Nucleobase Lesions and Strand Breaks in Dry DNA Thin Film

Nov 12, 2009 - Phone No.: +81-29-284-3516. Fax No.: +81-29-284-3516. E-mail address: [email protected]. Cite this:J. Phys. Chem. B 113, 49, 160...
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J. Phys. Chem. B 2009, 113, 16007–16015

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Nucleobase Lesions and Strand Breaks in Dry DNA Thin Film Selectively Induced by Monochromatic Soft X-rays Kentaro Fujii,* Naoya Shikazono, and Akinari Yokoya AdVanced Science Research Center, Japan Atomic Energy Agency, 2-4 Shirakatashirane, Tokai, Naka, Ibaraki 319-1195, Japan ReceiVed: July 3, 2009; ReVised Manuscript ReceiVed: October 1, 2009

To verify the possibility of “selective damage induction” in DNA, the yields of base lesions as well as strand breaks have been measured in dry plasmid DNA films irradiated with highly monochromatized soft X-rays in the energy region of 270-760 eV, which includes the carbon, nitrogen, and oxygen K-edges. The yields of both pyrimidine and purine base lesions, observed as Nth-sensitive and Fpg-sensitive sites, respectively, are strikingly high at the oxygen K-edge (560 eV) but extremely low at an energy just below the nitrogen K-edge (380 eV) as compared with the yields observed at other photon energies. The yields at 560 eV are enhanced 9.6-fold and 27-fold for Nth-sensitive and Fpg-sensitive sites, respectively, compared with those at 380 eV. The yield of prompt single strand breaks is also enhanced at the oxygen K-ionization energy, but only 2-fold, as compared with that at 380 eV. Our results strongly suggest that (1) the K-shell ionization of oxygen in both the nucleobases as well as in other parts of DNA and in the hydrating water molecules bound to DNA, but not the K-shell ionization of nitrogen in the nucleobases, most likely contributes to the induction of nucleobase lesions and that (2) migration of electrons and holes is involved differentially in the production of each type of DNA lesion. These results could potentially lead to new methods for “partially selective induction” of specific types of DNA damage through tuning the energy of soft X-rays. Introduction Site-selective energy deposition in DNA, which is thought to be a major target molecule of radiation-induced cell lethality, mutation, and carcinogenesis, is desirable for exploration of the fundamental processes involved in radiobiological effects. Highenergy particles obtained from accelerator facilities for radiation therapy as well as environmental radiation from radioactive isotopes or cosmic radiation cause a variety of molecular changes in genomic DNA through energy deposition to the molecules by ionization. The ionization of DNA results in single-strand breaks (SSBs), double-strand breaks (DSBs), oxidatively and reductively generated nucleobase lesions, nucleobase release, and interstrand or intrastrand cross-linkages.1 Characteristic carbon K-shell (0.28 keV), aluminum K-shell (1.49 keV), and titanium K-shell (4.55 keV) soft X-rays are expected to be sources that partially restrict energy deposition to a narrow area in a cell.2 Soft X-ray photons mainly interact with the matter in living cells through a photoelectric process, and as a consequence, a photoelectron and Auger electrons are ejected from an atom that absorbs a soft X-ray photon. The biological effects induced by soft X-ray irradiation are thought to arise from the formation of DNA damage through both the ionization of DNA and the impact of the secondary electrons which ionize or excite nearby molecules through inelastic scattering.3 Since the 1990s, synchrotron radiation has been widely used as an intense soft X-ray source in molecular sciences, and many studies have verified “atom selective” innershell photoionization and the induction of scission of the desired chemical bond in a molecule.4 Tinone et al.5 reported the first evidence that the site of bond scissions in poly(methyl meth* To whom correspondence should be addressed. Phone No.: +81-29284-3516. Fax No.: +81-29-284-3516. E-mail address: fujii.kentaro@ jaea.go.jp.

acrylate) (PMMA) on a surface can be selectively induced by photoexcitation of a K-shell electron of an oxygen atom to a specific antibonding orbital. To introduce the “molecular scalpel” concept to the field of biological science, various radiobiological end points resulting from inner-shell photoionization of particular atoms in a living system6 have been studied. These studies used monochromatic (∆E < 1 eV) soft X-ray photons tuned to the specific energies around the K-shell ionization thresholds of carbon (284 eV), nitrogen (410 eV), oxygen (543 eV), and phosphorus atoms (2.15 keV). Using wild-type and repairdeficient cell lines of bacterial and mammalian cells, enhancements in the efficiencies of cell killing, mutation induction, and chromatid breaks were observed by K-shell ionization of phosphorus (2.15 keV) in DNA.7 Penhoat et al.8 reported that carbon K-ionization induced by 340 eV soft X-rays results in a highly efficient killing effect in Chinese hamster V79 cells. On the basis of the results of these studies, the damage induced in DNA is expected to be specific to the ionization of a particular element. To explore the nature of DNA damage by inner-shell ionization, the photon energy dependence of the yields of DNA strand breaks, particularly double strand breaks, which are thought to be one of the major forms of cell damage that result in cell death, was measured around the K-shell ionization threshold energies of the constituent elements of DNA. Early studies, however, have reported that the yields of DNA strand breaks are almost constant at energies below 1 keV or only slightly enhanced at the oxygen K-edge energy.9,10 Recent studies11,12 have also shown a similar enhancement in the yield of DSBs above the oxygen K-edge region by a factor of 1.4-2. Hieda et al.13 reported that the yields of the strand breaks do not strongly depend on the photon energy around the phosphorus K-edge when normalized by the photoabsorption cross section. Thus, the energy dependence of these yields of DNA strand breaks was not larger than that initially expected

10.1021/jp9062737 CCC: $40.75  2009 American Chemical Society Published on Web 11/12/2009

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from the cellular effects as final end points. On the basis of these results, Goodhead questioned whether the inner-shell ionization can actually cause specific biological and biochemical effects.14 To verify the possibility of site-selective energy deposition and induction of specific cellular effects, the yields of not only DNA strand breaks but also nucleobase lesions produced in DNA should be examined. As a result of irradiation, nucleobase lesions are produced in considerably greater numbers than strand breaks (e.g., the yield of total base lesions induced in a dry DNA film is about three times larger than that of SSBs15,16). It has been hypothesized that two or more nucleobase lesions, in some cases, with SSBs or an abasic site formed within one or two DNA helical turns (∼10 nm) are less repairable than isolated nucleobase lesions and are particularly harmful to cells.17,18 Recently, the baseexcision repair proteins endonuclease III (Nth), formamidopyrimidine-DNA glycosylase (Fpg), and endonuclease IV (Nfo) have been widely used as enzymatic probes to detect the nucleobase lesions induced by various radiation sources.19-22 The Nth protein excises mainly pyrimidine base lesions (ring-saturated pyrimidines, e.g. 5,6-dihydrothymine (DHT), thymine glycol, and abasic sites (AP sites)).23-26 The Fpg protein, on the other hand, excises mainly purine base lesions such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 8-oxo-7,8-dihydroguanine (8-oxoGua), and AP sites.27-29 The major substrates of Nfo are oxidized abasic sites.30 These enzymatic probes convert base lesions into readily detectable SSBs. However, the yields of nucleobase lesions have not been extensively investigated in studies of DNA damage resulting from soft X-ray irradiation. Only the yields of nucleobase lesions detected by Fpg treatment have been reported for DNA films that were irradiated with monochromatic soft X-rays (250, 380, and 760 eV).12 We still lack clear evidence of whether the photoelectric effect of the DNA constituent atoms induces particular types of nucleobase lesions as well as DNA strand breaks. In this study, we provide the first evidence of “partially selective damage induction” by determining the yields of pyrimidine and purine base lesions as well as single- and doublestrand breaks induced by irradiation of dry plasmid DNA films. The selectivity was realized using highly monochromatized soft X-rays using an undulator beamline in SPring-8 in the carbon, nitrogen, and oxygen K-edge regions (270-760 eV). The role of the photoelectric effect of the DNA constituent atoms and the contribution of water molecules tightly bound to DNA in the damage induction process are discussed in terms of the relative yields of nucleobase lesions and strand breaks. Materials and Methods Preparation of Dry Plasmid DNA Thin Films. Plasmid DNA (pUC18, 2686 bp) was retrieved from a 3 L overnight culture using a plasmid purification kit (QIAfilter plasmid Mega kit, QIAGEN, Japan). The plasmid, of which over 90% was in the supercoiled form, was subsequently stored at -20 °C in TE buffer (10 mM Tris, (Wako Pure Chemical Industries Ltd., Japan), 1 mM ethylene diamine tetraacetic acid (EDTA, Wako)), at pH 8.0 and a concentration of 1.35 mg/mL. The stock solution of DNA was diluted with TE buffer to give a final DNA concentration of 1.05 mg/mL in TE buffer. This plasmid solution was spotted at 4 °C in 5 µL aliquots onto a coverslip (Matsunami Glass Ind., Ltd., Japan) of 12 mm × 12 mm and dried by blown N2 gas (Tatsumi Sangyo, Japan) for 30 min at 4 °C to avoid crystallization of the buffer solute. After drying, a uniform film of DNA/buffer solutes having a diameter of 5.5 mm formed on the coverslip. Typically, four plasmid DNA films were prepared on a coverslip. In preliminary experiments, we tested mica as

Fujii et al. a plasmid substrate, as it had been used in a previous study.11 When we attempted to recover hydrated plasmid DNA samples from the mica surface, about 40% of the supercoiled plasmid DNA was transferred as an open circle, which would not be relevant for the present irradiation experiments. To avoid these substrate effects, we used cover glass as the substrate. We monitored damage caused by the substrate to ensure that strand breaks were less than 10%. We also carefully monitored damage under the irradiation conditions in a vacuum. Strand breaks induced under these conditions without irradiation were not detectable. The DNA films were introduced into the highvacuum chamber (base pressure: 2 × 10-6 Pa) after being kept in a low-vacuum chamber (1 × 10-4 Pa) for 30-60 min. Under the above conditions, degradation of the supercoiled DNA was observed to be less than 5%. Soft X-ray Irradiation of the DNA Samples. The pUC18 plasmid DNA film was irradiated at room temperature (25 ( 2 °C) in a vacuum chamber. Irradiation was carried at the soft X-ray beamline (BL23SU) in SPring-8 (Hyogo, Japan). Monochromatic soft X-rays were obtained using varied line spacing grating.31 The irradiation of the sample at the selected energies around the carbon, nitrogen, and oxygen K-edges (270, 380, 435, 560, and 760 eV) was carried out. The calibration of photon energy was performed by measuring the total ion yield spectrum of N2 gas (Tatsumi Sangyo) around the nitrogen K-edge. To obtain uniform irradiation, the sample holder was moved up and down in the vacuum chamber using a motor-drive manipulator. The sample was recovered with 150 µL of TE buffer and then stored at -20 °C after the irradiation, prior to determination of the yield of strand breaks by agarose gel electrophoresis or enzymatic treatments as described below. The photon flux of the beam was measured using a photodiode (AXUV-100, International Radiation Detectors Inc., USA) at the sample position. The photon fluxes were almost constant with the topup electron injection to the storage ring of the SPring-8 during the irradiation.32 Three or four independent irradiation experiments were performed during at least two beamtimes on different days. The mass absorption coefficients of pUC18 at each soft X-ray energy, which were calculated using reference data of the mass absorption coefficient of each DNA constituent atom,33 are listed in Table 1. The photon flux to an irradiated sample is on the order of 1017 photons per sample. The correction factors to estimate the penetration of the soft X-rays into the sample were calculated with the following equation.

( ( µF ) ) µ σ( ) F

1 - exp -σ fcorr )

sample

(1)

sample

where σ is the surface density (0.122 kg/m2) of the sample. Under the present conditions, we obtained the values 0.53, 0.47, 0.49, 0.51, and 0.71 for 270, 380, 435, 560, and 760 eV, respectively. Therefore 47-71% of entering photons are transferred to the sample. The absorbed doses are estimated based on the mass absorption cross sections and the atomic composition of the sample. The methods for quantifying the absorbed doses of the samples containing hydrated water molecules (2.5 mol of water per mole of nucleotide) are the same as those reported previously.9 Treatments of the Irradiated DNA Samples. The irradiation of supercoiled DNA in aqueous solution34 at 4 °C in the presence of Tris (see Table 1) followed by incubation at 37 °C or the irradiation of cellular DNA35 resulted in significant yields of

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TABLE 1: Mass Absorption Coefficienta for the Relevant Atoms, for pUC18 Plasmid DNA, and for Tris EDTA Buffer Solute mass absorption coefficient (µ/F) (m2/kg) photon energy (eV)

H

C

N

O

P

Na

Cl

pUC18b (0.36)

Tris EDTA buffer solutesc (0.64)

270 380 435 560 760

50.07 16.52 10.61 4.64 1.69

213.8 2706 1937 1028 471.3

402.9 146.4 2678 1492 685.7

643.4 273.8 193.9 1981 935.7

4303 2193 1633 898.6 412.0

1905 809.6 570.5 298.5 134.3

5190 2938 2192 1208 568.2

305 454 466 470 219

86.4 1011 883 819 381

a Based on Henke et al.31 b The values of pUC18 (sodium salute) are calculated using the values of each element and the abundance of the elements per nucleotide with 2.5 water molecules hydrated per nucleotide (see Materials and Methods): H, 28; C, 20; N, 7.4; O, 16; P, 2.0; Na, 2.0. The mass fraction of pUC18 plasmid in the sample is shown in parentheses. c The values of Tris EDTA buffer solutes are calculated using the values of each element and the atomic compositions of Tris and EDTA: H, 14; C, 5.0; N, 1.2; O, 4.0; Na, 0.2; Cl, 1.0. The mass fraction of the solutes in the sample is shown in parentheses.

additional strand breaks as a result of the presence of heat-labile sites. To distinguish the prompt strand breaks and enzyme sensitive sites from the heat-labile sites, as well as other additional effects on irradiated DNA by postirradiation treatment, we categorized the irradiated samples into four groups. (1) The first group was not subjected to any treatments and kept at a constant temperature of -20 °C just before agarosegel-electrophoresis analysis (see below) (hereafter denoted as “prompt SSB (or DSB)”). (2) The second was subjected to chemical treatments including ethanol precipitation and then kept below 4 °C in the absence of enzymes (“prompt + chemical treatment SSB (or DSB)”) prior to the agarose-gel-electrophoresis analysis. (3) The third was subjected to the chemical treatments and then incubated at 37 °C for 30 min in the absence of enzymes (“prompt + chemical treatment + heat-labile SSB (or DSB)”). (4) The fourth was subjected to the chemical treatments and was incubated at 37 °C for 30 min in the presence of Fpg or Nth (“Fpg + chemical treatment + heat-labile SSB (or DSB)” or “Nth + chemical treatment + heat-labile SSB (or DSB)”). For the chemical treatments including ethanol precipitation, 5 µL of sodium acetate (Wako) (3M, pH 5.5) and 150 µL of chilled ethanol (Wako) were added to 50 µL aliquots of the recovered solutions. The solutions were left at -20 °C for 30 min to precipitate the DNA and subsequently centrifuged (CF15RXII, HITACHI, Japan) at 15 000 rpm for 30 min at 4 °C. After decanting the liquid, the DNA pellets were rinsed with 70% ethanol. Following centrifugation, the resulting DNA pellets were dried for 10 min to remove any excess ethanol using an evaporator (VA-250F and TAITEC, Japan), and the plasmid DNA was dissolved in 98 µL of reaction buffer (0.5 mM EDTA, 0.1 mM KCl (Wako), 0.5 mM dithiothreitol (DTT, Nacalai Teque, Japan) and 0.2 mg/mL of bovine serum albumin (BSA) (New England Biolabs, USA)) at pH 8.0. The solution was then divided into four tubes, two 19 µL samples for incubation in the absence of enzyme or two 19 µL samples for incubation in the presence of Nth and Fpg, respectively. One of the former samples was kept below 4 °C during the enzyme treatment, and the other was incubated for 30 min at 37 °C to estimate the net yield of heat-labile strand breaks. Purified proteins, Nth and Fpg, were purchased from NEB (New England Biolabs Japan, Japan). Nth and Fpg at pH 6.6 and concentrations of 2.5 U/µL and 0.031 U/µL, respectively, were stored at -20 °C in stock solutions (50% glycerol (Wako), 100 mM potassium phosphate (Wako), 100 mM DTT, and 0.005% Triton X-100 (Wako)). The incubation conditions (37 °C for 30 min) and the optimal concentrations of Nth and Fpg (5.7 U and 0.071 U per 1 µg of DNA, respectively) were determined by enzymatic treatment of DNA samples that were either nonirradiated or

irradiated with 4 kGy soft X-rays (150 kVp) (M-150WE, Softex, Japan). At these concentrations, the enzymes did not degrade the supercoiled form of the plasmid DNA. The irradiation of supercoiled DNA in aqueous solution34 at 4 °C in the presence of Tris (see Table 1) followed by incubation at 37 °C or the irradiation of cellular DNA35 resulted in significant yields of additional strand breaks as a result of the presence of heat-labile sites. Quantification of the Yields of Strand Breaks in Irradiated Plasmid DNA. The methods for quantification of the yields of strand breaks are the same as those reported previously.16 Prior to agarose gel electrophoresis, 5 µL of the loading buffer (0.1% w/v bromophenol blue (Wako), 0.1% w/v xylene cyanol (Wako), 30% v/v glycerol (Wako)) was added to the solutions containing irradiated or control DNA. Either 19 µL of solutions containing DNA treated with an enzyme or 4 µL of nonenzymatically treated solutions was placed into the well of a 1% agarose (Type 1-A, Sigma-Aldrich Japan, Japan) gel in TBE buffer (90 mM Tris, 90 mM boric acid (Wako), 2 mM EDTA) at pH 7.1. The electrophoresis was conducted at 1.4 V/cm for 18 h at less than 6 °C. Following electrophoresis, the gel was stained with 25 µL of ethidium bromide (10 mg/mL, Bio-Rad Japan, Japan) in 500 mL of TBE buffer for 1 h at room temperature and destained with TBE buffer for 0.5 h. The separated supercoil, open circular, and linear forms of the plasmid DNA in the gel were visualized using a charge-coupled device (CCD, Ettan DIGE, GE Healthcare UK Ltd., England) and analyzed by analysis software (ImageQuant TL, GE Healthcare UK). The relative amount of DNA in each form was then quantified as described previously.36 The effects of super helical density on the correction factor for ethidium binding to supercoiled DNA have been discussed previously.36,37 The dose response was determined from the logarithmic loss of supercoiled plasmid DNA on radiation dose. From the slope of this response, a D37 value was obtained which, assuming a Poisson distribution of SSB induction, represents the radiation dose required to give on average one SSB per plasmid molecule. Using the D37 value, an average number of SSBs/Gy/Da (n(SSB)) was obtained (eq 1), assuming an average mass of a base pair of 650 Da and knowing that pUC18 DNA contained 2686 base pairs.

n(SSB) )

1 2686 × 650 × D37

(2)

The average number of DSB/Gy/Da (n(DSB)) was determined from the dose dependence of the fractional abundance of the number of DNA plasmids in the linear form, which is given by

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n(DSB) )

b 2686 × 650

Fujii et al.

(3)

where b is obtained from the slope of the dose response. Calculations of the radiation chemical yields, the G values (mol J-1), of SSBs in the hydrated DNA samples have been described previously.16 These are given by

G(SSB) )

(1000/mwt) D37

(4)

where mwt is the molecular weight of the pUC18 plasmid DNA (1.746 M Dalton). Results Dependence of the Yields of SSBs and Nucleobase Lesions on Soft X-ray Energy. A typical example of the relation between the amount of supercoiled DNA and the radiation dose is shown in Figure 1 for the irradiation of pUC18 plasmid DNA with soft X-rays (560 eV) in a vacuum. Postirradiation incubation of the plasmid DNA with either Nth or Fpg at 37 °C results in a greater loss of supercoiled DNA. The yields of Nth + chemical treatment + heat-labile SSB and Fpg + chemical treatment + heat-labile SSB, as determined from the dose dependence of the loss of supercoiled DNA, are also shown in Figure 1. From these dependences, the yields of SSBs and Nth + heat-labile SSB and Fpg + heat labile SSB, n(SSB)/Gy/Da, were calculated from the D37 values (see eq 2) in addition to the yields determined similarly at various soft X-ray energies, which are shown in Figure 2 and listed in Table 2. Additional SSBs are about 10% of the yield of prompt SSB by the heat treatment (37 °C, 30 min) following irradiation with soft X-rays, indicating that the yields obtained by enzymatic treatments include the heat-labile sites (10%). On the other hand, somehow the yield of prompt + chemical treatment SSB was 30% lower than the prompt SSB for the all photon energy tested. The net yields of enzyme-sensitive sites observed as additional SSBs, n(ESSSSB), were obtained using the following equation.

)Nth (or Fpg) ) n(SSB)Nth (or Fpg)+heat-labile SSB n(SSB)prompt+heat-labile SSB

SSB

n(ESS

(5)

These yields are shown in Figure 2. The values of n(ESSSSB) for Nth and Fpg treatment and the ratios of n(ESSSSB)/ n(SSB)prompt are also listed in Table 3 for various soft X-ray energies. Recently, Agrawala et al.12 also reported similar values for Fpg-sensitive sites for three soft X-ray energies (shown in Table 3 for comparison). The values of n(ESSSSB) for Nth and Fpg treatments are strikingly enhanced by irradiation at the oxygen K-absorption energy (560 eV). The ratios between n(ESSSSB) at 560 eV and n(ESSSSB) at 380 eV are 9.6 and 27 for treatment with Nth and Fpg, respectively. On the other hand, the yields obtained at 380 eV are very low following the Nth or Fpg treatment. For samples irradiated at 760 eV, n(ESSSSB) shows significantly smaller yields for Fpg treatment than those for Nth treatment. The yield of prompt SSBs obtained at the oxygen K-absorption (560 eV) is about two times larger than that at 380 eV. The enzyme-sensitive sites revealed by both enzymes are preferentially produced at 560 eV. On the other hand, the number of prompt SSBs at 380 eV is considerable. Nth-sensitive sites, namely, pyrimidine lesions, are most likely damage-induced by irradiation with 760 eV photons.

Figure 1. Relation between the loss of supercoiled DNA and absorbed dose after exposure of dry pUC18 plasmid DNA to 560 eV photons at room temperature (9) or following postirradiation incubation for 30 min at 37 °C in the absence (b) or presence of either Nth (b) or Fpg (2). Straight lines are drawn by the least-squares method. Vertical error bars are the standard error for the values determined from four independent experiments.

Figure 2. Relation between the yield of SSBs and photon energy (eV) for dry plasmid (pUC18) DNA thin films irradiated with monochromatic soft X-rays (270, 380, 435, 560, and 760 eV) at room temperature (9) or following postirradiation incubation for 30 min at 37 °C in the presence of Nth (2) or Fpg (b).Vertical error bars are the standard error for the values of SSB determined from the slope of dose-response curves for four independent experiments. Solid lines are drawn by connecting the data points.

TABLE 2: Yields of SSB and DSB Induced in the Dry pUC18 Plasmid DNA by Irradiation with Monochromatic Soft X-rays at 25 °C (with Standard Deviations Shown in Parentheses)a photon energy (eV)

n(SSB) (× 10-11 SSB/ Gy/Da)

G(SSB) (× 10-7 mol/J)

n(DSB) (× 10-12 DSB/ Gy/Da)

n(SSB)/ n(DSB)

250b 270 380b 380 435 560 760b 760

3.6 1.87(0.25) 3.7 1.85(0.19) 2.11(0.10) 3.79(0.21) 4.4 4.11(0.24)

0.187(0.025) 0.185(0.019) 0.211(0.010) 0.379(0.021) 0.411(0.024)

5.6 0.66(0.06) 6.3 0.93(0.14) 0.90(0.01) 1.68(0.06) 8.5 1.41(0.18)

6.4 28 5.8 20 23 23 5.2 29

a The yields of Fpg- and Nth-sensitive sites were obtained as the average value of three or four individual enzymatic experiments, and the error bar corresponds to the standard deviation of these values. b Cited from Eshenbrenner et al.11

Relation between the Yields of Prompt DSB and Additional DSB after Nth and Fpg Treatment. The induction of DSBs by soft X-ray irradiation of plasmid DNA under vacuum or following incubation in buffer for 30 min at 37 °C increases linearly with the radiation dose as shown in Figure 3. The yields of DSBs, n(DSB)/Gy/Da, were calculated from the dose dependences using eq 2 and are shown in Table 2; the

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TABLE 3: Yields of Base Lesions Induced in the Dry pUC18 Plasmid DNA by Irradiation with Monochromatic Soft X-rays at 25 °C (with the Standard Deviations Shown in Parentheses), Followed by Incubation with Nth and Fpg Proteinsa treatment of sample prompt + chemical treatment

prompt + chemical treatment + heat

Nth

Fpg

photon energy (eV)

n(ESSSSB) (× 10-11 ESSSSB/Gy/Da)

n(ESSSSB)/ n(SSB)prompt

n(ESSDSB) (× 10-11 ESSDSB/Gy/Da)

n(ESSDSB)/ n(DSB)prompt

270 380 435 560 760 270 380 435 560 760 270 380 435 560 760 250 270 380 380 435 560 760 760

1.22(0.10) 0.96(0.07) 1.49(0.28) 2.49(1.11) 2.48(0.42) 1.38(0.19) 1.09(0.03) 1.69(0.03) 2.78(0.38) 2.71(0.41) 1.55(0.11) 0.60(0.07) 1.38(0.08) 5.77(0.77) 4.09(0.38) 1.05b 1.07(0.07) 0.49b 0.17(0.05) 0.70(0.02) 4.52(0.40) 0.92b 1.12(0.40)

0.83 0.32 0.65 1.52 1.00 0.29 0.57 0.13 0.09 0.33 1.19 0.21 0.27

0.58(0.02) 0.63(0.13) 0.70(0.26) 1.35(0.12) 0.61(0.39) 0.51(0.05) 0.75(0.17) 0.75(0.19) 1.25(0.04) 0.89(0.46) 0.25(0.05) 0.03(0.01) 0.15(0.01) 0.63(0.01) 0.52(0.38) 0.15b 0.44(0.07) 0.20b 0.19(0.01) 0.22(0.05) 0.59(0.02) 0.18b 0.29(0.42)

0.38 0.03 0.17 0.38 0.37 0.28b 0.67 0.32b 0.20 0.24 0.35 0.22b 0.21

a The yields of Fpg- and Nth-sensitive sites were obtained as the average value of three or four individual enzymatic experiments, and the error bar corresponds to the standard deviation of these values. b Cited from Agrawala et al.12

Figure 3. Relation between the fraction of linear DNA and radiation dose after exposure of dry pUC18 plasmid DNA to 560 eV photons at room temperature (9) or following postirradiation incubation for 30 min at 37 °C in the absence (b) or presence of Nth (b) or Fpg (2). Straight lines are drawn by the least-squares method. Vertical error bars are the standard error for the values determined from four independent experiments.

ratios of n(SSB) to n(DSB) are also shown. Significant levels of additional DSBs are not induced by heat treatment since the yields of prompt DSB and prompt + heat-labile DSB at the various soft X-ray energies are similar, as shown in Figure 4. Postirradiation incubation of the plasmid DNA with either Nth or Fpg at 37 °C results in linear induction of DSBs as a function of radiation dose, which is also shown in Figure 3. The yields of enzymatically induced additional DSBs, which are indicative of clustered DNA damage, are plotted in relation to photon energy in Figure 4. The net yields of enzyme-sensitive sites observed as additional DSB, n(ESSDSB), are given by

n(ESSDSB)Nth (or Fpg) ) n(DSB)Nth (or Fpg)+heat-labile site n(DSB)prompt+heat-labile site

(6)

Figure 4. Relation between the yield of DSBs and photon energy (eV) for dry plasmid (pUC18) DNA thin films irradiated with monochromatic soft X-rays (270, 380, 435, 560, and 760 eV) at room temperature (9) or following postirradiation incubation at 37 °C in the presence of Fpg (b) or Nth (2).Vertical error bars are the standard error for the values of SSB determined from the slope of dose-response curves of four independent experiments. Solid lines are drawn by connecting the data points.

values of n(ESSDSB) and the ratio of n(ESSDSB) to n(DSB) are also listed in Table 3. To compare the damage yields among the photon energies based on the number of photoabsorption events, we converted the SSB (or ESS) yield (per Gy per Da) to ΦSSB (or ΦESS), which is the yield of the damage produced in a plasmid by one photoabsorption, corresponding to the quantum efficiency.

Φ) PD37

Nplasmid µ × σsample × F

()

sample

(7) × fcorr

DSB

The values of n(ESS ) as well as the yield of the prompt DSB reach a maximum at 560 eV. The values of n(ESSDSB)Nth are smaller than those of n(ESSDSB)Fpg except at 435 eV. The

where NPlasmid is the number of plasmids irradiated; PD37 is the number of photons that gives D37; σsample is the surface density

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TABLE 4: Calculated Yields of SSB (or ESSSSB) per Photoabsorption (SSB/Plasmid or ESS/Plasmid)a photon ΦSSB ΦSSBFpg ΦSSBNth energy (eV) (× 10-2 SSB/plasmid) (× 10-2 ESS/plasmid) (× 10-2 ESS/plasmid)

270 380 435 560 760

1.4(0.17) 1.5(0.043) 2.7(0.18) 5.7(0.76) 7.6(1.2)

0.84(0.35) 0.11(0.096) 0.88(0.17) 6.7(0.88) 2.5(0.91)

1.2(0.44) 0.42(0.082) 1.7(0.29) 6.7(0.82) 9.5(2.1)

a The yields of Fpg- and Nth-sensitive sites were obtained as the average value of three or four individual enzymatic experiments, and the error bar corresponds to the standard deviation of these values.

of the sample; (µ/F)sample is the photoabsorption cross section of the sample; and fcorr is the correction factor (eq 1). The calculated quantum efficiencies were shown in Table 4. The values for ESS as well as SSB are less than 0.5. Discussion The damaging effects of soft X-rays below 1 keV were investigated by exposing a model DNA system to monochromatic synchrotron radiation. Previous studies9-11 have mainly focused on the formation of DNA strand breaks induced by soft X-rays in the energy region around the carbon, nitrogen, and oxygen K-edges. These studies reported relatively constant or slightly larger DNA strand break yields for oxygen K-ionization. The yields of strand breaks, particularly prompt DSB yields, are lower than those reported by Eschenbrenner et al.11 as shown in Table 2. Differences in the experimental conditions used by the two groups, such as the substrate on which the plasmid DNA was prepared or buffer solutes in the DNA films, possibly affect the strand break efficiency.38 To obtain experimental evidence regarding the amount of nucleobase damage, we studied the base lesions in plasmid DNA using two base excision repair proteins, Nth and Fpg, as enzymatic probes. These proteins convert the nucleobase lesions to detectable SSBs via their glycosylase activity. The main findings from this study on the direct ionization of the constituent atoms of DNA are as follows: (1) the yields of both pyrimidine and purine base lesions, revealed as Nth-sensitive and Fpg-sensitive sites, respectively, are strikingly enhanced by oxygen K-ionization at 560 eV and show considerably lower yield at the photon energy just below the nitrogen K-edge (380 eV) compared to those observed at the other photon energies tested; (2) the yields of prompt SSBs are also enhanced by oxygen K-ionization but not by a considerable amount when compared with the yields of nucleobase lesions; (3) the yield of the purine lesions (Fpg-sensitive sites) induced by 760 eV photons is significantly smaller than that of pyrimidine lesions (Nth-sensitive site); and (4) the number of clustered lesions detected as DSBs after treatment with Nth or Fpg is similarly enhanced by oxygen K-ionization (560 eV) and also increases for 270 eV photons, although the yield of prompt DSB is only enhanced by oxygen K-ionization. The difference between the ionization efficiency of nucleobases and 2-deoxyribose is too small to induce significantly different ionization efficiencies between them.39 Thus, our results in this study cannot be simply rationalized in terms of ionization efficiency. Recently, we have studied short-lived unpairedelectron species produced in DNA films by the soft X-ray irradiation using an EPR spectrometer installed in a synchrotron soft X-ray beamline. We reported that a significant EPR signal of the short-lived transient species is observed only during soft X-ray irradiation.40 Although this short-lived EPR spectrum has not been assigned yet to any specific molecular species, the EPR

Fujii et al. signal intensity is strongly enhanced at the oxygen K-edge but is not significant at the nitrogen K-edge. We also observed the short-lived species in evaporated guanine and adenine films.41 In this case, the spectra of the photon energy dependence of EPR intensity of the short-lived species coincided with the photoabsorption spectra of the nucleobases, showing significant enhancement of the EPR intensity at the nitrogen K-edge region. Interestingly, the enhancement of the EPR intensity of the evaporated adenine film at the nitrogen K-edge region is almost reduced completely following slight exposure to water vapor in a vacuum. Instead, a significant oxygen K-edge structure appears in the spectrum of the photon energy dependence, even though adenine does not have any oxygen atoms. These pieces of evidence obtained by EPR experiments also support the hypothesis that the water molecules bound to DNA play an important role in the induction of purine base lesions. We have reported that the hydrating water molecules surrounding DNA efficiently increase the yields of base lesions but not considerably for the yield of SSBs when irradiated with γ-radiation.16 Irradiation of nucleobases with ionizing radiation is known to produce a variety of unpaired-electron species observed as free radicals by electron paramagnetic resonance (EPR) or the electron and nuclear double resonance (ENDOR) method (see review).42 Purkayastha et al.43 have studied the G-values of free radicals trapped in hydrated DNA directly ionized by X-ray irradiation (70 kV, tungsten target) using a low-temperature (4 K) EPR technique. They suggested that the observed free radicals are mainly localized in the nucleobases (80-90%). Two species of trapped radicals are formed: a purine radical cation formed by one-electron oxidation or hole formation and a pyrimidine radical anion formed by one-electron reduction.42 However, the pathways that produce these chemically stable nucleobase lesions via oxidative or reductive radical productions are not completely understood. The yield of prompt SSBs is also enhanced by oxygen K-ionization, although not considerably when compared with the yields of nucleobases lesions. Ejected electrons due to the photoelectric effect in DNA are also expected to cause molecular damage, not only through normal ionization of DNA but also through a resonantly dissociative attachment process. The latter process preferentially induces DNA strand breaks.44 The effect by electron impacts would be nonspecifically induced regardless of the atom from which the secondary electrons are ejected. In our previous studies, the prompt SSB yield for dry pUC18 plasmid DNA is almost constant for a variety of radiation energies (0.5-1.2 × 10-10 SSB/Gy/Da), indicating that the DNA strand breaks are mainly induced by the random collisions of the secondary electrons. Ito et al.45 have reported that the main target of SSBs in solid-state DNA is the pentose ring (2deoxyribose). We also concluded that the 2-deoxyribose is a more fragile site than the nucleobases, as the result of an ion desorption mass spectroscopy study using soft X-rays of the oxygen K-edge.39 The induction of SSBs is not expected to be much more sensitive to irradiation with soft X-ray at this energy level. The yield of Nth-sensitive sites is higher than that of Fpgsensitive sites induced by irradiations for all photon energies, particularly for 760 eV (n(ESSSSB)Nth/n(ESSSSB)Fpg is about 3.3). We have reported that the significant excess of Nth-sensitive sites compared with Fpg-sensitive sites is induced in hydrated pUC18 plasmid DNA film by He2+ ions and 150-kVp X-ray irradiation. The ratio between the two nucleobase lesions (n(ESSSSB)Nth/n(ESSSSB)Fpg) is typically 2.6 for He2+ (12.5 MeV/ nucleon)46 and 1.9 for X-ray irradiation.47 Soft X-ray irradiation

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Figure 5. Schematic illustrations of photoionization of DNA or hydrating water and ejected low-energy electrons.

around the phosphorus K-edge region (2.1 keV) also induces excess Nth-sensitive sites depending on both photon energy and hydration conditions (1.3-2.8). γ-Irradiation of the hydrated plasmid DNA results in a slight excess of Nth-sensitive sites rather than Fpg-sensitive sites; the ratio is about 1.2.16 On the other hand, the differences in yields are not always observed for irradiation with 150-kVp X-rays in solutions with low scavenging capacities less than 108 s-1.47 On the basis of this experimental evidence, the photoelectric effect, particularly oxygen K-ionization arising in the DNA molecule or the hydrating water layer, causes purine and pyrimidine base lesions with different efficiencies, even though these lesions are produced almost equally by reaction of diffusible OH radicals induced in bulk water or inelastic scattering of Compton electrons ejected by irradiation of the sample with γ-rays. As already discussed above, Nth-sensitive sites such as DHT are likely produced by addition of H atoms or H+ ions to the C5or C6-position of thymine or thymine anion radical. These H atom or H+ ions could be easily produced by soft X-ray irradiation of the sample. Thus, this type of lesion might be induced more efficiently than the purine base lesions that require binding of an oxygen atom to produce the oxidative guanine lesions observed as Fpg-sensitive sites. One of the major nucleobase radicals is the cytosine anion radical, which forms by trapping one electron at low temperatures (4-10 K). At higher temperatures (>180 K), thymine primarily traps the electron to produce the thymine anion radical. This radical easily converts to a 5,6-dihydrothymin-5-yl radical (5-thymyl radical), which has a specific eight-line EPR spectrum.48 The 5-thymyl radical is likely a precursor of 5,6dihydrothymine (DHT), which is one of the major substrates of the Nth protein. When a K-shell electron of an atom in thymine is ionized by soft X-ray irradiation, one or two holes are left in the thymine as a consequence of the Auger process, and an electron adduct is not likely to be produced at the thymine. Nevertheless, in our previous EPR study, the 5-thymyl radical is one of the major products formed by oxygen K-shell photoabsorption in a thymine thin film that was irradiated with 538 eV soft X-rays in a vacuum at 77 K.49 The dose response curve of the total spin number obtained from the EPR spectrum has a slope similar to that of 407 eV photoirradiation (nitrogen K-shell photoabsorption) in the low dose range.49 We also reported that the amount of DHT produced by irradiating a

thymine-pellet sample in a vacuum with soft X-rays having energies of 538 eV, as determined by HPLC analysis, is slightly lower than the amount of DHT produced by irradiation with soft X-rays below the oxygen K-edge (395 or 407 eV).50 This experimental evidence suggests that the production of DHT is not enhanced by oxygen K-ionization when thymine exists alone, that is, when it is not present as part of DNA. In other words, oxygen K-ionization affects not only thymine but also the sugar-phosphate backbone of DNA or the hydrating water molecules that tightly bind to DNA even in a vacuum and is responsible for the strong enhancement of the yield of Nthsensitive sites. This hypothesis is supported by the fact that 435 eV irradiation causing K-ionization of nitrogen atoms, which in DNA are present only in the nucleobases, induces the formation of the Nth-sensitive site in greatly lower yield than oxygen K-ionization at 560 eV as shown in Figure 2. Furthermore, it is necessary to determine the source of hydrogen atoms that add to the C6 or both the C5 and C6 positions in thymine to produce a 5-thymyl radical or DHT. We have reported that H+ is the major ion desorbed by 538 eV soft X-ray irradiation in thin films of thymine as well as 2-deoxy-D-ribose, thymidine (dThd), and thymidine 5′-monophosphate (dTMP).39 These results suggested that H+ is overwhelmingly produced in the sample by oxygen K-ionization. In addition, the 2.5 hydrating water molecules per nucleotide, which inevitably are present in DNA samples even under high vacuum,51 could be the hydrogen source. Thus, we propose a model of the production of Nth-sensitive site enhanced by oxygen K-ionization, which is shown in Figure 5. Similarly, oxygen K-ionization in the functional groups surrounding guanine or adenine in DNA is expected to be responsible for the production of Fpg-sensitive sites. Guanine is known to be a major hole trapping site in DNA since guanine has the lowest ionization potential (in the gas phase) among the nucleobases (see review42). A positive charge would migrate from H2O+ or H2O2+, which is produced by Kionization of oxygen in hydrating water, to DNA, and the resulting 8-hydroxy-7,8-dihydroguanyl radical may react with the water molecule concomitantly with the charge migration to rise to 8-OHGua which exists in a dynamic equilibrium with 8-oxoGua, the predominant 6,8-diketo tautomer. Competitive reduction of the 8-hydroxy-7,8-dihydroguanyl radical

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Fujii et al.

Figure 6. Schematic illustrations of photoionization of DNA or hydrating water and ejected low-energy photo or Auger-electrons or transferred holes.

leads to the formation of FapyGua52,53 which is also a substrate for Fpg protein (Figure 6). When a nucleobase lesion is induced proximally to other lesions (nucleobase lesions or SSBs) within a separation of about ten base pairs in a DNA molecule, the clustering lesions are known to be converted to DSB by the base excision repair enzymes.21 Enzymatically induced DSBs in genomic DNA in a living cell are a possible cause of cell lethality as induced by directly radiation-induced DSBs (prompt DSBs). In this study, both types of DSBs show one-order smaller yields than those of SSBs. However, the yields of DSBs show a characteristic photon energy dependence as seen for nucleobase lesions and SSBs. The yields of enzymatically induced DSBs are enhanced not only by oxygen K-ionization at 560 eV but also by irradiation of 270 eV (below the carbon K-edge) photons as shown in Figure 4. On the other hand, prompt DSBs are significantly enhanced by oxygen K-ionization. As presented above, oxygen K-ionization increases the nucleobase lesions. Consequently, the yields of the clustering of the nucleobase lesions likely increase above the oxygen K-edge energy. The clustering of nucleobase lesions might be induced through valence electron ionization or phosphorus L-ionization by 270 eV photons (below the carbon K-edge). The ionization of the phosphorus L-shell electrons produces phosphorus LMM Auger electrons (∼120 eV). We recently reported that these LMM Auger electrons are responsible for the clustering of the nucleobase lesions induced by phosphorus K-ionization.54 However, irradiation with 380 eV photons also produces similar energy photoelectrons (∼100 eV) from the carbon K-shell. Although a detailed mechanism has not yet been elucidated, it is possible that not only core ionizations but also valence electron ionizations and the resulting formation of holes in DNA are involved in causing serious DNA damage such as the clustering of nucleobase lesions. It should be noted that the final degradation products formed upon irradiation of solid-state films in a vacuum possibly differ

from degradation products reported in previous studies for irradiation of DNA in aerated aqueous solutions. 5,6-Dihydrothymine is the main radiation-induced pyrimidine base degradation product in the dry state,55,56 whereas 5,6-dihydroxy5,6-dihydrothymine is predominantly generated as an oxidatively formed lesion in aerated aqueous solution of DNA and in cells.52 This is related primarily to the fact that molecular oxygen does not diffuse in the solid state and does not react with DNA radicals. In contrast, evidence has been provided for the occurrence of O2 addition reactions to pyrimidine radicals in aerated aqueous solutions and in cellular DNA. This is also likely to apply to the radiation-induced mechanisms of degradation of the 2-deoxyribose moiety that is involved in the formation of SSBs and DSBs. In future studies, we plan to investigate whether the results obtained in the present study using “dry” samples can be applied to cell mimetic conditions. Nevertheless, the atom selective photoelectric effect is attractive for studying the technical possibility of selective damage induction in DNA. In summary, the ratio of DNA strand breaks and base lesions strongly depends on the photoabsorption site. Strand breaks can be specifically induced below the nitrogen K-edge, and base lesions can be specifically induced above the oxygen K-edge. Oxygen K-ionization was induced in not only the nucleobases but also other parts of the DNA as well as in hydrating water molecules. This oxygen K-ionization may be substantially involved in the production of nucleobase lesions not through simple bond scission at the K-ionized oxygen but through a complex process of binding protons or oxygen atoms to the nucleobases. These unique features of monochromatic soft X-ray irradiation in regard to biological samples will facilitate the development of techniques for the analysis of DNA damage and its enzymatic repair system. Acknowledgment. This work was supported by a Grant-inAid for Young Scientists (B) (17710051) from The Ministry of

Selective DNA Damage Induction by Soft X-rays Education, Culture, Sports, Science and Technology (MEXT) and a Grand-in-Aid for Scientific Research (B) (18310041) from Japan Society for the Promotion of Science (JSPS). We gratefully acknowledge Ken Akamatsu and Ritsuko Watanabe for their help with dosimetry and fruitful discussions. We gratefully acknowledge Yoshihiko Hatano for helpful discussions regarding oxygen K-ionization. We also thank Yoshihiro Fukuda and Yuji Saitoh of SPring-8, JAEA, for operating the synchrotron soft X-ray beamline. References and Notes (1) Free-Radical-Induced DNA Damage and Its Repair; Sonntag, C. v., Ed.; Springer: Muhlheim, 2006. (2) de Lara, C. M.; Hill, M. A.; Jenner, T. J.; Papworth, D.; O’Neill, P. Radiat. Res. 2001, 155, 440. (3) Nikjoo, H.; Bolton, C. E.; Watanabe, R.; Terrissol, M.; O’Neill, P.; Goodhead, D. T. Radiat. Prot. Dosim. 2002, 99, 77. (4) Tanaka, K.; Sako, E. O.; Ikenaga, E.; Isari, K.; Sardar, S. A.; Wada, S.; Sekitani, T.; Mase, K.; Ueno, N. J. Electron Spectrosc. Relat. Phenom. 2001, 119, 255. (5) Tinone, M. C. K.; Tanaka, K.; Maruyama, J.; Ueno, N.; Imamura, M.; Matsubayashi, N. J. Chem. Phys. 1994, 100, 5988. (6) Hieda, K.; Ito, T. Handbook on Synchrotron Radiation; Elsevier: Amsterdam, 1991; Vol. 4. (7) Kobayashi, K. Charged Particle and Photon Interaction with Matter; Marcel Dekker: New York, 2004. (8) du Penhoat, M. A. H.; Fayard, B.; Abel, F.; Touati, A.; Gobert, F.; Despiney-Bailly, I.; Ricoul, M.; Sabatier, L.; Stevens, D. L.; Hill, M. A.; Goodhead, D. T.; Chetioui, A. Radiat. Res. 1999, 151, 649. (9) Yokoya, A.; Watanabe, R.; Hara, T. J. Radiat. Res. 1999, 40, 145. (10) Fayard, B.; Touati, A.; Abel, F.; Penhoat, M. A. H. d.; DespineyBailly, I.; Gobert, F.; Ricoul, M.; L’hoir, A.; Politis, M. F.; Hill, M. A.; Stevens, D.; Sabatier, L.; Sage, E.; Goodhead, D. T.; Chetioui, A. Radiat. Res. 2002, 157, 128. (11) Eschenbrenner, A.; Herve Du Penhoat, M. A.; Boissiere, A.; EotHoullier, G.; Abel, F.; Politis, M. F.; Touati, A.; Sage, E.; Chetioui, A. Int. J. Radiat. Biol. 2007, 83, 687. (12) Agrawala, P. K.; Eschenbrenner, A.; du Penhoat, M. A.; Boissiere, A.; Politis, M. F.; Touati, A.; Sage, E.; Chetioui, A. Int. J. Radiat. Biol. 2008, 84, 1093. (13) Hieda, K.; Hirono, T.; Azami, A.; Suzuki, M.; Furusawa, Y.; Maezawa, H.; Usami, N.; Yokoya, A.; Kobayashi, K. Int. J. Radiat. Biol. 1996, 70, 437. (14) Goodhead, D. T. Synchrotron Radiation in the Biosciences; Oxford Science Publications: New York, 1994. (15) Nikjoo, H.; O’Neill, P.; Terrissol, M.; Goodhead, D. Radiat. EnViron. Biophys. 1999, 38, 31. (16) Yokoya, A.; Cunniffe, S.; O’Neill, P. J. Am. Chem. Soc. 2002, 124, 8859. (17) Goodhead, D. T. Int. J. Radiat. Biol. 1994, 65, 7. (18) Ward, J. F. Int. J. Radiat. Biol. 1994, 66, 427. (19) Melvin, T.; Cunniffe, S. M.; O’Neill, P.; Parker, A. W.; RoldanArjona, T. Nucleic Acids Res. 1998, 26, 4935. (20) Prise, K. M.; Pullar, C. H.; Michael, B. D. Carcinogenesis 1999, 20, 905. (21) Sutherland, B.; Bennett, P.; Sidorkina, O.; Laval, J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 103. (22) Milligan, J. R.; Aguilera, J. A.; Nguyen, T. T.; Paglinawan, R. A.; Ward, J. F. Int. J. Radiat. Biol. 2000, 76, 1475. (23) Demple, B.; Linn, S. Nature 1980, 287, 203. (24) Breimer, L. H.; Lindahl, T. J. Biol. Chem. 1984, 259, 5543. (25) Dizdaroglu, M.; Laval, J.; Boiteux, S. Biochemistry 1993, 32, 12105.

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