Association of XRCC1 and tyrosyl DNA phosphodiesterase (Tdp1) for the repair of topoisomerase I-mediated DNA lesions

DNA Repair 2 (2003) 1087–1100

Association of XRCC1 and tyrosyl DNA phosphodiesterase (Tdp1) for the repair of topoisomerase I-mediated DNA lesions Isabelle Plo a,1 , Zhi-Yong Liao a , Juana M. Barceló a , Glenda Kohlhagen a , Keith W. Caldecott b , Michael Weinfeld c , Yves Pommier a,∗ a

Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institute of Health, Building 37, Room 5068, Bethesda, MD, USA b University of Sussex, Falmer, Brighton BN1 9RR, UK c University of Alberta, Edmonton, Alberta, Canada T6G 1Z2 Received 27 February 2003; accepted 4 June 2003

Abstract DNA topoisomerase I (Top1) is converted into a cellular poison by camptothecin (CPT) and various endogenous and exogenous DNA lesions. In this study, we used X-ray repair complementation group 1 (XRCC1)-deficient and XRCC1-complemented EM9 cells to investigate the mechanism by which XRCC1 affects the cellular responses to Top1 cleavage complexes induced by CPT. XRCC1 complementation enhanced survival to CPT-induced DNA lesions produced independently of DNA replication. CPT-induced comparable levels of Top1 cleavage complexes (single-strand break (SSB) and DNA–protein cross-links (DPC)) in both XRCC1-deficient and XRCC1-complemented cells. However, XRCC1-complemented cells repaired Top1-induced DNA breaks faster than XRCC1-deficient cells, and exhibited enhanced tyrosyl DNA phosphodiesterase (Tdp1) and polynucleotide kinase phosphatase (PNKP) activities. XRCC1 immunoprecipitates contained Tdp1 polypeptide, and both Tdp1 and PNKP activities, indicating a functional connection between the XRCC1 single-strand break repair pathway and the repair of Top1 covalent complexes by Tdp1 and PNKP. © 2003 Elsevier B.V. All rights reserved. Keywords: Camptothecin; Topoisomerase I; XRCC1; Transcription-mediated damage; Replication-mediated DSB

Abbreviations: APE1, AP endonuclease; BER, base excision repair; BRCT, BRCA1 carboxyl terminal; BSA, bovine serum albumin; CPT, camptothecin; DPC, DNA–protein cross-links; DSB, double-strand break; FBS, fetal bovine serum; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PARP, poly(ADP-ribose)polymerase; PNKP, polynucleotide kinase phosphatase; SSB, single-strand break; SSBR, single-strand break repair; Tdp1, tyrosyl DNA phosphodiesterase; Top1, topoisomerase I; XRCC1, X-ray cross-complementing group 1 ∗ Corresponding author. Fax: +1-301-402-0752. E-mail address: [email protected] (Y. Pommier). 1 Present address: L’Association pour la Recherche sur le Cancer, Laboratory of Recombination Mechanisms, DRR/DSV/CEA, 68 Avenue du G´en´eral Leclerc, 92265 Fontenay-aux-Roses, France.

1. Introduction DNA topoisomerase I (Top1) is ubiquitous [1] and essential [2]. It relaxes DNA supercoiling ahead of replication and transcription complexes by inducing transient single-strand breaks (SSB) in the phosphate-deoxyribose backbone, thereby allowing rotation of the DNA double helix around the intact phosphodiester bonds opposite the enzyme-mediated DNA cleavages. Once the DNA has been relaxed, Top1 religates the breaks and regenerates intact duplex DNA. Biochemically, the Top1-mediated DNA breaks

1568-7864/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1568-7864(03)00116-2

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result from the reversible transfer of a DNA phosphodiester bond to the enzyme catalytic tyrosine (Tyr723) (for review see [3–5]). The covalent Top1-cleaved DNA intermediates are referred to as “cleavage complexes” [6]. In cells, cleavage complexes can be detected as protein-linked DNA single-strand breaks and DNA–protein cross-links (DPC) by the alkaline elution technique [7,8]. Identification of these unique DNA lesions led to the discovery that DNA topoisomerases are the targets for some of the most effective anticancer and antimicrobial drugs [8–10]. Recently, Top1 cleavage complexes have been detected after formation of endogenous and exogenous DNA lesions (for review see [11]), including UV-induced base modifications, guanine methylation and oxidation, polycyclic aromatic carcinogenic adducts [12], base mismatches, abasic sites, cytosine arabinoside or gemcitabine incorporation [13] and DNA nicks. Topoisomerase I (Top1) inhibitors such as camptothecin (CPT) convert Top1 into a cellular poison by inhibiting the religation step of the Top1 nickingclosing reaction, and thereby trapping Top1 cleavage complexes (for review see [9,14–17]). In cells, the Top1 cleavage complexes induced by CPT and its derivatives can be detected as protein-linked SSB [7]. Top1 cleavage complexes are converted to cytotoxic lesions primarily by collisions between replication forks and drug-stabilized Top1 cleavage complexes. These collisions generate replication-mediated DNA double-strand breaks (DSB) [18–20]. DNA damage can also result from replication-independent DNA lesions [21–25], such as collisions between transcription complexes and CPT-stabilized cleavage complexes [26,27]. Tyrosyl DNA phosphodiesterase (Tdp1) catalyzes the removal of the Top1 polypeptide linked to the 3 -DNA terminus by hydrolyzing the covalent bond between the Top1 tyrosyl residue and the 3 -DNA– phosphate [5,28–32]. Povirk and coworkers also reported that Tdp1 can remove 3 -phosphoglycolate from DNA ends generated by oxidative DNA damage [33]. Tdp1-mediated hydrolysis produces a DNA break terminated with a 3 -phosphate. Polynucleotide kinase phosphatase (PNKP) can remove this phosphate and produce a 3 -hydroxyl terminus that can be extended by DNA polymerase ␤ and/or ligated by DNA ligases [34,35]. The conservation of Tdp1

and PNKP from yeast to humans is suggestive of the importance of this repair pathway [28,36]. Furthermore, genetic studies recently demonstrated the role of Tdp1 for survival of yeast in the presence of Top1-mediated DNA damage [29,37,38]. Cellular resistance or sensitivity to Top1 inhibitors is determined by pre- and post-cleavage complex mechanisms. The latter are related to the multiple pathways leading to apoptosis, cell cycle regulation and checkpoints, and DNA repair [15,39]. Alteration of these pathways, which is a common characteristic of transformed cells, probably accounts for the selectivity of camptothecins for cancer cells. Previous studies have shown that several DNA repair-deficient cell lines are hypersensitive to camptothecins (for review see [15,40]). Among these cell lines, the Chinese hamster cell line EM9, which is defective in SSB repair (SSBR), is hypersensitive to CPT [41,42]. Its DNA repair complementation group has been designated X-ray cross-complementing group 1 (XRCC1), and EM9 cells have an inactivating mutation of the XRCC1 gene [43]. Other characteristics of the EM9 cells include hypersensitivity to DNA methylating agents and ionizing radiation, and a high frequency of spontaneous sister chromatid exchanges [44]. The XRCC1 gene product is a scaffolding protein containing two BRCA1 carboxyl terminal (BRCT) domains, which are binding motifs for DNA repair and cell cycle checkpoint proteins [45]. XRCC1 is implicated in SSBR (for review see [46,47]). XRCC1 forms a multimeric repair complex with several enzymes implicated in base excision and gap repair: DNA ligase III [48,49], poly(ADP-ribose)polymerase 1 (PARP-1) [50,51], poly(ADP-ribose)polymerase 2 (PARP-2) [52], DNA polymerase ␤ [50,53,54], polynucleotide kinase phosphatase (PNKP) [55], and AP endonuclease (APE1) [56]. In addition, the XRCC1 N-terminal region interacts with DNA containing a single-stranded gap [57,58]. The XRCC1 complex therefore represents what could be a remarkably coherent repair system (for review see [46,47]): PARP would bind to the break and recruit the XRCC1 complex to the DNA lesion; then, PNKP could process the ends of the broken DNA generating a 3 -hydroxyl and a 5 -phosphate; DNA polymerase ␤ could then extend the 3 -end and fill the gap, and ligase III could complete the reaction by joining the

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newly synthesized patch to the 5 -end of the broken DNA. The aim of the present study was to investigate the role of XRCC1 in the repair of Top1-mediated DNA lesions and in the cytotoxic response induced by camptothecins. We compared CPT-induced cytotoxicity, SSBR, as well as Tdp1 and PNKP activities in XRCC1-complemented and XRCC1-deficient EM9 cells [59]. The results presented here demonstrate the involvement of XRCC1 in the cytotoxic response to CPT, and suggest that XRCC1 is involved in the repair of SSB resulting from Top1 cleavage complexes in association with Tdp1 and PNKP. 2. Materials and methods 2.1. Cells

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2.3. Top1 Western blot analyses Cells (5 × 105 ) were washed with phosphate buffered saline (PBS) [BioWhittaker Molecular Applications, Rockland, ME] and lyzed for 30 min in 250 ␮l of buffer containing 1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 2 ␮g/ml aprotinin, and 1 mM sodium orthovanadate. Cell extracts were clarified and supernatants (25 ␮l) were added to 25 ␮l 2× SDS loading buffer (Invitrogen Corporation, Carlsbad, CA). Two microliters aliquots of each sample were loaded in SDS–PAGE (8%), transferred onto nitrocellulose and probed with a mouse monoclonal antibody directed against human Top1 (Ab C-21, kindly provided by Dr. Yung-Chi Cheng, New Haven, CT). Proteins were detected by chemiluminescence.

The Chinese hamster ovary (CHO) EM9 cell line was isolated on the basis of its sensitivity to EMS [44]. The EM9 cells were transfected by calcium phosphate co-precipitation either with empty (control) pcD2E expression vector or pcD2E construct encoding histidine-tagged wild-type human XRCC1, as reported recently [59]. The resulting cell lines are referred to as EM9-V (XRCC1-deficient with empty vector) and EM9-XH (EM9 cells complemented with XRCC1). Both cell lines were cultured in DMEM with 10% heat inactivated fetal bovine serum (FBS) complemented with 250 ␮g/ml glutamine, 110 ␮g/ml sodium pyruvate, and 1.5 mg/ml G418.

Sub-confluent proliferating cells were labeled with [14 C]-thymidine (0.02 ␮Ci/ml) (Perkin-Elmer Life Science Co., Boston, MA) for 48 h, chased for 2 h in isotope-free medium, and exposed to CPT. Equal numbers of cells were loaded onto polycarbonate or PVC filters, lyzed and subjected to alkaline elution [7,8,60]. Radioactivity in the elution fractions was counted and the filter retention rate was calculated. Computations were performed according to the formulas (units in Rad-equivalents): For SSB “long method”:

2.2. MTT assays

SSB =

This assay is based on the ability of mitochondria to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, St Louis, MI), a soluble tetrazolium salt into an insoluble formazan precipitate. This precipitate was dissolved in dimethyl sulfoxide and quantified by spectrophotometry. Approximately 2000 cells were seeded in 96-well dishes for 24 h, then treated with CPT in the presence or absence of aphidicolin (APD) (10 ␮M, 1 h) (Sigma, St Louis, MI), post-incubated for 72 h, and tested for MTT conversion. Absorbance was read at two wavelengths 540 and 690 nm.

2.4. DNA filter elution assays

log(retentiontreated cells /retentioncontrol cells ) log(retention300 rad /retentioncontrol cells ) × 300

For SSB “short method”: SSB =

log(retentiontreated cells /retentioncontrol cells ) log(retention2000 rad /retentioncontrol cells ) × 2000

For DNA–protein cross-links (DPC): DPC = [(1 − retentiontreated cells )−1 − (1 − retentioncontrol cells )−1 ] × 3000

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2.5. Preparation of nuclear extracts Approximately 107 cells grown in culture to 80–90% confluence were harvested and washed twice by centrifugation-resuspension in ice-cold nucleus buffer [150 mM NaCl, 1 mM KH2 PO4 pH 6.4, 5 mM MgCl2, 1 mM EGTA, supplemented with CompleteTM Mini (Roche, Indianapolis, IN) protease inhibitor cocktail tablets]. Cells were then exposed to 0.3% Triton X-100 and washed through centrifugation. The nuclear pellets were mixed gently with an equal volume of nucleus buffer containing 0.4 M NaCl at 4 ◦ C for 30 min. The nuclear protein extracts, obtained as the supernatant fractions after centrifugation, were stored at −80 ◦ C until use. 2.6. Tdp1 and PNKP assays HPLC purified oligonucleotides were purchased from the Midland Certified Reagent Co. (Midland, TX). The 5 -32 P-end radio labeled oligonucleotides were prepared by incubation with T4 polynucleotide kinase (Life Technologies, Rockville, MD) and [␥32 P]ATP (Perkin-Elmer Life Science Co., Boston, MA) according to the manufacturer’s protocols. Unincorporated nucleotides were removed by Sephadex G-25 spin-column chromatography (Mini Quick Spin Oligo Columns, Roche, Indianapolis, IN). For the production of oligonucleotide duplexes, complementary oligonucleotides were mixed in equal molar ratios in the presence of annealing buffer (10 mM Tris–HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2 ), heated to 96 ◦ C, and allowed to cool slowly (over 2 h) to room temperature. For Tdp1 assays with recombinant enzyme (a gift from Dr. Howard Nash, NIMH [29]), 4 ␮l aliquots of 5 -32 P-end-labeled oligonucleotide duplex substrate (14-Y) at a 0.12 ␮M concentration were reacted for 15 min at 30 ◦ C with 10 ng Tdp1 in 50 mM Tris–HCl, pH 8.0, 80 mM KCl, 2 mM EDTA, 1 mM dithiothreitol (DTT), and 40 ␮g/ml bovine serum albumin (BSA). Assays with recombinant PNKP were performed with 10 ng purified PNKP [34] at 37 ◦ C for 15 min in the same buffer as for Tdp1 except for the addition of 25 ␮M MgCl2 (final concentration). Reactions were stopped by addition of 40 ␮l of Maxam-Gilbert loading buffer (80% (v/v) formamide, 10 mM NaOH,

1 mM EDTA, 1% (w/v) xylene cyanol, 1% (w/v) bromophenol blue). For Tdp1 and PNKP assays with nuclear extracts, the 32 P-end-labeled oligonucleotide duplex substrate (14-Y) was mixed in 36 ␮l reactions containing 6 ␮l of nuclear extract (with 5–10 ␮g/␮l of protein) and incubated at 25 ◦ C over a time-course. Successive 6 ␮l aliquots were taken at defined time-points and mixed with 30 ␮l of Maxam-Gilbert loading buffer. For assays used for the detection of activity in relation to protein amount (Fig. 6), the nuclear extract volumes were doubled in succession from 1.5 to 6 ␮l. Reactions were then incubated at 25 ◦ C for the indicated times prior to mixing with gel loading buffer. Four micoliters aliquots were loaded onto gel electrophoresis. Gel electrophoreses were performed with 20% polyacrylamide gels. After drying, gels were exposed to PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA) overnight. Screens were scanned, and images obtained with the Molecular Dynamics software (Sunnyvale, CA). 2.7. XRCC1 immunoprecipitation and Western blotting for XRCC1 and Tdp1 For immunoprecipitation, 5 ␮g anti-human XRCC1 mouse monoclonal antibody Ab-3 (NeoMarkers, Fremont, CA) were added to each nuclear extract sample (400 ␮g in 400 ␮l) and incubated at 4 ◦ C for 1–2 h. Sixty ␮l Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added per sample and incubated overnight at 4 ◦ C. The beads were then washed in 500 ␮l nucleus buffer three times, spun (6000 rpm × 2 min), and collected. Samples were resuspended in 20 ␮l nucleus buffer before testing Tdp1 activity. For Western blotting, 40 ␮l nucleus buffer were added to 60 ␮l from the immunoprecipitates. Samples were mixed and 40 ␮l 2× SDS loading buffer added. Mixtures were boiled for 5 min, and 40 ␮l samples were loaded in 4–12% Tris–glycine gel (Invitrogen Corporation, Carlsbad, CA). Proteins were electrotransferred overnight at 5 V to Hybond-P membranes (Amersham Pharmacia Biotech, Piscataway, NJ) using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA). The membranes were probed with polyclonal rabbit anti-human Tdp1 antibodies (a gift from Dr. Howard Nash, NIMH) or mouse monoclonal

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anti-XRCC1 Ab-3 antibody (NeoMarkers, Fremont, CA). These antibodies were diluted 1:1000 in PBS containing 3% Membrane Blocking Agent (Amersham Biosciences UK Limited, Little Chalfont Buckinghamshire, UK) and 0.05% Tween 20 (B3T5 buffer). After 3 h incubation at 25 ◦ C, the membranes were washed three times in PBS containing 0.05% Tween 20, and were incubated for 1 h at 25 ◦ C with donkey anti-rabbit Ig (Amersham Biosciences UK Limited, Little Chalfont Buckinghamshire England) for Tdp1 or sheep anti-mouse Ig (Amersham Biosciences UK Limited, Little Chalfont Buckinghamshire England) for XRCC1. Secondary antibodies were both diluted 1:50 000 in B3T5 buffer. Proteins were detected by chemiluminescence using the SuperSignal West Pico Chemiluminescence Substrate (PIERCE, Rockford, IL).

3. Results 3.1. XRCC1-complementation reduces CPT-induced cytotoxicity in EM9 cells XRCC1-deficient (EM9-V) or XRCC1-complemented (EM9-XH) EM9 cells were treated with a range of CPT concentrations for 72 h. Cellular viability was determined either by counting live cells or by MTT assays. As shown in Fig. 1, XRCC1complemented EM9 cells were more resistant to

Viable cells (% of control)

100 EM9-XH + APD EM9-XH

30

EM9-V + APD 10 EM9-V 3 0

100

200

300

400

500

CPT (nM)

Fig. 1. XRCC1 complementation confers resistance to CPT in non-replicating and replicating EM9 cells. XRCC1-deficient (squares) and XRCC1-complemented (circles) EM9 cells were treated with the indicated concentrations of CPT for 72 h in the absence or presence of 10 ␮M aphidicolin. Aphidicolin (APD; filled symbols and solid lines) was added 15 min before CPT.

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CPT than the XRCC1-deficient EM9 cells. The IC50 were 280 and 25 nM for EM9-XH cells and EM9-V cells, respectively. This difference demonstrates that XRCC1 complementation increases resistance to CPT. Replication-dependent and independent DNA damage induced by CPT can be distinguished by treating the cells with aphidicolin, a specific inhibitor of DNA polymerases ␣, ␦, and ε [61], prior to exposure to CPT [18,62–65]. By inhibiting the movement of replication forks, aphidicolin prevents the formation of replication-mediated DNA double-strand breaks [18]. Therefore, under these conditions, it is assumed that most Top1 cleavage complexes are converted into DNA damage primarily by transcription-mediated DNA lesions. To investigate the role of the replication component in the repair of residual SSB following CPT treatment, XRCC1-deficient (EM9-V) and XRCC1-complemented (EM9-XH) cells were pre-treated with aphidicolin (10 ␮M) for 15 min before the addition of CPT. Fig. 1 shows that aphidicolin only partially protected the XRCC1-deficient (EM9-V) cells [42], and that XRCC1 complementation enhanced both replication-independent and dependent survival. 3.2. XRCC1-complementation has a minimal effect on the formation of CPT-induced Top1 cleavage complexes in EM9 cells We next evaluated Top1 expression in XRCC1deficient and XRCC1-complemented EM9 cells by Western blotting. Fig. 2A shows that Top1 levels were comparable in both cell lines, with slightly higher level in XRCC1-deficient EM9 cells than in XRCC1-complemented EM9 cells. The cytotoxicity of CPT and its clinical derivatives is directly related to the levels of Top1 cleavage complexes in tumors [66], in human cell lines [15,67–69], and in yeast [70,71]. To investigate the mechanisms by which XRCC1 reduced CPT-induced cytotoxicity, we evaluated the formation of Top1 cleavage complexes using the alkaline elution assay. Top1–DNA covalent complexes can be measured as DPC. As shown in Fig. 2B, CPT treatment induced a dose-dependent increase in DPC in both cell lines. No significant difference in DPC was found between XRCC1-deficient and XRCC1-complemented EM9 cells. To further investigate the frequency of Top1

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Fig. 2. Top1 expression and Top1 cleavage complexes induced by CPT in XRCC1-complemented (EM9-XH) and XRCC1-deficient (EM9-V) cells. (A) Top1 expression was determined by Western blot analysis with an anti-Top1 antibody in XRCC1-deficient (EM9-V; lane 1) or XRCC1-complemented (EM9-XH; lane 2) EM9 cells. (B) XRCC1 complementation does not affect CPT-induced DPC formation in EM9 cells. XRCC1-deficient (EM9-V), or XRCC1-complemented (EM9-XH) cells were treated with CPT (0.1, 0.3, or 1 ␮M) for 1 h and DPC were measured by alkaline elution assays. Results are mean ± S.D. of at least three independent experiments. (C) XRCC1 complementation does not affect CPT-induced SSB in EM9 cells. DNA elution curves are shown for XRCC1-deficient (EM9-V; left panel) and XRCC1-complemented (EM9-XH; right panel) cells treated with CPT (1 ␮M) for 1 h (filled circles) or 3 h (open circles). Cells that had been irradiated with 20 Gy (filled squares) were used as calibration controls. Elution curves of untreated cells are shown as open squares. Results are mean ± S.D. of at least three independent experiments.

cleavage complexes, CPT-induced DNA SSB were measured in XRCC1-deficient and complemented EM9 cells. Top1-mediated SSB can be detected as an increase in the DNA elution rate in alkaline elution assays [8,60]. In the absence of DNA damage, most of the cellular DNA remains in the filter, whereas, in the presence of DNA breaks, the DNA elutes from the filter. SSB frequencies can be determined from the elution slopes, and expressed in Rad-equivalents [8,60]. Fig. 2C shows comparable elution rates for the XRCC1-deficient (Fig. 2C, left panel) and XRCC1-complemented cells (Fig. 2C,

right panel) treated with CPT (1 ␮M; at both time points examined). Topoisomerase cleavage complexes are characterized by equal frequencies of SSB and DPC because, in a topoisomerase cleavage complex, each SSB is associated with a DPC (i.e. a topoisomerase covalent complex) [7,8,60,72]. Table 1 shows that CPT produced similar frequencies of SSB and DPC in both cell lines, indicating that the SSB and DPC corresponded to Top1 cleavage complexes. Furthermore, Table 1 shows that both XRCC1-complemented and XRCC1-deficient cells produced similar frequencies

I. Plo et al. / DNA Repair 2 (2003) 1087–1100 Table 1 CPT induces similar DPC and SSB in XRCC1-deficient and XRCC1-complemented EM9 cells

DPC SSB

EM9-V

EM9-X

3311 ± 1553 4088 ± 470

3621 ± 1369 3929 ± 937

XRCC1-deficient (EM9-V) or XRCC1-complemented (EM9-XH) cells were treated with CPT (1 ␮M, 1 h). DPC or SSB were calculated using alkaline elution as described in Section 2. Results (in Rad-equivalents) are mean ± S.D. of at least three independent experiments.

of SSB and DPC. Thus, treatment with CPT generated similar levels of Top1 cleavage complexes in XRCC1-deficient and XRCC1-complemented EM9 cells. Moreover, no significant difference in SSB and DPC was found up to 6 h after addition of CPT in both cell lines (data not shown). These results show that XRCC1 does not affect the production of Top1 cleavage complexes. 3.3. XRCC1 enhances SSB repair (SSBR) in CPT-treated EM9 cells To determine whether XRCC1 complementation affected the repair of Top1 cleavage complexes, the reversal kinetics of CPT-induced SSB were examined using the high sensitivity alkaline elution SSB assay. XRCC1-deficient (EM9-V) and XRCC1complemented (EM9-XH) cells were treated with 1 ␮M CPT for 1 h, washed with fresh medium and incubated in CPT-free medium for 15, 30, and 60 min. Fig. 3A shows a more rapid normalization of the DNA elution rates in the XRCC1-complemented (EM9-XH) cells than in the XRCC1-deficient (EM9-V) cells. The corresponding elution rates were computed and converted to SSB Rad-equivalents (Table 2). One SSB Rad-equivalent corresponds to approximately 1 SSB per 109 nucleotides [8]. For both cell lines, about 95% of the SSB reversed rapidly (within 15 min) after drug removal [7], which can be interpreted as indicating that only a small fraction (approximately 5%) of the cleavage complexes corresponded to DNA lesions in replicating and transcribing DNA [9,11,14]. Fig. 3B shows that the CPT-induced SSB reversed more rapidly in the XRCC1-complemented (EM9-XH) cells than in the XRCC1-deficient EM9-V cells. This difference was also observed in

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Table 2 XRCC1 complementation enhances SSBR in EM9 cells Time after CPT (min)

15 30

SSB (Rad-equivalents) EM9-V

EM9-XH

222 ± 33 161 ± 15

127 ± 28∗ 51 ± 27∗

XRCC1-deficient (EM9-V) or XRCC1-complemented (EM9-XH) cells were treated with CPT (1 ␮M) for 1 h, washed with fresh medium and incubated in CPT-free medium for 15 or 30 min. SSB were determined by alkaline elution as described in Section 2. Results are mean ± S.D. of at least three independent experiments. ∗ Significant differences (P < 0.05).

aphidicolin-treated cells, suggesting that XRCC1 is implicated in the repair of Top1-mediated DNA lesions produced independently of DNA replication. 3.4. XRCC1-complementation enhances Tdp1 activity The enzymatic activities of Tdp1 and PNKP were analyzed by measuring the successive specific modifications of an oligonucleotide substrate that mimics a Top1–DNA adduct repair intermediate [31,73]. Tdp1-mediated hydrolysis produces a DNA break terminated with a 3 -phosphate (Fig. 4A). PNKP can remove this phosphate and produce a 3 -hydroxyl terminus that can be extended by DNA polymerase ␤ and/or ligated by DNA ligases [34,35]. The upper strand was end-labeled at the 5 -end with [␥32 P]ATP (Fig. 4B) for the purpose of detecting the reaction products in DNA sequencing gels [32]. The 14-P product migrates more rapidly than the 14-Y oligopeptide substrate in DNA sequencing gels. The 3 -OH product resulting from hydrolysis of the 3 -phosphate migrates more slowly than the corresponding 3 -phosphate because of its reduced negative charge due to the loss of the phosphate (Fig. 4C) [29,32]. Fig. 4C shows that PNKP is unable to remove the 5 -tyrosyl conjugate (lane 6), indicating that production of a 3 -hydroxyl DNA terminus from a 3 -tyrosyl oligopeptide requires the prior activity of Tdp1. To determine whether XRCC1 could influence the repair of Top1 covalent complexes, the Tyr–DNA conjugate (14-Y oligopeptide) was incubated with nuclear extracts obtained from XRCC1-deficient (EM9-V) and XRCC1-complemented (EM9-XH) cells (Fig. 5). Nuclear extract from XRCC1-deficient

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(A)

Fraction of DNA remaining on filter

EM9-V (XRCC1-deficient)

EM9-XH (XRCC1-complemented)

1

1

0,1

0,1

0,01

0,01 0

5

10

15

0

5

10

15

Elution time (hours) EM9-V

(B)

EM9-XH

300

300

SSB (Rad-equivalents)

- APD + APD 200

200

100

100

0

0 15

30

15

30

Time after CPT removal (min) Fig. 3. XRCC1 enhances SSB repair in EM9 cells independently of DNA replication. (A) DNA elution curves of XRCC1-deficient (EM9-V; left panel) and XRCC1-complemented (EM9-XH, right panel) cells treated with CPT (1 ␮M) for 1 h (open circles). The high sensitivity long elution method was used [60]. Elution curves for untreated cells are shown as open squares. CPT was removed by two centrifugations and resuspension in fresh medium. The figure shows elution curves at various times after CPT removal: 15 min (filled circles), 30 min (filled triangles), and 60 min (open triangles). Cells that had been irradiated with 3 Gy (filled squares) were used as calibration controls. Elution curves of untreated cells are shown as open squares. Results are mean ± S.D. of at least three independent experiments. (B) Reversal of CPT-induced SSB in the absence and presence of aphidicolin (APD). XRCC1-deficient and XRCC1-complemented EM9 cells were pre-treated with 10 ␮M aphidicolin (filled rectangles) or without aphidicolin (gray rectangles) for 15 min prior to the addition of 1 ␮M CPT. After 1 h, cells were washed with fresh medium and incubated in the absence of drug for 15 or 30 min. SSB (expressed in Rad-equivalents) were determined using the high sensitivity alkaline elution long method. A representative experiment is shown.

(EM9-V) cells showed reduced processing of the 14-Y substrate to the 14-OH product when compared to nuclear extracts from XRCC1-complemented (EM9-XH) cells (Fig. 5). These results indicate that XRCC1 complementation enhances Tdp1 and PNKP activities.

3.5. Tdp1is associated with XRCC1 Because XRCC1 is known to act as a scaffold for the binding of several enzymes implicated in SSBR [48–54,56], including PNKP [55], we tested its possible association with Tdp1. Fig. 6A shows that Tdp1

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Fig. 4. Repair of Tyr–DNA complexes by Tdp1 and PNKP. (A) Schematic representation for the activities of Tdp1, and subsequently of PNKP on a tyrosyl DNA adduct. PNKP can also phosphorylate the 5 -hydroxyl end of a DNA break generated by Top1 (not shown). (B) The oligopeptide 14-Y was used as a substrate. Radiolabeling was at the 5 -terminus of the upper stand (indicated as 32 P). (C) Consecutive processing of the 14-Y oligopeptide substrate (14-Y; shown in (A) and (B)), and 3 -phosphate oligonucleotide (14-P; see (A)) by Tdp1 and PNKP, respectively. The resulting 3 -OH oligonucleotide (14-OH; see (A)) runs slower than the corresponding 14-P through polyacrylamide gels. T4 kinase, the bacteriophage equivalent of PNKP [34] also hydrolyzes the 3 -phosphate generated by Tdp1 (lane 5). PNKP requires Mg2+ as a cofactor (lane 4). PNKP alone does not process the original 14-Y oligopeptide unless the 3 -Tyr has previously been hydrolyzed by Tdp1 (lane 6).

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Fig. 6. Tdp1 and PNKP activities are associated with XRCC1. (A) XRCC1 immunoprecipitate from XRCC1-complemented (EM9-XH) nuclear extract were reacted for 20 min with the 14-Y substrate (shown in lane 1) in the absence (lane 5) or presence of sodium orthovanadate (lane 6), a reported Tdp1 inhibitor [74]. Lane 4 shows a comparable reaction with nuclear extracts run on protein A/G beads in the absence of XRCC1 antibodies (mock reaction). Lane 3 shows the activity of EM9-XH nuclear extract before immunoprecipitation. Lane 2 is a marker for the 14-P product. Reaction products were separated in a DNA sequencing gel and visualized after PhosphorImager analysis. A representative experiment is shown. (B) Western blotting for Tdp1 (upper panel) and XRCC1 (lower panel). Lane 1: nuclear extract from EM9-XH cells before immunoprecipitation; lane 3: XRCC1 immunoprecipitate and lane 2 mock precipitation as described for (A).

Fig. 5. XRCC1-complemented EM9 cells have enhanced Tdp1 and PNKP activities. (A) The 14-Y substrate shown in Fig. 4B was incubated with nuclear extracts (6 ␮l per reaction) from XRCC1-deficient (EM9-V) and XRCC1-complemented (EM9-XH) cells for the indicated times. Reaction products were separated in DNA sequencing gels and visualized by PhosphorImager analysis. A representative experiment is shown. (B) Quantitation of 14-OH product from experiment shown in (A). (C) The 14-Y substrate shown in Fig. 5B was incubated for 5 min with increasing volumes of nuclear extracts from XRCC1-deficient (EM9-V) and XRCC1-complemented (EM9-XH) cells. Reaction products were separated in DNA sequencing gels and formation of the 14-OH product was quantitated after PhosphorImager analysis.

activity co-immunoprecipitated with XRCC1 antibodies. This activity was inhibited in the presence of sodium orthovanadate, a reported inhibitor of Tdp1 [74] (Fig. 6A, lane 6). Finally, Western blotting showed the presence of Tdp1 in the XRCC1 immunoprecipitates (Fig. 6B, lane 3). These results demonstrate an interaction between Tdp1 and XRCC1. 4. Discussion In this study, we show that XRCC1 complementation protects against CPT-induced cytotoxicity and enhances the repair of replication-independent SSB induced by Top1 cleavage complexes. We also find that XRCC1 complementation enhances Tdp1 and PNKP activities, and that Tdp1 is associated with XRCC1.

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Hypersensitivity of the EM9 cells to CPT [41,42] is due to XRCC1-deficiency since XRCC1-complementation of EM9 cells is sufficient to confer marked resistance to CPT. Similarly, Cheng and coworkers [75] reported a correlation between CPT resistance and XRCC1 levels in human epidermoid carcinoma KB cells. CPT-induced DNA damage consists of Top1-linked DNA breaks with Top1 covalently linked to 3 -DNA termini. These lesions are referred to as “Top1 covalent complexes” [9,11,14,16,17,40]. In order to investigate whether the hypersensitivity of XRCC1-deficient cells was related to an increase in cleavage complexes, we measured CPT-induced DPC and SSB. Top1 cleavage complexes are characterized by an equal frequency of DPC and SSB [7,8]. Although Top1 levels were slightly lower in the XRCC1-complemented cells than in the XRCC1deficient cells, CPT produced comparable levels of DPC and SSB in both cell lines. Moreover, different times of incubation with CPT had no influence on the levels of DPC and SSB. These results indicate that XRCC1 does not affect the generation of Top1 cleavage complexes by CPT. We then investigated the role of XRCC1 in the repair of Top1-mediated DNA damage. Approximately 95% of the SSB associated with Top1 cleavage complexes induced by CPT reversed within 15 min independently of XRCC1. These rapidly reversible complexes correspond to the religation of cleavage complexes once CPT is removed [7]. XRCC1 enhanced the reversal of the SSB that remain after 15, 30, and 60 min following CPT removal, as the XRCC1-complemented (EM9-XH) cells repaired these SSB more efficiently than the XRCC1-deficient (EM0-V) cells. These results are consistent with a role for XRCC1 in the repair in the DNA lesions induced by CPT. It is well-established that a fraction of the druginduced Top1 cleavage complexes are converted into cytotoxic lesions after replication forks collide with the cleavage complexes [62–64]. Aphidicolin, an inhibitor of DNA polymerases ␣, ␦, and ε [61], has been shown to protect cells from CPT by preventing the formation of replication-mediated DNA double-strand breaks [18]. Other DNA lesions occur independently of replication because CPT can kill non-replicating cells such as neurons [24]. In agreement with our results, D’Arpa and coworkers [42] showed that aphidicolin only partially inhibits

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CPT-induced cytotoxicity in EM9 cells suggesting that lethal damage is produced independently of DNA replication. In the present study, we found that the repair defect in XRCC1-deficient cells was comparable in the presence and in the absence of aphidicolin. It is noticeable that aphidicolin does not inhibit DNA polymerase ␤ [61], the base excision repair (BER) polymerase, which interacts with XRCC1 and is involved in SSBR [47]. aphidicolin was only able to partially inhibit the cytotoxicity of CPT in XRCC1-deficient EM9 cells, suggesting that part of the difference between the two cell lines is independent of DNA replication. At this point, we may

Fig. 7. Schematic representation for the proposed repair of a Top1 covalent complex by the XRCC1-dependent pathway. The XRCC1 complex including the associated repair enzymes is shown at the top. Tdp1 hydrolyzes the Top1–DNA–phosphotyrosyl bond. PNKP hydrolyzes the resulting 3 -phosphate and phosphorylates the 5 -hydroxyl. ␤-Polymerase fills the gap and ligase III seals the DNA.

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hypothesize that the replication-independent DNA lesions could result from transcription-mediated DNA damage [27]. Thus, our results suggest that XRCC1 is involved in the repair of replication-independent SSB, presumably associated with stalled transcription complexes induced by Top1 cleavage complexes. The experiments performed with a substrate mimicking a Top1 covalent complex intermediate [29,32] indicate that XRCC1 enhances the activity of the Tdp1/PNKP pathway, which has been implicated in the repair of Top1 covalent complexes [29,31,36– 38,76]. Our finding that Tdp1 co-immunoprecipitates with XRCC1 indicates that XRCC1 could act to recruit Tdp1 to the DNA single-strand break sites produced by Top1 cleavage complexes. Fig. 7 shows a proposed scheme for the role of XRCC1 DNA repair complexes for the repair of Top1-mediated DNA lesions. Such complexes would contain the enzymes required to excise the Top1–DNA adduct (Tdp1), process the DNA ends (PNKP), replace the missing DNA segment (␤-polymerase), and seal (ligase III) the DNA. Although poly(ADP-ribose)polymerase (PARP) [50–52] is known to form complexes with XRCC1 and to contribute to the survival of CPT-treated cells [77], further investigations are needed to determine its functional role in the repair of Top1-mediated DNA damage.

Acknowledgements We wish to thank Dr. Feridoun Karimi-Busheri and Mr. Mesfin Fanta for technical assistance. We wish to thank Dr. Yung-Chi Cheng (Yale University, New Haven, CT) for the kind gift of the Top1 monoclonal antibody, and Dr. Howard A. Nash (NIMH, Bethesda, MD) for the kind gift of the Tdp1 antibodies. Dr. Michael Weinfeld was supported by a grant from the Canadian Institutes of Health Research (MOP-15385).

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