched bases by DNA glycosylases. MutY homolog (MYH, MUTYH, or Myh1) is a DNA glycosylase which excises adenine paired with the oxidative lesion 8-oxo-7,8-dihydroguanine (8-oxoG, or G°), thus reducing G:C to T:A mutations. The resulting apurinic/apyrimidinic (AP) site is processed by an AP-endonuclease or a bifunctional glycosylase/lyase. We show here that the major Schizosaccharomyces pombe AP endonuclease, Apn2, binds to the inter-domain connector located between the N- and C-terminal domains of Myh1. This Myh1 inter-domain connector also interacts with the Hus1 subunit of the Rad9–Rad1–Hus1 checkpoint clamp. Mutagenesis studies indicate that Apn2 and Hus1 bind overlapping but different sequence motifs on Myh1. Mutation on I²⁶¹ of Myh1 reduces its interaction with Hus1, but only slightly attenuates its interaction with Apn2. However, E²⁶² of Myh1 is a key determinant for both Apn2 and Hus1 interactions. Like human APE1, Apn2 has 3′-phosphodiesterase activity. However, unlike hAPE1, Apn2 has a weak AP endonuclease activity which cleaves the AP sites generated by Myh1 glycosylase. Functionally, Apn2 stimulates Myh1 glycosylase activity and Apn2 phosphodiesterase activity is stimulated by Myh1. The cross stimulation of Myh1 and Apn2 enzymatic activities is dependent on their physical interaction. Thus, Myh1 and Apn2 constitute an initial BER complex.     

Correlation between checkpoint activation and in vivo assembly of the yeast checkpoint complex Rad17-Mec3-Ddc1

Rad17-Mec3-Ddc1 forms a proliferating cell nuclear antigen-like complex that is required for the DNA damage response in Saccharomyces cerevisiae and acts at an early step of the signal transduction cascade activated by DNA lesions. We used the mec3-dn allele, which causes a dominant negative checkpoint defect in G1 but not in G2, to test the stability of the complex in vivo and to correlate its assembly and disassembly with the mechanisms controlling checkpoint activation. Under physiological conditions, the mutant complex is formed both in G1 and G2, although the mutant phenotype is detectable only in G1, suggesting that is not the presence of the mutant complex per se to cause a checkpoint defect. Our data indicate that the Rad17-Mec3-Ddc1 complex is very stable, and it takes several hours to replace Mec3 with Mec3-dn within a wild type complex. On the other hand, the mutant complex is rapidly assembled when starting from a condition where the complex is not pre-assembled, indicating that the critical factor for the substitution is the disassembly step rather than complex formation. Moreover, the kinetics of mutant complex assembly, starting from conditions in which the wild type form is present, parallels the kinetics of checkpoint inactivation, suggesting that the complex acts in a stoichiometric way, rather than catalytically.     

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.     

Origin of endogenous DNA abasic sites in Saccharomyces cerevisiae

Abasic (AP) sites are among the most frequent endogenous lesions in DNA and present a strong block to replication. In Saccharomyces cerevisiae, an apn1 apn2 rad1 triple mutant is inviable because of its incapacity to repair AP sites and related 3'-blocked single-strand breaks (M. Guillet and S. Boiteux, EMBO J. 21:2833, 2002). Here, we investigated the origin of endogenous AP sites in yeast. Our results show that the deletion of the UNG1 gene encoding the uracil DNA glycosylase suppresses the lethality of the apn1 apn2 rad1 mutant. In contrast, inactivation of the MAG1, OGG1, or NTG1 and NTG2 genes encoding DNA glycosylases involved in the repair of alkylation or oxidation damages does not suppress lethality. Although viable, the apn1 apn2 rad1 ung1 mutant presents growth delay due to a G(2)/M checkpoint. These results point to uracil as a critical source of the formation of endogenous AP sites in DNA. Uracil can arise in DNA by cytosine deamination or by the incorporation of dUMP during replication. Here, we show that the overexpression of the DUT1 gene encoding the dUTP pyrophosphatase (Dut1) suppresses the lethality of the apn1 apn2 rad1 mutant. Therefore, this result points to the dUTP pool as an important source of the formation of endogenous AP sites in eukaryotes.     

Repair of DNA strand breaks by the overlapping functions of lesion-specific and non-lesion-specific DNA 3' phosphatases

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 of TPP1 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.