Effects of 5′,8-Cyclodeoxyadenosine Triphosphates on DNA

Nov 12, 2012 - Hydroxyl radicals generate a broad range of DNA lesions in living cells. Cyclopurine deoxynucleosides (CPUs) are a biologically signifi...
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Effects of 5′,8-Cyclodeoxyadenosine Triphosphates on DNA Synthesis Naoto Kamakura,† Junpei Yamamoto,† Philip J. Brooks,‡ Shigenori Iwai,† and Isao Kuraoka*,† †

Graduate School of Engineering Science, Osaka University Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan ‡ Section on Molecular Neurobiology, Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892, United States S Supporting Information *

ABSTRACT: Hydroxyl radicals generate a broad range of DNA lesions in living cells. Cyclopurine deoxynucleosides (CPUs) are a biologically significant class of oxidative DNA lesions due to their helical distortion and chemically stability. The CPUs on DNA are substrates for the nucleotide excision repair (NER) but not for base excision repair or direct damage reversal. Moreover, these lesions block DNA and RNA polymerases, resulting in cell death. Here, we describe the chemical synthesis of 5′S and 5′R isomers of 5′,8-cyclodeoxyadenosine triphosphate (cdATP) and demonstrate their ability to be incorporated into DNA by replicative DNA polymerases. DNA synthesis assays revealed that the incorporation of the stereoisomeric cdATPs strongly inhibits DNA polymerase reactions. Surprisingly, the two stereoisomers had different mutagenic profiles, since the S isomer of cdATP could be inserted opposite to the dTMP, but the R isomer of cdATP could be inserted opposite to the dCMP. Kinetic analysis revealed that the S isomer of cdATP could be incorporated more efficiently (25.6 μM−1 min−1) than the R isomer (1.13 μM−1 min−1) during DNA synthesis. Previous data showed that the S isomer in DNA blocked DNA synthesis and the exonuclease activity of DNA polymerase and is less efficiently repaired by NER. This indicates that the S isomer has a tendency to accumulate on the genome DNA, and as such, the S isomer of cdATP may be a candidate cytotoxic drug.

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INTRODUCTION A variety of DNA lesions are caused by environmental and endogenous factors. Hydroxyl radical can induce more than 30 different base lesions in genomic DNA, many of which are ringsaturated or ring-condensed derivatives of pyrimidines.1−3 However, oxidation of purines can also occur, particularly in association with saturation or fragmentation of the guanine imidazole ring. Many DNA lesions induced by hydroxyl radicals (e.g., thymine glycol and 8-oxoguanine) are removed by base excision repair (BER) using specialized DNA glycosylases, such as endonuclease III (NTH1) and 8-oxoguanine-DNA glycosylase (OGG1) found in human cells.4,5 The cyclopurine deoxynucleosides (CPUs) are a structurally unusual type of lesion induced by the hydroxyl radical. After exposure of cells or DNA solutions to hydroxyl radical, 5′,8cyclodeoxyadenosine (cdA) is formed slightly more frequently than 5′,8-cyclodeoxyguanosine (cdG).6−9 These biologically significant, chemically stable lesions include a covalent bond that is formed between the C-8 position of the purine and the C-5′ residue of the adjacent deoxyribose. Consequently, the base is attached to the DNA backbone by two covalent bonds, one of which is the normal glycosyl bond. The presence of the C8−C5′ covalent bond prevents DNA repair by glycosylases © 2012 American Chemical Society

during in BER, and direct damage reversal is unable to remove the lesion. Therefore, nucleotide excision repair (NER) is the only known mechanism that can remove the CPU lesions from DNA.10−12 Lesion accumulation is thought to be one potential cause of the neurodegeneration seen in NER-deficient xeroderma pigmentosum (XP). Elevated levels of these lesions have also been reported in other pathologic conditions,15,16 including transcription-coupled NER-deficient Cockayne syndrome.13,14 The CPU lesion can occur in two stereoisomeric forms, 5′S and 5′R. Both isomers are generated by oxygen free radicals in similar amounts in double-stranded DNA, whereas the 5′R isomer is predominant in single-stranded DNA.8,17 The cdA lesions inhibit DNA synthesis by microbial, replicative mammalian, and translesion DNA polymerases and are resistant to the exonucleolytic activity of DNA polymerase. The S isomer of CPU lesions blocks transcription in mammalian cells and is less efficiently repaired by NER. Thus, the S isomer on the DNA is thought to have a potential cytotoxic effect greater than that of the R isomer in vivo.18 Received: August 9, 2012 Published: November 12, 2012 2718

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purity of the major product was analyzed by HPLC, using a TSKgel DEAE-2SW column (4.6 mm × 250 mm) with a linear gradient of ammonium formate in 20% acetonitrile (0−0.8 M for 20 min) at a flow rate of 1.0 mL/min. The fractions containing the major product were concentrated in vacuo, and after triethylammonium hydrogencarbonate was removed by coevaporation with water, the countercation was exchanged with sodium using the Bio-Rad AG 50W-X2 resin. The sodium salt of (5′S)-5′,8-cyclo-2′-deoxyadenosine 5′-triphosphate was obtained as white powder after evaporation. Yield, 19.2 mg (33.3 μmol, 31%). 1H NMR (500 MHz, D2O): δ 8.11 (s, 1H, H2), 6.45 (d, 1H, H1′), 5.80 (dd, 1H, H5′), 5.11 (d, 1H, H4′), 4.87 (q, 1H, H3′), 2.65 (dd, 1H, H2′), 2.22 (m, 1H, H2″). 31P NMR (203 MHz, D2O, trimethyl phosphate): δ −11.5 (d, Pa), −14.6 (d, Pg), −24.9 (t, Pb). The (5′R) isomer was synthesized in the same manner, using 33.3 mg (71 μmol) of (5′R)-N6-benzoyl-3′-O-(tert-butyldimethylsilyl)-5′,8cyclo-2′-deoxyadenosine26 as the starting material. In this case, the final product was purified further by anion-exchange HPLC, under conditions similar to those for the above-mentioned analysis, after separation on DEAE cellulose. Yield, 2.6 mg (4.5 μmol, 6%). 1H NMR (500 MHz, D2O): δ 8.21 (s, 1H, H2), 6.63 (d, 1H, H1′), 5.53 (d, 1H, H5′), 5.19 (s, 1H, H4′), 4.57 (q, 1H, H3′), 2.60 (dd, 1H, H2′), 2.23 (m, 1H, H2″). 31P NMR (203 MHz, D2O, trimethyl phosphate): δ −10.6 (d, Pa), −15.3 (d, Pg), −24.8 (t, Pb). DNA Synthesis Assays. DNA synthesis assays were performed as previously described.23,27,28 Briefly, the 5′-32P-labeled primer-template complex was prepared by mixing the primer with a template containing the indicated sequence context at a molar ratio of 1:1 (Figure 2A). Reaction mixtures of 10 μL containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, and 40 nM labeled primertemplate complex, the indicated deoxyribonucleotide triphosphates, and 0.1 unit of Klenow fragment (KF) (New England Biolabs) were incubated at 30 °C. The reactions were terminated by the addition of 10 μL of stop solution containing 95% formamide, 10 mM EDTA,

The widely used anticancer drugs 2′,2′-difluoro-2′-deoxycytidine (gemcitabine) and 1-β-D-arabinofuranosylcytosine (cytarabine) are nucleoside analogues.19−22 In living cells, these analogues are metabolized to biologically active deoxynucleotide triphosphates, which can be used as substrates for DNA replication. Incorporation of the analogues by DNA polymerase primarily leads to the inhibition of DNA synthesis. Anticancer drugs such as cis-diamminedichloroplatinum(II) (cisplatin) also act by inducing the formation of platinum− DNA adducts that block replication.23,24 In both cases, blocking DNA replication by anticancer drugs is thought to induce cell death. Stemming from this concept, we report the chemical synthesis of 5′S and 5′R isomers of 5′,8-purine cyclodeoxyadenosine triphosphate (cdATP) as a new nucleotide analogue compound. We further assess their ability to be incorporated into duplex DNA by replication. In vitro primer extension reactions revealed that both stereoisomeric cdATPs inhibited DNA synthesis reactions, but the S isomer of cdATP is more efficiently incorporated into DNA than the R isomer. Previous data showed that the S isomer in DNA blocked DNA synthesis and the exonuclease activity of DNA polymerase and is less efficiently repaired.11,18 Thus, drugs based on the S isomer of cdATP may be potential cytotoxic compound candidates.

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MATERIALS AND METHODS

Synthesis of the Two Stereoisomers of 5′,8-Cyclo-2′deoxyadenosine 5′-Triphosphate (Figure 1). To a solution of

Figure 1. 5′R and 5′S diastereoisomers of 5′,8-purine cyclodeoxyadenosine triphosphates (cdATP). (5′S)-N6-benzoyl-3′-O-(tert-butyldimethylsilyl)-5′,8-cyclo-2′-deoxyadenosine25 (50.3 mg, 107 μmol) in anhydrous pyridine (107 μL) and 1,4-dioxane (321 μL), a 1 M solution (182 μL, 182 μmol) of 2-chloro4H-1,3,2-benzodioxaphosphorin-4-one in pyridine was added. After 20 min, another 100 μL (100 μmol) of the reagent was added, and the mixture was allowed to stand for 20 min. Then, a mixture of a 0.5 M solution of bis(tributylammonium) pyrophosphate in N,N-dimethylformamide (364 μL, 182 μmol) and tributylamine (120 μL) was added, and the resultant mixture was stirred for 20 min. After the completion of the reaction was confirmed by thin-layer chromatography on a silica gel RP-18W F254S plate (Merck) using a 1:1 mixture of acetonitrile and 0.1 M triethylammonium acetate (pH 7.0) as a mobile phase, 1% iodine (3.0 mL, 230 μmol) in pyridine−water (98/2, v/v) was added. After 20 min, 5% sodium hydrogensulfite was added dropwise until the solution discolored. The solution was concentrated on a rotary evaporator, and the residue was dried over phosphorus oxide in a desiccator equipped with a vacuum pump. This residue was dissolved in 1 M tetrabutylammonium fluoride in tetrahydrofuran (1.0 mL, 1.0 mmol), and the solution was allowed to stand for 24 h. Water (1.5 mL) was added to the solution, and the solvent was removed by evaporation. The residue was dissolved in 28% ammonia−water (3.0 mL) and heated to 60 °C in a sealed vial. After 6 h, the solution was concentrated in vacuo, and the product was purified on a Bio-Rad BioLogic LP system, using a column (1.5 cm × 50 cm) of DEAE cellulose with a linear gradient of triethylammonium hydrogencarbonate (0.1−1.0 M for 360 min) at a flow rate of 2.0 mL/min. The

Figure 2. (A) Schematic drawing of a series of 30-mer oligonucleotide templates (A, C, G, or T) annealed to a 32P-labeled 16-mer primer. (B) KF was incubated with one of the indicated DNA template-primer complexes at 37 °C for 5 min in the presence of 40 μM 5′R-cdATP (lanes 1−4) or 40 μM 5′S-dATP (lanes 5−8) and then were analyzed using 15% polyacrylamide/7.5% M urea gel electrophoresis. 2719

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0.025% bromphenol, and 0.025% xylene cyanol. The products were separated by electrophoresis on a denaturing 15% polyacrylamide gel, dried, and analyzed using a FUJIFILM BAS 1800 bioimage analyzer. Data Analysis. Kinetic parameters were determined from primer extension reactions.28 The velocity of each deoxynucleotide incorporation, V, was determined by dividing the amount of the reaction product by the reaction time. The relationship between V and dNTP concentration conformed to a Michaelis−Menten equation. Vmax (the maximum value of reaction velocity) and Km (dNTP concentration at which the reaction velocity is half-maximal) were determined from the nonlinear least-squares fit (Figure S1 in the Supporting Information). These parameters were used to calculate the frequency of deoxynucleotide insertion ( f ins). Triplet determinations of the velocities were performed for each template dNTP combination at six different dNTP concentrations. Less than 20% of the primers were extended under the steady-state conditions ensuring single hit conditions. Here, kcat was presented by utilizing the equation kcat = [Vmax(mol of primer-template)]/[(mol of polymerase)]. Model Construction. The model in Figure 5 was visualized by Pymol ver. 0.99 (DeLano Scientific LLC, South San Francisco, CA) and was constructed using coordinates deposited in the Protein Data Bank with the accession number of 1QSY (ref PMID: 10449720). Three-dimensional structures of 5′R- and 5′S-cdA were constructed by ChemBio3D Ultra (Cambridgesoft, Cambridge, MA) and were followed by structural optimization with density functional theory (DFT). The calculations based on DFT were carried out using Gaussian03W at the B3LYP/6-31G(d) level. The four atoms (C1′, N7, N3, and C6) within these nucleosides were fitted with those of the incoming dATP using the pair-fitting wizard equipped in Pymol.

2B, lanes 1, 3, and 4). This result indicates that 5′R-cdATP, unlike dATP, is unable to form a normal Watson−Crick base pair with dTMP during DNA polymerase reactions. In contrast, 5′S-cdATP was preferentially inserted opposite dTMP or dCMP (Figure 2B, lanes 6 and 8), indicating that 5′S-cdATP can form a normal Watson−Crick base pair with dTMP. Interestingly, 5′S-cdATP can be inserted opposite first dCMP and second dTMP (Figure 2B, lane 6) in the template-primer (Figure 2A, template C) without pause, thus indicating that 5′S-cdATP does not strongly inhibit DNA polymerase activity after insertion of 5′S-cdATP opposite dCMP. To further analyze the insertion of cdATPs in the DNA polymerase reaction, primer extension reactions were performed at various cdATP concentrations (Figure 3). At

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RESULTS Synthesis of (5′S)- and (5′R)-5′,8-Cyclo-2′-deoxyadenosines and Their 5′-Triphosphates. According to the literature (25), N6- and 3′-O-protected (5′S)-5′,8-cyclo-2′deoxyadenosine was prepared first, and this compound was converted to the (5′R) isomer using the method developed by the same group.25 Both compounds were characterized by nuclear Overhauser effect spectroscopy (NOESY) experiments after assignment of the 1H NMR signals by correlation spectroscopy (COSY). In the NOESY spectrum of the first obtained product, a cross-peak was observed between the H4′ and the H5′ but not between the H3′ and the H5′. In contrast, the second compound showed cross-peaks not only between the H4′ and the H5′ but also between the H3′ and the H5′, with the latter being stronger. These results, as well as the chemical shifts reported for the two isomers,25,26 demonstrated that the configuration at the 5′-position of each compound was correct. The two isomers were separately converted to the 5′triphosphate using the method described by Ludwig and Eckstein.29 Following this, the protecting groups were removed. The final products were purified by performing chromatography on DEAE cellulose and analyzed by anion-exchange HPLC, as described in the Materials and Methods. Because of impurities detected for the (5′R)-triphosphate after chromatography on DEAE cellulose, further purification by HPLC was required, resulting in a reduced yield. The obtained products had retention times similar to those of dATP in anion-exchange HPLC analysis and showed three signals, which could be assigned to Pa, Pg, and Pb from the lower field, in the 31P NMR measurement. cdATPs Incorporation in DNA. To investigate which template deoxyribonucleoside monophosphate cdATP is inserted opposite, we employed the KF of DNA polymerase I, utilizing the indicated template-primer complexes (Figure 2A). 5′R-cdATP was inserted opposite dCMP in the template (Figure 2B, lane 2), but not opposite other DNA bases (Figure

Figure 3. (A) Schematic drawing of the 30-mer oligonucleotide template C, annealed to a 32P-labeled 16-mer primer (top panel). KF and primer-template C were incubated with increasing amounts of 5′R-cdATP (lanes 1−6) at 30 °C for 5 min (bottom panel). (B) Schematic drawing of the 30-mer oligonucleotide template T, annealed to a 32P-labeled 16-mer primer (top panel). KF and primer-template T were incubated with increasing amounts of 5′S-cdATP (lanes 1−6) at 30 °C for 5 min (bottom panel). In each group of six lanes, the cdATP concentrations were 0.5, 1, 5, 10, 50, and 100 μM.

concentrations between 5 and 100 μM, very little 5′R-cdATP was inserted opposite dCMP in template (Figure 3A, lanes 3− 6). In contrast, when 5′S-cdATP was inserted opposite dTMP (Figure 3B, lanes 3−6; 5−100 μM), the products were increased in a concentration-dependent manner. The data suggested that 5′S-cdATP is more efficiently inserted than 5′RcdATP during the DNA polymerase reaction. To quantitatively assess the efficiency and fidelity of 5′R- and 5′S-cdATPs incorporation by KF, we measured the kinetics of nucleotide triphosphate insertion during DNA synthesis. The Km and kcat parameters were determined and used to calculate the efficiency of the reaction (kcat/Km) and the frequency of nucleotide incorporation (Fins) (Table. 1). 2720

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Table 1. Kinetic Parameters of the Nucleotide Insertion Reaction by KFa substrate dATP:dT 5′R-cdATP:dC 5′S-cdATP:dT

Km (μM) −3

4.11 ± 1.13 × 10 3.80 ± 0.61 2.57 ± 0.82

kcat (min−1)

kcat/Km (μM−1 min−1)

f insb

79.5 ± 6.14 4.28 ± 0.30 65.9 ± 2.89

1.93 × 10 1.13 25.6

1 5.85 × 10−5 1.33 × 10−3

4

a Data are expressed as the values ± standard deviations obtained from three independent experiments. bFrequency of insertion as compared to that for dATP opposite dTMP.

As indicated by the kcat/Km values, DNA polymerase incorporates dATP opposite the dTMP (1.93 × 104), 5′RcdATP opposite the dCMP (kcat/Km = 1.13), and 5′S-cdATP opposite the dTMP (kcat/Km = 25.6). Thus, the efficiency to incorporate 5′R-cdATP or 5′S-cdATP was about 17000- or 750-fold lower than that of dATP incorporation, respectively. This is consistent with the results showing that 5′S-cdATP was inserted more frequently than 5′R-cdATP (Figure 3A,B). Next, to investigate whether DNA polymerase can insert deoxynucleotides opposite a DNA template after incorporation of cdATP during a DNA extension reaction, primer extension reactions were performed at indicated nucleotide combinations (Figure 4). When KF was incubated with the template and dATP, dCTP, and dTTP, DNA synthesis was undetectable (Figure 4A,B, lanes 2 and 3). In the presence of dGTP (Figure 4A) or dATP (Figure 4B), KF catalyzed the synthesis of complementary strands of DNA from parent strands (Figure 4A,B, lanes 4 and 5). Using 5′R-cdATP, instead of dGTP, the polymerase paused at 19-mer and 22-mer opposite dCMPs on the template (Figure 4A, lanes 6 and 7). In the case of 5′ScdATP, KF paused at 20-mer, 26-mer, and 29-mer opposite dTMPs (Figure 4B, lanes 6 and 7). The data indicate that KF is able to catalyze DNA synthesis after the incorporation of both cdATPs. The DNA synthesis products of 5′S-cdATP were longer than that of 5′R-cdATP, indicating that 5′S-cdATP was more readily incorporated and extended in DNA than 5′RcdATP was.

C8−C5′ covalent bond, which stabilizes the glycosidic bond against acid-induced hydrolysis.30 Therefore, once in the genome, they can accumulate in NER-deficient or TC-NERdeficient cells. This accumulation may be involved in the progressive neurodegeneration seen in XP individuals.10−12 In this study, we report that chemically synthesized cdATP isomers differentially inhibit DNA synthesis reactions at the site of incorporation, using a replicative DNA polymerase. After incorporation of the damaged nucleotides, further DNA synthesis reactions were able to continue with the incorporation of other nucleotides. Notably, stereospecific differences between the 5′R- and the 5′S-cdATP affected the relative incorporation of DNA polymerase. This could be due to the differential recognition of the DNA polymerase active site. Covalent 8,5′-bond of isomers cause the restricted rotations near the active site in DNA polymerase. Figure 5A shows one model of the isomers and the active site of KF in the closed form viewed from different positions. Thermus aquaticus KF reportedly exists in either open or closed forms in the ternary complex.31 The triphosphate moiety of an incoming nucleotide is first captured in the open form without forming its base pair, and then, the polymerase transitions to the closed form and transfers the nucleotide at the appropriate position to catalyze the extension reaction, suggesting that base recognition would be performed in the closed form. A model structure of KF in the closed form, in which 5′R- and 5′S-cdA were superimposed on the incoming dATP, suggests that the phosphate site of 5′RcdA is away from the DNA polymerase active site while that of 5′S-cdA is oriented to the active site (Figure 5B,C). The model also implies that the incoming 5′R-cdATP barely forms hydrogen bonds with dTMP upon the transition to the closed form, and this may be one of the reasons that DNA polymerase incorporates the S-isomer into DNA more than the R-isomer in addition to misincorporation of the R-isomer opposite dCMP. Expected Biological Properties of cdPu. Nucleoside analogues are an important class of cancer chemotherapeutics that are used for the treatment of various malignancies. Two drugs in this class, cytarabine and gemcitabine, are analogues of 2′-deoxycytidine.19−22 Cytarabine is successfully used in the treatment of acute myeloid leukemia and hematological malignances.32 Gemcitabine is also successful in the treatment of nonsmall cell lung,33 pancreatic,34 ovarian,35 breast cancer,36 and hematological malignances.37 The mechanism of action of each drug is multifaceted but primarily involves inhibiting DNA synthesis. The triphosphates of cytarabine and gemcitabine are incorporated into DNA opposite dG, but they then inhibit DNA synthesis. As compared to the incorporation efficiency of gemcitabine, the incorporation of 5′S-cdATP or 5′R-cdATP is approximately 1.7 or 40 times lower, respectively. Thus, rather than 5′R-cdATP, 5′S-cdATP in cells might be incorporated as nucleoside analogues into DNA, which then inhibit DNA synthesis. When the damaged nucleotides incorporated in DNA become a DNA lesion, cdA, in the genomic DNA, the lesion can inhibit DNA polymerase reactions during subsequent

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DISCUSSION In this work, we report the synthesis of 5′R- and 5′S-cdATP, which are the triphosphate forms of cdA. The biochemical properties of these cdATP isomers were characterized in vitro by DNA synthesis reactions. We report that both 5′R- and 5′ScdATP were incorporated opposite dCMP and dTMP in the template, respectively. Thus, the incorporation of 5′R-cdATP is an error-prone event, which is not the case with 5′S-cdATP. Furthermore, the nucleotide selection of 5′R- and 5′S-cdATP was ∼17000- and ∼750-fold less efficient than that of dATP, respectively. Although the DNA replication reaction is inhibited by incorporation of both cdATP isomers, both lesions only slightly inhibited the incorporation of the second downstream nucleotide. Previous data revealed that the S-isomer in DNA blocked DNA polymerase exonuclease activity18 and that it was less efficiently repaired by NER (that can remove the lesions),12 and it blocked DNA synthesis by replicative DNA polymerases.12 Therefore, 5′S-cdATP is likely to be a more cytotoxic DNA lesion in the genome than the 5′R-cdATP, during or after replication. Biochemical Properties of cdATP. The cyPy lesions induced by hydroxyl radicals exert a cytotoxic effect by inhibiting DNA replication and transcription. In human cells, the genome DNA lesions can be removed by NER and/or transcription coupled NER but not by BER or direct reversal reaction. These lesions are chemically stable because of the 2721

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Figure 4. continued incubated with an indicated set of dNTPs at 30 °C for 5 (lanes 2, 4, and 6) or 10 min (lanes 3, 5, and 7). Lane 1, 32P-labeled 16-mer primer. Lanes 2 and 3, incubation with dATP, dCTP, and dTTP. Lanes 4 and 5, incubation with dATP, dCTP, dTTP, and dGTP. Lanes 6 and 7, incubation with dATP, dCTP, dTTP, and 5′S-dATP. Each dNTP (2 μM), 5′R-dATP (2 mM), and 5′S-dATP (500 μM). The band indicated with an asterisk is a nonspecific band.

Figure 5. Overhead model (A) and its detail (B) of the active site DNA polymerase KF with a template dT to an incoming 5′R-cdA and 5′S-cdA (blue, 5′S-cdA; red, 5′R-cdA). Note that these cdAs lack a phosphate moiety. (C) Schematic diagram of this model structure between an incoming cdA pair with dT and a putative active site containing Mg2+.

Figure 4. (A) Schematic drawing of the 30-mer oligonucleotide template C, annealed to a 32P-labeled 16-mer primer (top panel). KF and primer-template C were incubated with an indicated set of deoxynucleoside triphosphate (dNTP) at 30 °C for 5 (lanes 2, 4, and 6) or 10 min (lanes 3, 5, and 7). Lane 1: 32P-labeled 16-mer primer. Lanes 2 and 3, incubation with dATP, dCTP, and dTTP. Lanes 4 and 5, incubation with dATP, dCTP, dTTP, and dGTP. Lanes 6 and 7, incubation with dATP, dCTP, dTTP, and 5′R-dATP. (B) Schematic drawing of the 30-mer oligonucleotide template T, annealed to a 32Plabeled 16-mer primer (top panel). KF and primer-template C were

rounds of replication. This inhibition by cdA resembles the DNA synthesis inhibition induced by the DNA adduct generated by the cancer drug cisplatin. Moreover, like cisplatin, CPU lesions inhibit not only DNA replication but also transcription. Both lesions are removed by NER, but repair efficiency of cdA is lower than that of cisplatin adducts.12 In addition, the damaged residues are resistant to exonucleolytic 2722

dx.doi.org/10.1021/tx300351p | Chem. Res. Toxicol. 2012, 25, 2718−2724

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(8) Dirksen, M. L., Blakely, W. F., Holwitt, E., and Dizdaroglu, M. (1988) Effect of DNA conformation on the hydroxyl radical-induced formation of 8,5′-cyclopurine 2′-deoxyribonucleoside residues in DNA. Int. J. Radiat. Biol. 54, 195−204. (9) Schroder, E., Budzinski, E. E., Wallace, J. C., Zimbrick, J. D., and Box, H. C. (1995) Radiation chemistry of d(ApCpGpT). Int. J. Radiat. Biol. 68, 509−523. (10) Brooks, P. J. (2008) The 8,5′-cyclopurine-2′-deoxynucleosides: candidate neurodegenerative DNA lesions in xeroderma pigmentosum, and unique probes of transcription and nucleotide excision repair. DNA Repair 7, 1168−1179. (11) Brooks, P. J., Wise, D. S., Berry, D. A., Kosmoski, J. V., Smerdon, M. J., Somers, R. L., Mackie, H., Spoonde, A. Y., Ackerman, E. J., Coleman, K., Tarone, R. E., and Robbins, J. H. (2000) The oxidative DNA lesion 8,5′-(S)-cyclo-2′-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells. J. Biol. Chem. 275, 22355−22362. (12) Kuraoka, I., Bender, C., Romieu, A., Cadet, J., Wood, R. D., and Lindahl, T. (2000) Removal of oxygen free-radical-induced 5′,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells. Proc. Natl. Acad. Sci. U.S.A. 97, 3832−3837. (13) D'Errico, M., Parlanti, E., Teson, M., Degan, P., Lemma, T., Calcagnile, A., Iavarone, I., Jaruga, P., Ropolo, M., Pedrini, A. M., Orioli, D., Frosina, G., Zambruno, G., Dizdaroglu, M., Stefanini, M., and Dogliotti, E. (2007) The role of CSA in the response to oxidative DNA damage in human cells. Oncogene 26, 4336−4343. (14) Kirkali, G., de Souza-Pinto, N. C., Jaruga, P., Bohr, V. A., and Dizdaroglu, M. (2009) Accumulation of (5′S)-8,5′-cyclo-2′-deoxyadenosine in organs of Cockayne syndrome complementation group B gene knockout mice. DNA Repair 8, 274−278. (15) Rodriguez, H., Jaruga, P., Leber, D., Nyaga, S. G., Evans, M. K., and Dizdaroglu, M. (2007) Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5′-Cyclopurine-2′deoxynucleosides and 8-hydroxy-2′-deoxyguanosine. Biochemistry 46, 2488−2496. (16) Kirkali, G., Tunca, M., Genc, S., Jaruga, P., and Dizdaroglu, M. (2008) Oxidative DNA damage in polymorphonuclear leukocytes of patients with familial Mediterranean fever. Free Radical Biol. Med. 44, 386−393. (17) Dizdaroglu, M., and Jaruga, P. (2012) Mechanisms of free radical-induced damage to DNA. Free Radical Res. 46, 382−419. (18) Kuraoka, I., Robins, P., Masutani, C., Hanaoka, F., Gasparutto, D., Cadet, J., Wood, R. D., and Lindahl, T. (2001) Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues. J. Biol. Chem. 276, 49283−49288. (19) Gandhi, V., Mineishi, S., Huang, P., Chapman, A. J., Yang, Y., Chen, F., Nowak, B., Chubb, S., Hertel, L. W., and Plunkett, W. (1995) Cytotoxicity, metabolism, and mechanisms of action of 2′,2′difluorodeoxyguanosine in Chinese hamster ovary cells. Cancer Res. 55, 1517−1524. (20) Huang, P., Chubb, S., Hertel, L. W., Grindey, G. B., and Plunkett, W. (1991) Action of 2′,2′-difluorodeoxycytidine on DNA synthesis. Cancer Res. 51, 6110−6117. (21) Prakasha Gowda, A. S., Polizzi, J. M., Eckert, K. A., and Spratt, T. E. (2010) Incorporation of gemcitabine and cytarabine into DNA by DNA polymerase beta and ligase III/XRCC1. Biochemistry 49, 4833−4840. (22) Richardson, K. A., Vega, T. P., Richardson, F. C., Moore, C. L., Rohloff, J. C., Tomkinson, B., Bendele, R. A., and Kuchta, R. D. (2004) Polymerization of the triphosphates of AraC, 2′,2′-difluorodeoxycytidine (dFdC) and OSI-7836 (T-araC) by human DNA polymerase alpha and DNA primase. Biochem. Pharmacol. 68, 2337−2346. (23) Chijiwa, S., Masutani, C., Hanaoka, F., Iwai, S., and Kuraoka, I. (2010) Polymerization by DNA polymerase eta is blocked by cisdiamminedichloroplatinum(II) 1,3-d(GpTpG) cross-link: implications for cytotoxic effects in nucleotide excision repair-negative tumor cells. Carcinogenesis 31, 388−393.

activity and inhibit DNA synthesis by translesion DNA polymerase.18 Thus, the lesions are likely to be highly cytotoxic in vivo. These features of cdATP may be suitable as an anticancer candidate drug. This is especially the case for tumors that lack NER. One notable difference between cdA and cisplatin is that, at least at present, NER is the only known repair mechanism for cdA, whereas cisplatin activates the Fanconi anemia-BRCA DNA damage response pathway.38 Several nucleoside analogues are used as antiviral or anticancer agents. Because nucleotides are charged molecules that cannot easily cross cell membranes to enter cells, analogues are administered as nucleosides. While we have described the chemical synthesis of 5′S-cdATP and 5′R-cdATP, to determine whether cdA can be used as a drug, future studies will be needed to assess the ability of cells to convert cdA into cdATP.

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ASSOCIATED CONTENT

S Supporting Information *

Figure of Michaelis−Menten kinetics for dATP:dT, 5′RcdATP:dC, and 5′S-cdATP:dT. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-6-6850-6250. Fax: +81-6-6850-6240. E-mail: [email protected]. Funding

This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS NER, nucleotide excision repair; NTH1, endonuclease III; OGG1, 8-oxoguanine-DNA glycosylase; XP, xeroderma pigmentosum; BER, base excision repair; gemcitabine, 2′,2′difluoro-2′-deoxycytidine; cytarabine, 1-β-D-arabinofuranosylcytosine; cisplatin, cis-diamminedichloroplatinum(II); KF, Klenow fragment; NOESY, nuclear Overhauser effect spectroscopy; COSY, correlation spectroscopy

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REFERENCES

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