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Photochemical crosslinking is a method for studying the molecular details of protein-nucleic acid interactions. In this study, we describe a novel strategy to localize crosslinked amino acid residues that combines laser-induced photocrosslinking, proteolytic digestion, Fe3+-IMAC (immobilized metal affinity chromatography) purification of peptide-oligodeoxynucleotide heteroconjugates and hydrolysis of oligodeoxynucleotides by hydrogen fluoride (HF), with efficient matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The new method is illustrated by the identification of the DNA-binding site of the restriction endonuclease MboI. Photoactivatable 5-iododeoxyuridine was incorporated into a single site within the DNA recognition sequence (GATC) of MboI. Ultraviolet irradiation of the protein-DNA complex with a helium/cadmium laser at 325 nm resulted in 15% crosslinking yield. Proteolytic digestion with different proteases produced various peptide-oligodeoxynucleotide adducts that were purified together with free oligodeoxynucleotide by Fe3+-IMAC. A combination of MS analysis of the peptide-nucleosides obtained after hydrolysis by HF and their fragmentation by MS/MS revealed that Lys209 of MboI was crosslinked to the MboI recognition site at the position of the adenine, demonstrating that the region around Lys209 is involved in specific binding of MboI to its DNA substrate. This method is suitable for the fast identification of the site of contact between proteins and nucleic acids starting from picomole quantities of crosslinked complexes.

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PURPOSE: To test the integrity of the thymine molecule that experiences an increasing number of charges due to the loss of Auger electrons emitted by the decay of incorporated 125I. Besides the radiation action of these electrons, Coulomb explosion is suspected to be an additional mechanism responsible for the strong radiotoxic effect of decaying DNA-incorporated 125I. The two-step decay process initiates a first Auger cascade within 10(-16) to 10(-14) s resulting in the release of about 7 electrons on average and a corresponding large positive charge on the 125Te daughter atom. Being part of iododeoxyuridine (125IUdR), the analogue of the DNA base thymine, the base is suddenly confronted with this charge. Experimentally, the situation was investigated with small molecules (CH3(125)I and C2H5(125)I) resulting in ion fragmentation in agreement with a Coulomb explosion model (Carlson and White, 1963, 1966). MATERIALS AND METHODS: Semi-empirical quantum mechanical calculations on the Parametric Method 3 (PM3) level (Stewart, 1989a, 1989b) were performed and geometry optimisation was applied for the identification of stable molecule conformations. Subsequently, semiempirical molecular dynamics simulations allowed changes in the conformations to be studied as a function of time. RESULTS: First results show that there is no stable molecular configuration with a total charge of > or = +5e. PM3 calculations will not converge for such a charge located at the 125I/125Te position. This finding is supported by total energy considerations, which begin to favour a system of isolated atoms versus molecular bound atoms when the molecular charge is greater than +4e. The distribution of the partial charges indicates that most of the charge will remain on the tellurium atom with slight increases of charge at the other molecular partners within 125IUdR. Moreover, the molecular dynamics simulations reveal a breaking of chemical bonds between those atoms with the strongest charge increase. CONCLUSIONS: Coulomb explosion must be taken into account as a possible damaging mechanism following the decay of DNA-incorporated Auger electron emitters. Lobachevsky and Martin (2000) have identified the same mechanism to be responsible for part of strand breakage in oligo-deoxynucleotides. To elucidate a possible link between both damage patterns the molecular mechanics simulations have to be extended to larger parts of the DNA molecule.

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PURPOSE: To estimate the enhancement of DNA strand breaks induced by low linear energy transfer (LET) radiation in the presence of halogenated pyrimidines and to examine complexity and clustering properties of damage that could provide a correlation between DNA damage and lethality. MATERIALS AND METHODS: Monte Carlo track structure methods were used to model and estimate the induction of strand breakage by X-ray photons with and without the incorporated Br/I deoxyuridine in cell-mimetic conditions. The increase of DNA strand break induction was modelled by taking into account the direct energy deposition and the reactions of radicals. The yield and spectrum of strand breaks were calculated at various degrees of Br/IdU incorporation. The excess strand breaks due to Br/IdU incorporation was assumed to be induced by highly reactive uracilyl radicals. Four mechanisms were considered for the production of uracilyl radicals classified into three groups, by hydrated electrons, by direct energy deposition, and by both hydrated electrons and direct energy depositions. In total, nine different models were considered to test the excess strand breaks by incorporated Br/IdU assuming different pathways. RESULTS: Model calculations show the following: the yield of strand breaks is enhanced by both the e(aq)(-) reaction and the direct energy deposition on base moiety; there is a significant contribution to the enhancement of yield of strand breaks due to energy transfer about four bases along the DNA to Br/IdU and DNA strand break complexity increases with degree of Br/IdU incorporation. Enhancement ratios of 1.8 and 2.5 for 40% Br/IdU substitution were obtained for single- and double-strand breaks, respectively. CONCLUSIONS: The increase in the yield of strand breaks due to Br/IdU incorporation could be explained by the mechanism of uracilyl radical production by e(aq)(-) and direct energy deposition. The importance of energy transfer along the DNA is demonstrated. It is shown that the incorporation of Br/IdU causes a spectral shift towards a greater complexity of clustered DNA damage.

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Photon activation therapy is a binary system being investigated as a potential therapeutic modality to improve the treatment of malignancies, particularly the highly lethal and malignant brain tumor, glioblastoma multiforme. Its success relies upon the incorporation of a target atom in the immediate vicinity of a tumor cell's critical site, followed by the activation of this atom with photons of energies suitable for the induction of the photoelectric effect and its concomitant Auger cascades. The collective action of the Auger electrons imparts high-LET type damage at the critical site. Photon activation therapy uses iodine from stable iododeoxyuridine (IdUrd) as the target atom, and monochromatic photons above the K absorption edge of iodine (33.2 keV) as the activating agent. Although IdUrd is a cell-sensitizing agent, work described was designed to separate the biological efficacy due to sensitization from that of the Auger effect. Chinese hamster V79 cells with and without IdUrd in cellular DNA were irradiated at the X17B1 beam line in the National Synchroton Light Source of Brookhaven National Laboratory. Monochromatic photons above (33.4 keV) and below (32.9 keV) the K absorption edge were used to determine if any additional biological damage would accrue from the Auger cascades. The 33.4-keV photons were found to be a factor of 1.4 times more effective than 32.9-keV photons in damaging iodinated cells. The sensitizing effect, evaluated separately, was found to be a factor of 2.2 at 10% survival, regardless of photon energy. Thus the total therapeutic gain was 1.4 x 2.2 = 3.1. Irradiations of noniodinated control cells showed no difference in their response to energies above and below the iodine K edge.

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Halogenated pyrimidines such as iododeoxyuridine (IdUrd), when incorporated into cellular DNA, sensitize cells to radiation. A technique known as photon activation therapy (PAT) has recently been proposed to further enhance IdUrd radiosensitization. This approach relies on the emission of high linear energy transfer Auger electrons as a result of photoelectric absorption of photons with energies slightly above the K shell binding energy for iodine. Thus, additional IdUrd radiosensitization has been predicted for this technique using photons either generated externally from orthovoltage units or with implants of radioisotopes with emissions of the appropriate energy range. We have tested this prediction by comparing the radiosensitization of Chinese V79 cells, which have incorporated IdUrd (16% replacement of thymidine with IdUrd), to megavoltage (15 MV) versus orthovoltage (100 KVp) radiation. Enhancement ratios at 1% survival levels for 15 MV vs. 100 KVp radiation were 1.8 and 1.95, respectively. This modest 10-15% additional enhancement was much less than that predicted by PAT. The discrepancy between experimental data and the theoretical predictions of PAT are discussed.

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It is suggested here that significant advantages should accrue from the use of 40 keV photons from implanted sources of 145Sm. These energies should stimulate Auger electron cascades from IdUrd, as well as produce non-repairable damage from radiosensitization. The use of low dose rates (approximately 10 rd/hr) should allow repair in normal tissues exposed to the activating photons. Utilization of this technique with brain tumors should minimize problems associated with radiosensitization of normal tissues, as CNS tissues do not synthesize DNA. The deposition of high LET radiations selectively in tumor cells provides unique advantages not available to either conventional therapy or other forms of particle therapy (fast neutrons, protons, pions, heavy ions).

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X-irradiation of single crystals of 5-iododeoxyuridine (IUdR) in the temperature range 8-300 K produces mainly four different radicals which have been studied by electron spin resonance (e.s.r.) and electron nuclear double resonance (ENDOR)-spectroscopy. At low temperatures, a pi-anion is formed which shows predominantly an interaction of the unpaired electron with a proton at carbon C6 of the base (-11.8 G, -23.9 G, -4.6 G). Above 10-20 K, the anion protonates at C6 to yield a RC-I(CH2)-R' radical comprising alpha-iodo and beta-methylene proton hyperfine interactions. The primary oxidation product is an O5'-situated alkoxy radical RCH2O which shows inequivalent beta-proton couplings of about 100 G and 35 G together with a highly anisotropic g-tensor. Upon warming to 265 K, a C2'-located radical on the deoxyribose is formed which is stable at room temperature. A detailed account of its spectral features as obtained by ENDOR exhibits three different alpha-type couplings, two small beta-protons and a dipolar interaction. Other radicals, not reproducibly observed, involve a C5'-hydroxyalkyl radical and a species related to the base cation at low temperatures.

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Two hypotheses have been advanced to explain the extreme biological toxicity of DNA associated 125I: (a) high local concentrations of radiation energy from low energy Auger electron; (b) charge-induced molecular fragmentations in the DNA. To distinguish between these two hypotheses an attempt was made to evaluate the molecular events associated with electron capture decay, to calculate the microdistribution of radiation energy after 125I decay, and to relate the microdosimetry data to the biological toxicity of 125I and 3H. The cellular damage produced by the two radionuclides was evaluated on synchronized Chinese hamster ovary cells (CHO) labeled with various doses of 3H-thymidine or 125I-iododeoxyuridine. As expected, 125I (LD50: 45 rad; D0: 74 rad) proved much more toxic to CHO cells than either 3H (LD50: 380 rad; D0: 250 rad) or external X-irradiation (LD50: 330 rad; D0: 230 rad). To evaluate the molecular mechanism of 125I toxicity, iododeoxyuridine labeled with both 125I and 14C was synthesized and the effect of 125I decay on the molecular structure of iododeoxyuridine was studied by monitoring the fate of 14C activity after 125I decay. The results of this experiment indicate that 125I decay does not cause molecular fragmentation in iododeoxyuridine, only deiodination. Moreover, microdosimetry calculations show that at least in small target spheres more radiation energy is deposited on the average by decaying 125I than by a high LET alpha-particle traversing a sphere of equal diameter. These findings greatly strengthen the hypothesis that the high LET-type damage produced by Auger emitters results from high local concentrations of radiation energy rather than from charge-induced fragmentation of the DNA.

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Wild-type M. radiodurans and two radiosensitive mutants were used to study the lethal effects of 125I disintegrations in their DNA. The relative sensitivities of these three strains to inactivation by gamma-radiation were reflected in their relative sensitivities to inactivation by 125I decay. The number of double-strand (ds) breaks in the DNA appeared to be similar at levels of gamma-radiation and of 125I decay that reduced survival to 10%. All three strains of M. radiodurans rapidly repaired ds breaks produced in their DNA by either gamma-radiation or 125I disintegrations. If one ds break per is a lethal event [Kirsch et al., 1975], cells of the three strains tested would die only when they had left unrepaired one ds break out of an initial 45,600 or 1800 ds breaks per single cell.

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In view of the enhanced biological damage caused by the "Auger nuclide" iodine-125, we have carried out quantitative ESR-studies of the radical formation in polycrystalline 5-iododoeoxyuridine (IUdR) resulting from the following internal or external radiation sources: (1) Decay of 3H, 125I or 131I in labeled IUdR; (2) Lanthanum K-photons corresponding to the K-edge of iodine; (3) 60Co gamma-rays. The results clearly indicate that inner shell ionization with its accompanying Auger effect as caused by the lanthanum K X-ray produces about 30% more free radicals per unit dose absorbed than 60Co gamma-rays, when considering the long-lived secondary radicals. Similarly, the concentration of free radicals is by about 30% higher in 125I- than in 131I-labeled IUdR at comparable doses. In the case of 3H-labeled IUdR the dose curve is almost identical with that observed for 125I-labeled IUdR. The results are discussed in terms of a localized radiation damage from low energy electrons.

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Effects of 125I decay in DNA were investigated by measurements of strand breaks and lethal efficiencies of the decays. In bacteriophages T1 and T4, local decay effects were compared with effects of the emitted electrons by induction of both single (ssb) and double strand breaks (dsb) in the intact phage head and in extended free state DNA. Most dsbs were found to result from local decay effects whereas most real ssbs are caused by the electrons. A simple one-to-one relationship seems to exist in the phages between the decays of 125I, numbers of dsbs and lethal effects. In E. coli rec+ and recA repair of dsbs was studied in addition to lethal decay efficiencies. In rec+ more than 70% of the dsbs were repaired within 1 h at 37 degrees C. No repair was observed in recA. The probability of lethality per 125I decay per completed genome was found to be 0.37 for rec+ and 0.93 for recA cells. The number of lethal events per unrepaired dsb was found to be practically equal to unity. Unrepaired dsbs thus seem to be the primary mechanism of lethality caused by 125I decay, and all unrepaired dsbs seem to be lethal.

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Radioactive decay in a labelled molecule leads to specific chemical and biological consequences which are due to local transmutation effects such as recoil, electronic excitation, build-up of charge states and change of chemical identity, as well as to internal radiolytic effects. In the present paper these effects are reviewed emphasizing the relation of the chemical alterations on a molecular level to the biological manifestation. Potential importance of this type of research for biomedical applications is pointed out. In part 1 we review the underlying physical and chemical principles and consequences of beta-decay of 3H, 14C, 32P, 33P, 35S and 125I for gaseous and simple condensed organic systems. Part 2 which will appear in the next issue will include the discussion of biological effects associated with beta-decay.

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Variant clones were isolated from cultured Chinese hamster Don cells after treatment with irradiated 5-iodouridine. The following characters of a primary variant clone, C-11 and a secondary variant clone, C-24 were compared with those of the original clone C-1: colony-forming activity, growth rate in the presence of irradiated and unirradiated 5-iodouridine, distribution of chromosome numbers and cell cohesion. The variant clones C-11 and C-24 were partially resistant to unirradiated 5-iodouridine at lower concentration and C-24 cells were slightly resistant to short-term treatment with irradiated 5-iodouridine. Unlike clines C-1 and C-11 the variant clone C-24 showed no lag phase on growth in 5-iodouridine medium. The modal numbers of the chromosomes of all three clones were 22, like that of normal Chinese hamster diploid cells. Of the three clones, the variant C-24 cells showed the least mutual cohesion and the original C-1 cells showed the most. The possibility that an alteration in cellular membrane might be related to an increase in the resistance to radiosensitizing agents are discussed.

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