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Debije, M. G. and Bernhard, W. A. Electron Paramagnetic Resonance Evidence for a C3' Sugar Radical in Crystalline d(CTCTCGAGAG) X-Irradiated at 4 K. Radiat. Res. 155, 687-692 (2001). A neutral sugar radical formed by the net loss of hydrogen from C3' has been identified in crystalline DNA X-irradiated at 4 K. Crystals of duplex d(CTCTCGAGAG), known to be of B conformation, were studied using electron paramagnetic resonance (EPR) spectroscopy. The C3' radical was identified by using information from dose saturation, power saturation, thermal annealing, and spectrum simulation. The yield of the C3' radical, G(C3'), is 0.03 +/- 0.01 micromol/J, and its concentration does not appear to saturate up to at least 100 kGy. In the region in which total radical concentration increases linearly with dose, the C3' radical makes up about 4.5% of the total radical population trapped in the oligodeoxynucleotide crystal at 4 K. Based on free base release measured in other oligodeoxynucleotides, we suggest that in d(CTCTCGAGAG) the C3' radical is responsible for about one-third of the strand breakage events.

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The aim of this project was to gain an improved understanding of how the efficiency of hole and electron transfer from the solvation layer to DNA decreases as a function of distance from DNA. The packing of DNA in crystals of known structure makes it possible to calculate the degree of DNA hydration with a precision that is significantly greater than that achievable for amorphous samples. Previous work on oligodeoxynucleotide crystals has demonstrated that the efficiency of free radical trapping by DNA exposed to ionizing radiation at 4 K is relatively insensitive to base sequence, conformation, counterion, or base stacking continuity. Having eliminated these confounding variables, it is now possible to ascertain the degree of radical transfer that occurs from ionized water as a function of DNA hydration (Gamma, in mol water/mol nucleotide). EPR is used to measure the hydroxyl radical concentration in crystals irradiated at 4 K. From a lack of hydroxyl radicals trapped in the inner hydration mantle, we determine that hole transfer to DNA is complete for water molecules located within 8 A. This corresponds to Gamma = 9-11 and indicates that hole transfer is 100% (as efficient as direct ionization of DNA) for water molecules adjacent to DNA. Beyond approximately 8 A (Gamma > 10), hydroxyl radicals are observed; thus deprotonation of the water radical cation is seen to compete with hole transfer to DNA as soon as one water intervenes between the ionized water and DNA. The boundary for 0% hole transfer is projected to occur somewhere between 15 and 20 waters per nucleotide. Electron transfer, on the other hand, is 100% efficient across the entire range studied, 4.2

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The radiation chemical yields of unaltered base release have been measured in three crystalline double-stranded DNA oligomers after X irradiation at 4 K. The yields of released bases are between 10 and 20% of the total free radical yields measured at 4 K. Using these numbers, we estimate that the yield of DNA strand breaks due to the direct effect is about 0.1 micromol J(-1). The damage responsible for base release is independent of the base type (C, G, A or T) and is not scavenged by anthracycline drugs intercalated in the DNA. For these reasons, reactions initiated by the hydroxyl radical have been ruled out as the source of base release. Since the intercalated anthracycline scavenges electrons and holes completely but does not inhibit base release, the possibility for damage transfer from the bases to the sugars can also be ruled out. The results are consistent with a model in which primary radical cations formed directly on the sugar-phosphate backbone react by two competing pathways: deprotonation, which localizes the damage on the sugar, and hole tunneling, which transfers the damage to the base stack. Quantitative estimates indicate that these two processes are approximately equally efficient.

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The objective of this work is to determine the extent to which various structural factors influence the yield of trapped free radicals, G(tfr), in DNA irradiated at 4 K. G(tfr) was measured in a series of 13 different oligodeoxynucleotides using electron paramagnetic resonance (EPR) spectroscopy. Each sample consisted of crystalline duplex DNA for which the crystal structure was verified to be that reported in the literature. We find that the G(tfr) of these samples is remarkably high, ranging from 0.55 to 0.75 micromol/J. The standard deviation in G(tfr) for a given crystal structure is generally small, typically less than +/-10%. Furthermore, G(tfr) does not correlate with DNA base sequence, conformation, counterion or length of base stacking. Two observations point to the importance of DNA packing: (1) The radical yields in crystalline DNA are greater than those determined previously for DNA films (0.2 to 0.5 micromol/J); and (2) the variability in G(tfr) is less in DNA crystals than in DNA films. We conclude that closely packed DNA maximizes radical trapping by minimizing the interhelical solvent space. Furthermore, the high efficiency of electron and hole trapping at 4 K is not consistent with DNA possessing properties of a metallic conductor. Indeed, it behaves as an insulator, whether it is in A-, B-, or Z-form and whether base stacking is short- (8 bp) or long-range (>1000 bp).

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Radiation-induced free radical damage in solid-state DNA arises from direct-type damage mechanisms. In the present study, free radical yields in film and lyophilized Na DNA model systems are compared. The free radical yields in lyophilized samples are significantly greater than those in films. Since DNA conformation cannot account for the differences in free radical yields between different DNA preparations, it is proposed that the intermolecular spacing of DNA is a critical variable. The differences in the hydration dependence of free radical yields between the film and lyophilized DNA model systems are consistent with this thesis. The relatively large free radical yields observed in lyophilized DNA emphasize the fact that DNA is an extremely effective electron and hole scavenger, more so than previously thought.

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This work investigates the direct-type action of radiation (involving electron addition and electron abstraction) on DNA. Specifically, the effects of DNA hydration, conformation and packing on free radical yields are examined. The fact that these variables are interdependent complicates the analysis of how each variable affects free radical yields. The hydration dependence of free radical yields in films of both Li and Na DNA was examined. At low levels of DNA hydration (less than 25 waters per nucleotide), the relatively high free radical yields and the lack of water-derived radicals are evidence that damage transfer from the DNA solvation shell to the DNA molecule occurs. The scatter of measured free radical yields is significant (50-70%) in Li DNA, while in Na DNA it is much less (<25%). Our conclusions hinge upon two known differences between Li DNA and Na DNA: (1) At low DNA hydrations, the conformation of Na DNA undergoes several changes with increasing hydration, while the conformation of Li DNA is relatively constant over the same range. (2) Compared to Na DNA, Li DNA is more prone to self-associate, giving rise to macroscopic and microscopic crystalline domains in Li DNA films. The greater scatter of free radical yields in Li DNA films is therefore attributed to variability in packing. By virtue of the greater reproducibility of free radical yields in Na DNA films, the effects of DNA packing, conformation and hydration can be ascertained. In Na DNA, hydration-dependent changes in free radical yields are attributed primarily to changes in DNA packing.

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Electrons and holes generated in irradiated DNA migrate to stable trapping sites. Protonation and deprotonation reactions at these sites promote the trapping of electrons and holes, thereby inhibiting further migration. The extent of migration determines the final distribution of damage in irradiated DNA. In this study, electron and hole migration is investigated in a crystalline DNA hexamer intercalated with an anthracycline drug. The intercalator is no further than 2 base pairs away from any DNA base. From EPR measurements, there is no evidence of DNA-centered radicals in the irradiated DNA hexamer. The aromatic region of the anthracycline intercalator evidently sequesters most or all of the electrons and most of the holes. Further hole trapping and radical stabilization appear to occur on the anthracycline's amino sugar group, which is nestled in the minor groove of the hexamer. The relatively large yield of this proposed amino sugar radical suggests that holes generated in the DNA solvation shell migrate to the amino sugar, where they become trapped. This would be the first observation of a radical formed by the direct effect of low-dose, low-LET radiation that is trapped within the DNA helix, yet lies outside of the stacked bases. With respect to holes generated in the DNA bases at 4 K, we conclude that most, if not all, are capable of migrating to an intercalator < or = 2 base pairs away. With respect to dry electrons, we conclude that anthracycline competes effectively for electron trapping over a region of at least 2 base pairs; our experiments cannot distinguish between electron attachment to the bases followed by transfer to the intercalator and direct attachment to the intercalator.

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The hydration layer of DNA increases the target size of DNA with respect to the formation of direct-type damage by ionizing radiation. The mechanisms that give rise to this increase are being investigated by EPR spectroscopy. To determine these mechanisms, it is necessary to distinguish between the change in sample mass and changes in packing/conformation brought about by the change in the level of hydration. Certain model compounds that crystallize as hydrates provide a system where the effects of mass and packing can be discerned. Three such hydrate crystals were used in this work: barbituric acid dihydrate (BA:2H2O), inosine dihydrate (IR:2H2O) and thymine monohydrate (T:H2O). The free radical yields (+/-25%) in the native crystals at 7-11 K are 0.08, 0.03 and 0.02, respectively. Removal of the lattice water leaves behind an ordered lattice and results in free radical yields of 0.08, 0.03 and < 0.004, respectively. Thus removal of the lattice water does not affect the free radical yield in BA:2H2O or IR:2H2O but decreases the free radical yield in T:H2O by an order of magnitude. Based on these observations and the known crystal packing, we conclude that the hydrogen bonding network is a major factor in determining the distribution and yield of trapped free radicals. We ascribe this to the importance of proton transfer processes which act to reduce the probability of radical combination. Consistent with this conclusion are the types of free radicals trapped in these crystalline materials before and after dehydration. From these results, we argue that a major determinant of free radical yields in solidstate samples of DNA constituents is molecular packing. In addition, the absence of HO. radicals trapped in single crystals of BA:2H2O provides an upper limit for the yield of trapped HO. of less than 10(-4) mumol/J. This supports the thesis that at < 77 K direct ionization of those waters associated directly with a pyrimidine or purine results in hole transfer to that molecule. Hydroxyl radical formation on a water adjacent to a DNA base is predicted to be negligible.

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Powders and films of variably hydrated poly(U), poly(A) and poly(A):poly(U) were X-irradiated at 4 K. Spectra and free radical yields were acquired at 4 K using Q-band EPR spectroscopy. Evidence for electron transfer from the hydration layer to the RNA bases, supporting in part the damage transfer hypothesis of Gregoli et al. (Radiat. Res. 89, 238-254, 1985), is presented. Based on measurements of radical yield as a function of hydration, we propose that intermolecular packing and polymer conformation are dominant factors in determining free radical trapping ability in these polymers. Our annealing results indicate that increasing hydration facilitates intercluster combination reactions.

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A series of single- and double-stranded oligodeoxynucleotides of adenine and thymine, 8 to 12 nucleotides in length, were one-electron-reduced at 10 K in a > 8 M LiCl/H2O glass. The Q-band electron paramagnetic resonance (EPR) spectra of these radicals show that thymine is the dominant trapping site for mobile electrons in these oligomers. The spectra of the reduced oligomers in the series pd(AnT10-n).pd(A10-nTn) with n = 5-->10 showed a trend which is interpreted as either an increase in the probability of trapping at an adenine base in tracks of adenine > 7 base pairs in length, or the presence of different protonated states of the one-electron-reduced bases due to the adoption of a different conformational state for longer tracks of adenine, or a combination of these two possibilities. Analysis of the trends in the EPR spectra of the radicals as a function of sequence using multicomponent analysis is presented.

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The total free radical yield has been measured for crystals of five pyrimidine derivatives: thymine (T), 1-methylthymine (1MeT), 1-methyluracil (1MeU), 1-methylcytosine (1MeC) and cytosine monohydrate (C:HOH). Q-band EPR measurements were made on samples X-irradiated between 4 and 12 K. The G values in units of 10(-7) mol/J are 1MeC < 0.01, T < 0.04, 1MeU = 0.15, 1MeT = 0.25, and C:HOH = 0.8. The types of free radicals formed in these crystals are known through previous EPR investigations. A model is presented that attempts to identify the salient variables behind the large range in G values and, simultaneously, explain the variation in radical types. It is concluded that packing is a critical variable. Hydrogen-bonding networks promote the trapping of radicals through reversible proton transfer. In the absence of such a network less probable radical types are observed, such as radicals formed by irreversible protonation/deprotonation, higher-order reactions and homolytic bond cleavage. Crystals with low G values trap radicals formed predominantly by irreversible protonation/deprotonation at carbon positions and by excitation-spawned homolytic bond cleavage. In contrast, crystals with high G values trap radicals formed predominantly by reversible protonation/deprotonation at heteroatom positions. This model is extended to polynucleotides irradiated at low temperatures, where G values are typically 2-6. The high trapping efficiency seen in polynucleotides reflects highly efficient proton transfer. Consistent with this is the predominance of radicals formed by reversible protonation/deprotonation compared to those formed by irreversible protonation/deprotonation.

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An overview of the early processes initiated in DNA by ionizing radiation is given from the perspective of studies done by solid-state EPR with the focus on radical combination. Comparisons with free radical formation and trapping in crystalline pyrimidines (1-methylcytosine, thymine, 1-methylthymine, 1-methyluracil, and cytosine monohydrate) provide insight into the processes occurring in DNA. Between 25 and 50% of low LET ionizations in fully hydrated DNA at 4 K lead to trapped free radicals, the remaining unobserved radicals are assumed to have combined. The majority of the radicals trapped in DNA at 4 K (G approximately 0.3 mumol/J) are believed to be in clusters. Based on the value of G, it is argued that the range of holes and bound electrons in DNA at 4 K are, in the main, limited to within the cluster diameter, approximately 4 nm. Proton transfer across hydrogen bonds promotes radical trapping and inhibits combination but is thermally reversible. Warming to room temperature mobilizes the reversibly trapped radicals and gives additional combination (50-80% of those trapped at 4 K). The yield of free radicals, after anneal, is sufficient to account for the yield of single-strand breaks produced by direct effects.

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Variably hydrated sodium DNA was X-irradiated and observed at 4 K using Q-band EPR spectroscopy. The free radical yield, G, was measured as a function of the mass ratio of water:DNA, gamma. Both humidified and dehumidified samples showed large scatter when G was plotted against gamma in the range gamma < 1. For gamma > 1, the scatter in G(gamma) is greatly reduced. Also, as gamma is increased above 1.4, G(gamma) decreases toward the G of pure ice. There is no evidence of the ice radicals, OH. and H., until the water content exceeds 40 waters per nucleotide. The scatter in the G(gamma) data is suggested to be a consequence of DNA packing and/or conformation. The absence of ice radicals in DNA samples with twice the water content of fully hydrated DNA is suggested to be a consequence of (1) damage transfer from water of solvation to DNA and (2) differential recombination in which hydration water is less efficient at trapping free radicals.

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EPR measurements were made at Q-band microwave frequencies on powder samples pressed into pellets and X-irradiated at 4 K. Measurements were made at 4 K after no anneal, then after a 77 K anneal, and then after a 300 K anneal. In poly(dA):poly(T) the free radical distribution is approximately a simple sum of the distributions in the separate homopolymers. In poly(dA-T) the free radical distribution differs from that of poly(dA):poly(T). The clearest difference is that in poly-(dA):poly(T) the concentration of one-electron-reduced thymine (Tre.) is reduced relative to the total radical concentration. On warming the thymine-containing samples from 77 K to room temperature, the Tox. radical disappears and the (T-H5'). radical appears. Also, the Tre. radical disappears and the (T + H6). radical appears. There are three main conclusions. First, little or no transfer of free radicals between strands is needed to explain the data. Second, when A and T are interstacked, either the Tre. radical is less stable against recombination than other radical products or radical transfer occurs to an adjacent adenine. Third, in the poly- and oligonucleotides, the Tox. radical is a likely precursor to the (T-H5'). radical.

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Dose-response curves for free radical trapping in monomers, dimers, and polymers of dAMP and TMP have been measured. Powder samples pressed into pellets were X-irradiated and observed at 4 K using EPR at Q-band microwave frequencies. The initial free-radical yield (G value) and the destruction rate (k value) are reported for 22 samples. The absolute magnitude and relative changes in G are informative. For example, the duplex of homopolymers, poly(dA):poly(T)(Na), gives a large G of 6 while the closely related duplex of alternating ATs, poly(dA-T)(Na), gives a G value of 3. At G of around 6, the range of the bound electrons (e-) and electron loss centers (holes) is believed to be very limited. Two factors are suggested as important to limiting the range, proton transfer between strands and the low probability of radical transfer between strands. The lower G values are viewed as being due to relatively small increases in the range of e- and/or holes.

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Electron spin resonance was employed to study one-electron reduced cytosine stabilized in glasses at low temperatures. In a LiCl/H2O glass, deoxycytidine gives an extra approximately 1 mT splitting that is not observed in oligomers. To better understand the source of the extra splitting, 1-methylcytosine (1mC) and N,N-dimethyldeoxycytidine (dmC) were examined in an HCl/H2O glass. The spectrum of 1mC is a quartet and the spectrum of dmC is a triplet. A probable explanation for this is that in both cases N4 is fully protonated prior to electron addition. In the LiCl/H2O glass, monomeric cytosine, after one-electron reduction, appears to protonate at N4. However, oligomeric cytosine, after one-electron reduction, does not protonate at N4 and therefore must protonate at N3. This could be due to the exclusion of Li+ coordination at N3 and/or the constraining of N4 to a planar configuration via stacking interactions. These findings provide additional insight into why cytosine is the major site of electron capture in DNA. Proton transfer across the N1-H...N3 hydrogen bond is expected to stabilize electron addition to cytosine preferentially.

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ESR spectra have been recorded of one-electron reduced thymine (T.-) trapped in a variety of thymine derivatives and matrices. Most of the data were taken at Q-band microwave frequencies on 12 M LiCl glasses containing oligo(T) varying from 2 to 18 nucleotides in length. A nonlinear least-squares simulation program was used to simulate the ESR spectra, resulting in a parameter set that can be used to generate T.- powder spectra at X- and Q-band frequencies. Although it is not possible to identify a unique set of g and hyperfine tensors, it is possible to establish boundaries for these interactions and propose a parameter set that is likely and reasonable. As the trapping molecule is varied from TdR to oligo(T) to poly(T), there are small changes in the interaction parameters. The changes are not large enough to account for the differences between hfc values determined from DNA fiber data versus hyperfine coupling values measured from monomer systems. The analysis also indicates that, in these systems, T.- does not protonate between 4 and 77 K at neutral pH.

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Four free radicals are trapped in methyl alpha-D-mannopyranoside X-irradiated and maintained at 77 K. All four have been identified, with high confidence levels, using ESR and ENDOR spectroscopy. One, an alkoxy radical located at O4, is characterized by a gmax of 2.059, an isotropic beta hydrogen hyperfine coupling (hfc) of 98 MHz, and small interactions due to gamma or delta hydrogens. The second, a secondary dioxyalkyl radical due to loss of hydrogen from C1 is characterized by one beta hfc with an isotropic component of 19.03 MHz. The third, a secondary hydroxyalkyl radical due to loss of hydrogen from C2 is characterized by two nonexchangeable hydrogens with isotropic beta interactions of 22.45 and 6.44 MHz and one exchangeable hydrogen with an isotropic beta interaction of 9.88 MHz. The fourth is a .CH2OR radical that is formed by the net loss of hydrogen from the methyl group.

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