Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells*

Background: Tdp1 is a DNA repair enzyme conserved across eukaryotes. Results: Tdp1 repairs not only 3′-tyrosyl-DNA bonds and 3′-phosphoglycolates but also 5′-tyrosyl-DNA bonds and 3′-deoxyribose phosphates. Conclusion: The end processing functions of Tdp1 extend to the repair of Top2-DNA adducts and DNA breaks from base alkylation. Significance: Tdp1 has a broad range of DNA repair activities and is a potential drug target in anticancer therapy. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3′-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3′-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1−/−) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1−/− cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H2O2, and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5′-phosphotyrosyl DNA ends when they mimic the 5′-overhangs of Top2cc. Tdp1 also processes 3′-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1−/− cells to MMS and H2O2. Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.


Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1؊/؊) and
Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1؊/؊ cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H 2 O 2 , and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5-phosphotyrosyl DNA ends when they mimic the 5-overhangs of Top2cc. Tdp1 also processes 3-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1؊/؊ cells to MMS and H 2 O 2 . Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.
DNA topoisomerase I (Top1) 3 is an essential eukaryotic enzyme that regulates DNA topology by relaxing both positive and negative DNA supercoiling generated during replication and transcription (1)(2)(3). To untwist the DNA, Top1 nicks one DNA strand by covalently linking its catalytic tyrosine residue to a 3Ј-phosphate, which allows the controlled rotation of the broken strand around the intact strand (4,5). Once supercoiling is removed, Top1 is released by religation of the DNA 3Ј-phosphate with the 5Ј-hydroxyl end (2,6). Camptothecin (CPT) derivatives, such as topotecan and irinotecan, are Top1 inhibitors widely used in cancer chemotherapy (7). The indenoisoquinolines, another class of non-CPT Top1 inhibitors, are in clinical development (8). These agents kill cancer cells by stabilizing Top1 cleavage complex (Top1cc) (9) and inducing the formation of DNA double strand breaks (DSBs) upon replication fork collisions with Top1cc (3, 10 -13). Transcription has also been shown to contribute to the formation of DSBs (14 -16).
To better understand the role of Tdp1 in vertebrate cells, we took advantage of the relative ease to delete specific genes in chicken DT40 cells (43), which have well characterized repair pathways (44). We generated Tdp1-deficient chicken DT40 (Tdp1Ϫ/Ϫ) cell lines and Tdp1Ϫ/Ϫ cells complemented with human TDP1. Notably, there is a high degree of amino acid sequence identity and similarity between human and chicken Tdp1 (69 and 11%, respectively) with all catalytic residues conserved (45) (Fig. 1). We then examined the role of Tdp1 in repairing a broad range of DNA damages using the Tdp1Ϫ/Ϫ cells in cell survival assays and biochemical assays.
The role of Tdp1 in topoisomerase II cleavage complex (Top2cc) removal has been controversial. Etoposide, a clinically used Top2 inhibitor, stabilizes Top2cc, while the Top2 homodimers are covalently linked to extruding 4-nucleotide overhangs on the 5Ј-end of DNA (3,9,46). Some reports have suggested the involvement of Tdp1 in Top2cc repair (47,48), whereas others have provided contradictory data (18,30,32,49). The results of the present study clarify the involvement of Tdp1 in resolving Top2cc-mediated DNA damage. Finally, because CtIP (RBBP8) has recently emerged as a critical factor for the repair of topoisomerase-DNA complexes (26,27,50), we generated double mutant DT40 cells for Tdp1 and CtIP and investigated the relative contribution of the Tdp1-and CtIPdependent pathways in the cellular responses to Top1-and Top2-targeting drugs as well as methyl methanesulfonate (MMS).
Immunoblotting and Antibodies-To prepare whole cell lysates, cells were lysed by CelLytic TM M lysis reagent (C2978, Sigma-Aldrich). After thorough mixing and incubation at 4°C for 30 min, lysates were then centrifuged at 12,000 ϫ g at 4°C for 20 min. Supernatants were collected, aliquoted, and stored at Ϫ80°C. Preparation of mitochondrial and nuclear extracts was performed as described (6). Lysates were prepared in the same manner as whole cell lysates. Immunoblotting was carried out using standard procedures. Rabbit polyclonal anti-Tdp1 antibody was obtained from Abcam (Ab4166; Cambridge, MA). Mouse monoclonal anti-␥H2AX antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Actin antibodies were purchased from Sigma. Mouse monoclonal anti-Top1 antibody was purchased from BD Biosciences (556597). Rabbit polyclonal anti-Porin (AB-5; voltage-dependent anion channel) antibody was purchased from EMD Millipore (PC548T-5UG). Secondary antibodies were horseradish peroxidase (HRP)-conjugated antibodies to mouse or rabbit Ig (GE Healthcare).
DNA Reactions and Gel Analyses-Preparation of cell lysate was carried out as same as for immunoblotting described above. One nanomolar labeled DNA substrates in a 10-l reaction volume were incubated with the indicated concentration of recombinant human TDP1 (52) or cell lysate for the indicated time at 25°C in a buffer containing 80 mM KCl, 2 mM EDTA, 1 mM dithiothreitol (DTT), 40 g/ml bovine serum albumin, 50 mM Tris-HCl, pH 7.5, and 0.01% Tween 20. Reactions were terminated by adding 1 volume of gel loading buffer (96% (v/v) formamide, 10 mM EDTA, 1% (w/v) xylene cyanol, and 1% (w/v) bromphenol blue). Double-stranded substrates were heated at 95°C for 3 min before loading. Samples were subjected to 16% denaturing PAGE. Gels were dried and exposed on Phosphor-Imager screens. Imaging and quantification were done using a Typhoon 8600 and ImageQuant software (GE Healthcare).
Measurement of Cellular Sensitivity to DNA-damaging Agents-To assess IR sensitivity, 3 ϫ 10 5 cells in 10 ml of medium were irradiated with a 137 Cs source. To measure the sensitivity of cells to CPT, etoposide, MMS, bleomycin, and cisplatin, cells were continuously exposed to various concentrations of the drugs. Immunostaining-Cells were treated with or without 10 nM CPT for 2 h. After cytospin, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Primary antibody against ␥H2AX was detected using anti-mouse IgG secondary antibodies labeled with Alexa Fluor 488/568 (Invitrogen). Cells were mounted in antifade solution with DAPI (Vector Laboratories, Burlingame, CA) and examined using a laser-scanning confocal microscope (Zeiss LSM510) with a ϫ63 oil objective. Images were collected and processed using the Zeiss AIM software and sized in Adobe Photoshop 7.0.

RESULTS
Generation of Tdp1Ϫ/Ϫ DT40 Cells-We disrupted one of two Tdp1 alleles using targeting construct 1 (Tdp1-1-puro) carrying a puromycin resistance gene ( Fig. 2A) to generate Tdp1ϩ/Ϫ cells. Gene targeting was confirmed by Southern blotting (Fig. 2B). However, disruption of the other allele was not successful using targeting construct 1 carrying a hygromycin resistance gene. This indicated the possibility of an allelespecific mutation(s) between the two Tdp1 gene alleles. Therefore, we generated targeting construct 2 (Tdp1-2-hyg; Fig. 2A) in which the homology arms were amplified using the genomic DNA from the Tdp1ϩ/Ϫ cells as template. Targeting construct 2 successfully disrupted the other Tdp1 allele (Fig. 2B). We confirmed Tdp1 gene disruption by RT-PCR using paired primers a/b that were designed to flank both resistance genes containing stop codons. Other paired primers c/d that were designed from the 5Ј-side of the targeted sites were used as a control. As expected, primers a/b amplified the cDNA of wild type and Tdp1ϩ/Ϫ cells, whereas none of three independent Tdp1Ϫ/Ϫ cell clones yielded detectable product (Fig. 2C). To complement the Tdp1Ϫ/Ϫ cells, we transfected the FLAGtagged human TDP1 cDNA in Tdp1Ϫ/Ϫ cells. The chicken Tdp1Ϫ/Ϫ cells complemented with human TDP1 (hereafter referred to as Tdp1Ϫ/Ϫ;hTDP1 cells) showed a similar amount of TDP1 protein expression compared with human 293T cells (Fig. 2D). Several available antibodies raised against human TDP1 did not react with the chicken Tdp1 ( Fig. 2D and data not  shown). The proliferative properties of generated cells were indistinguishable from those of wild type cells as monitored by growth curves (Fig. 2E) and by cell cycle analysis (data not shown).
To examine the tyrosyl-DNA phosphodiesterase activity of the mutant cells, we performed gel-based assays using a 14-nt single-stranded DNA oligonucleotide bearing a 3Ј-phosphotyrosine (14Y) labeled at the 5Ј terminus with 32 P (19, 23, 49). Tdp1 activity was measured by the extent of substrate conversion into a 3Ј-phosphate DNA product (14P). The 14P product can be readily converted further to a 3Ј-hydroxyl DNA product (14OH) by polynucleotide kinase 3Ј-phosphatase (Fig. 2F, upper panel) (53). The 32 P-labeled 14Y substrate was incubated with serially diluted whole cell lysates from wild type, Tdp1Ϫ/Ϫ, and Tdp1Ϫ/Ϫ;hTDP1 cells in buffer without magnesium. Wild type and Tdp1Ϫ/Ϫ;hTDP1 cells showed similar processing activity that was detectable with as little as 0.1 ng/l whole cell lysate. By contrast, no processing activity was observed in Tdp1Ϫ/Ϫ cells extract even at 8,800-fold excess (880 ng/l) (Fig. 2F, lower panel). These results show that Tdp1 is the only factor able to process the 3Ј-phosphotyrosyl-DNA bond under divalent cation-free conditions and that human TDP1 can complement chicken Tdp1 for the 3Ј-phosphotyrosyl-DNA bond processing activity.
To determine whether Tdp1 was also functioning in DT40 cell mitochondria, we examined the Tdp1 activity of mitochondrial extracts by fractionating wild type and Tdp1Ϫ/Ϫ cells into nuclear and mitochondrial fractions and then performed the gel-based assays using 14Y substrates (supplemental Fig. S1). The mitochondrial fraction from wild type cells showed robust 3Ј-phosphotyrosyl diesterase activity, whereas no detectable processing activities were observed for the mitochondrial fraction of Tdp1Ϫ/Ϫ cells. These data demonstrate that the nuclear Tdp1 gene provides 3Ј-phosphodiesterase activity to both nuclear and mitochondrial DNA.
Broad Involvement of Tdp1 for DNA Repair-We examined the sensitivity of Tdp1Ϫ/Ϫ cells to various types of DNA-damaging agent (Fig. 3). As expected (31,32), Tdp1Ϫ/Ϫ cells showed hypersensitivity to CPT. Low dose CPT (10 nM) also induced high ␥H2AX signal (12) in the Tdp1Ϫ/Ϫ cells as detected by immunostaining and Western blotting (supplemental Fig. S2, A and B) and induced the accumulation of cells at the G 2 phase in Tdp1Ϫ/Ϫ cells (supplemental Fig. S2C).
Tdp1Ϫ/Ϫ cells showed consistent hypersensitivity to etoposide, a selective Top2 inhibitor (3,46), marginal sensitivity to IR, and strong sensitivity to bleomycin in agreement with the fact that both IR and bleomycin induce DSBs and/or single strand breaks with 3Ј-PG ends that can be processed by Tdp1 (6,37,39,54). Tdp1Ϫ/Ϫ cells also showed significant sensitivity to MMS and mild sensitivity to H 2 O 2 . Complementation of the Tdp1Ϫ/Ϫ cells with human TDP1 reduced the sensitivities to these treatments. These results suggest that Tdp1 has broad involvement in the repair of lesions induced by a variety of DNA-damaging agents.
Cleavage of Top2cc by Tdp1-Our cell survival results showing that Tdp1Ϫ/Ϫ cells are hypersensitive to etoposide prompted us to examine the involvement of Tdp1 for the repair of Top2cc in greater detail. To test the activity of Tdp1 for Top2cc removal, we performed gel-based cleavage assays using DNA substrates with 5Ј-phosphotyrosyl ends. In addition to a blunt-ended double strand substrate (Y40/40), we tested substrates with 5Ј-overhangs of 2, 4, or 6 bases (Y40/38, Y40/36, and Y40/34, respectively; Fig. 4). The internally labeled substrates contained phosphate groups on the 3Ј-end to prevent the 3Ј-nucleosidase activity of Tdp1 from removing the last

Tdp1-mediated DNA Repair
base at the 3Ј-end (19,49). Use of these particular substrates eliminated the confounding issue of multiple products generated by TDP1 and allowed us to unambiguously interpret our results of 5Ј-phosphotyrosine processing activity by TDP1. When these substrates were incubated with recombinant human TDP1, the 5Ј-phosphotyrosine was removed for the 2-, 4-, and 6-base overhang substrates (Y40/38, Y40/36, and Y40/ 34) but not for the blunt-ended substrate (Y40/40). Notably, the 4-and 6-base overhang substrates (Y40/36 and Y40/34) were processed more efficiently than the 2-base overhang substrate (Y40/38). TDP1 also cleaved a single-stranded DNA harboring 5Ј-phosphotyrosine efficiently in a dose-dependent manner (Fig. 5). Note the substrates labeled on the 3Ј-end generated one additional product due to the 3Ј-nucleosidase activity of TDP1 (Fig. 5). When we tested a 5Ј-fluorescein (6-FAM)-labeled oligonucleotide, only one product was generated from this substrate. Because TDP1 had full 3Ј-nucleosidase activity under the same conditions (Fig. 5B, compare left and right panels), we attributed the product to the 3Ј-nucleosidase activity of TDP1 (Fig. 5A). Consistent with our previously published results (49), we conclude that TDP1 cannot process a 5Ј-fluorescein (6-FAM)-labeled oligonucleotide. Additional experiments revealed that TDP1 did not process 5Ј-phosphate or 5Ј-hy-droxyl DNA ends to any appreciable degree (supplemental Fig.  S3). Altogether, these results demonstrate that human TDP1 has specific processing activity for 5Ј-phosphotyrosine linkage in the context of 5Ј-overhangs, which are landmarks of Top2cc.
Involvement of Tdp1 for Repair of Abasic Sites-Because the hypersensitivity of Tdp1Ϫ/Ϫ cells to MMS was strong and almost fully reverted by human TDP1 (Fig. 3), we investigated the biochemical basis of this hypersensitivity. MMS is a strong electrophile that attacks the most nucleophilic centers in DNA, resulting in DNA base methylation (55). Methylated DNA bases can be converted into abasic (AP) sites by DNA N-glycosylase. H 2 O 2 also generates modified bases such as 8-oxoguanine, which can lead to AP sites (56) (Fig. 6A). AP sites are then repaired by various pathways, including base excision repair, nucleotide excision repair, translesion synthesis, or homologous recombination. In the process of base excision repair, a 3Ј-dRP can form as a result of ␤-elimination by OGG1 and NTH1 that inhibits the gap filling by DNA polymerase ␤ (57). Although 3Ј-dRP is known to be removed by AP endonuclease 1, a recent study revealed that both an AP site and a 3Ј-dRP are also cleaved by Tdp1 (38). Consistent with these results, we also demonstrated that recombinant human TDP1 can process a 3Ј-dRP substrate. The processing efficiency of 3Ј-dRP by TDP1 was 17-fold less than that of 3Ј-phosphotyrosine in terms of EC 50 (the half-maximal effective concentration) (Fig. 6, B and C, and supplemental Fig. S4). Therefore, the sensitivity of Tdp1Ϫ/Ϫ cells to MMS and H 2 O 2 is consistent with impaired repair activity of AP sites and 3Ј-dRP in these cells.
Parallel and Overlapping DNA Repair Functions of Tdp1 and CtIP-CtIP is known to be involved in homologous recombination pathways. Recent studies show that CtIP, together with BRCA1, acts in the nuclease-mediated elimination of Top1cc and Top2cc (26,27,50). CtIP S332A/Ϫ/Ϫ DT40 cells, which harbor an alanine substitution at residue Ser-332 of CtIP that abrogates its interaction with BRCA1, are hypersensitive to CPT, etoposide, and MMS but remain proficient in homologous recombination (26). Because this function overlaps with that of Tdp1, we generated double mutant DT40 cells (referred to as CtIP S332A/Ϫ/Ϫ;Tdp1Ϫ/Ϫ) to examine the relationship between Tdp1 and CtIP in different types of DNA repair. Both

DISCUSSION
The role of Tdp1 is well established for the repair of Top1induced DNA damage (20,21,31,32,35,58,59). Because inactivation of Tdp1 augments the antiproliferative effect of Top1 inhibitors, Tdp1 inhibitors are being explored as anticancer drugs in cancer cells with preexisting Top1cc repair defects (45,60). Our results expand the spectrum of repair pathways that involve Tdp1, including the repair of DNA lesions produced by etoposide, IR, bleomycin, MMS, and H 2 O 2 . Biochemical assays corroborate this view given that human TDP1 can process 3Ј-PGs (6,37,39,54), 3Ј-dRP (present study), 5Ј-phosphotyrosine (present study), and AP sites (19,38). 3Ј-PG ends represent up to half of the breaks induced by IR (40) and are present at a large fraction of the DSB ends produced by bleomycin (37,54). Accordingly, cells from Tdp1 knock-out mice are defective in the repair of IR-and H 2 O 2 -induced breaks and hypersensitive to bleomycin (31,32). Spinocerebellar ataxia neuropathy 1 cells are also radiosensitive and unable to rapidly repair H 2 O 2induced single strand breaks (20). Consistent with these results, our finding that Tdp1Ϫ/Ϫ cells are hypersensitive to H 2 O 2 , bleomycin, and IR suggests a role for Tdp1 in the processing of 3Ј-PG and possibly AP sites. Moreover, oxidative lesions pro-   6-FAM19). A, scheme for the processing pathways of Y19 (left) and 6-FAM19 (right) by TDP1. Because of the nucleosidase activity of TDP1 (19), which removes the last 3Ј-end base, the Y19 substrate can be processed in two ways. One is that the 5Ј-tyrosine is removed first (P19) before the 3Ј-terminal adenine (P18) (clockwise). The other is that the 3Ј-adenine is removed first (Y18) before the 5Ј-tyrosine (P18) (counterclockwise). An asterisk indicates the radiolabeled site. B, each substrate was incubated with serial dilutions (1:3) of TDP1 at 25°C for 30 min with the highest TDP1 concentration starting at 1 M. Processing of 6-FAM19 by TDP1 yielded one product (right), whereas processing of Y19 yielded two distinct products (left), indicating that TDP1 processes 5Ј-phosphotyrosine but not 5Ј-fluorescein.

Tdp1-mediated DNA Repair
duced by IR and H 2 O 2 are known to lead to the formation of Top1cc (61), which would also require Tdp1 for their removal.
5Ј-Phosphodiesterase activity has been reported for yeast Tdp1. Moreover, deletion of the Tdp1 gene in yeast confers hypersensitivity to etoposide (47), and overexpression of human TDP1 in human cells counteracts the DNA damage mediated by etoposide (48). On the other hand, Tdp1 knockout mice and embryonic fibroblasts as well as spinocerebellar ataxia neuropathy 1 cells are not noticeably hypersensitive to etoposide (21,32). Thus, the potential role of Tdp1 in the repair of Top2-mediated DNA damage has been a long standing controversy (18,47). Our cell survival data with etoposide clearly indicate that Tdp1 can be involved in Top2cc repair in metazoans (Fig. 3). Why the sensitivity to etoposide is detectable in chicken Tdp1Ϫ/Ϫ cells but not in mouse and human Tdp1 mutant cells is unclear. One possibility is that chicken DT40 cells might be limited in their alternative repair pathways for Top2cc, whereas redundant pathways are present in human and murine cells. The repair protein complexes involved in the repair of Top2cc are not well understood in vertebrate cells except for the recent discovery of Tdp2 (62,63). It is also possible that DT40 cells show a differential response to etoposide because of the rapid cell cycle of DT40 cells (ϳ8 h for one cycle) with most of the cells in S phase. This may also contribute to the hypersensitivity of DT40 cells to CPT. The complementation by exogenous expression of human TDP1 in Tdp1Ϫ/Ϫ cells only partially restored the resistance of Tdp1Ϫ/Ϫ cells to etoposide, whereas it almost fully restored normal responses to CPT and MMS (Fig. 3). This difference might be due to speciesspecific protein-protein interactions whereby human TDP1 fails to interact fully with other chicken-specific repair complexes that are critical for Top2cc and alkylation damage removal. Despite these differences, our biochemical results using recombinant human TDP1 (Figs. 4 and 5) strongly support that Tdp1 plays a role in the removal of Top2cc and contributes to the response to Top2 inhibitors.
To our knowledge, the involvement of Tdp1 in the repair of the DNA lesions induced by the classical alkylating agent MMS has not been reported. MMS produces N7-guanine methyl . DNA adducts like methylated base and 8-oxoguanine (8-oxo) are converted into AP sites by DNA glycosylase. In one way, Tdp1 cleaves the AP site, directly generating the 3Ј-phosphate end. In the other way, the AP site turns out to be a single strand break with a 3Ј-dRP end after hydrolysis by AP lyase. Tdp1 cleaves the 3Ј-dRP, generating the 3Ј-phosphate end. B, processing activity of recombinant human TDP1 on the 14-nt 3Ј-phosphotyrosyl substrate (14Y) and the 14-nt 3Ј-dRP substrate (14dRP). Each substrate was incubated with serial dilutions (1:3) of TDP1 at 25°C for 30 min with the highest concentration starting at 30 nM. The 14-nt 3Ј-phosphate (14P) was used as a marker. A gel representative of consistent results in independent experiments is shown. C, quantification of TDP1 processing activity for each substrate from B. EC 50 values (the half-maximal effective concentration) for 14Y and 14dRP are 0.062 and 1.078 nM, respectively.  APRIL 13, 2012 • VOLUME 287 • NUMBER 16 adducts that are readily converted to AP sites by glycosylases or/and spontaneous base elimination. Such lesions are known to trap Top1cc (64 -66), which could explain the participation of Tdp1 in their repair. However, our present study also suggests a more direct involvement of Tdp1 in the repair of abasic sites by direct cleavage of the 3Ј-blocking dRP lesions (19,38).

Tdp1-mediated DNA Repair
The repair of Top1-and Top2-induced DNA lesions involves redundant pathways (46,66). A recent study revealed that CtIP, together with BRCA1, acts in the nuclease-mediated elimination of Top1cc and Top2cc (26). Deletion of Ctp1, the ortholog of CtIP in Schizosaccharomyces pombe, markedly sensitizes cells to CPT (50), and deletion of Sae2, the Saccharomyces cerevisiae ortholog of CtIP, produces a mild sensitization to CPT (36,67). Our data show at least an additive sensitization of Tdp1Ϫ/Ϫ;CtIP S322A/Ϫ/Ϫ double mutant cells compared with the single mutants, indicating parallel (redundant) activities of CtIP and Tdp1 for Top1cc repair in DT40 cells. By contrast, we found that Tdp1 and CtIP are epistatic for the repair of Top2cc, suggesting that Tdp1 and CtIP work together for the removal of Top2cc. Further investigations are warranted to elucidate the possible interactions of Tdp1 and CtIP for the repair of Top2-induced DNA lesions.