Electron injection from mitochondrial transcription factor A to DNA associated with thymine dimer photo repair.

Electron transfer through π-stacked arrays of double-stranded DNA contributes to the redox chemistry of bases, including guanine oxidation and thymine-thymine dimer repair by photolyase. 5-Bromouracil is an attractive photoreactive thymine analogue that can be used to investigate electron transfer in DNA, and is a useful probe for protein-DNA interaction analysis. In the present study using BrU we found that UV irradiation facilitated electron injection from mitochondrial transcription factor A into DNA. We also observed that this electron injection could lead to repair of a thymine-thymine dimer.

DHPLC and MS studies of a photoinduced intrastrand cross-link in DNA labeled with 5-bromo-2'-deoxyuridine.

It is well known that the replacement of thymidine with 5-bromo-2'-deoxyuridine (BrdU) in DNA sensitizes it to UVB light. Irradiation of a biopolymer substituted in such a way leads to manifold kinds of DNA damage, such as intrastrand cross-links, single- and double-strand breaks or alkali-labile sites that were studied in the past with a broad spectrum of analytical methods. Here, we demonstrate that completely denaturing high-performance liquid chromatography (DHPLC), underestimated so far in DNA damage studies, could act as an inexpensive, and high-resolution substitute for the commonly employed gel electrophoresis. We report on the DHPLC/mass spectrometry (MS) analyses of photolytes obtained with the UV irradiation of aqueous solutions containing 40 base pairs of a long, double-stranded oligonucleotide labeled with BrdU in one of its strands. The UV-product was detected by HPLC at a temperature of 70°C. Subsequent MS analysis with electrospray ionization (ESI-MS) of the photolyte, enzymatic digestion of the irradiated material and HPLC and MS analysis (LC-MS) of the digest demonstrated unequivocally that an intrastrand covalent dimer, involving adenine and uracil, is formed in the irradiated system.

A (6-4) photoproduct of 5-bromouracil.

The photoreactions of 5-bromouracil (BrUra) induced by 254 nm radiation have received a significant amount of scrutiny over the years, both when induced in liquid aqueous solution and in the frozen state. The characterized photoproducts in the liquid state include uracil, 5,5'-diuracil (I) and a variety of products arising from ring opening, while the photoreactivity of BrUra in ice using 254 nm radiation is very low. During examination of the photoreactions induced in BrUra in frozen aqueous solution by exposure to predominately UVB irradiation, we found that several products are formed. One of these products has an ultraviolet absorption spectrum similar to those associated with (6-4) adducts of the pyrimidine bases (λ(max) = 326 nm). Studies with (1)H and (13)C nuclear magnetic resonance spectroscopy and with mass spectrometry yielded data consistent with this compound indeed being a (6-4) product, namely 6-4'-(5'-bromopyrimidin-2'-one)-5,5-dihydroxy-5,6-dihydrouracil (IIa). The product IIa probably arises from an unstable precursor, namely 5-bromo-6-4'-(5'-bromopyrimidin-2'-one)-5-hydroxy-5,6-dihydrouracil (IV); this compound is a bromohydrin and, as such, is likely unstable to debromination to 6-4'-(5'-bromopyrimidin-2'-one)-5-oxo-5,6-dihydrouracil (V). Rehydration at the 5-position to form a gem diol would lead to IIa, the isolated product.

Damage induced by 1-30 eV electrons on thymine- and bromouracil-substituted oligonucleotides.

The impact of low-energy (1-30 eV) electrons on self-assembled monolayers of heterogeneous oligonucleotides chemisorbed on a gold surface has been investigated by mass spectrometry of desorbed neutral species in an attempt to understand the consequences of secondary electron damage in a short sequence of a DNA single strand. We demonstrate that the most intense observable neutral species (CN, OCN and/or H(2)NCN) desorbed from Cy(6)-Th(3) and Cy(6)-(BrdU)(3) oligos are related to primary fragmentation of the bases induced by electron impact. The dependence of the neutral species desorption on electron energy shows typical signatures of dissociative electron attachment initiated by the formation of shape- and core-excited resonances (i.e. single-electron and two-electron- one-hole transitory anions, respectively). Substitution of dTh by BrdU increases the production of neutral fragments by as much as a factor of about 3 for the entire electron energy range. When the distribution of secondary electrons along radiation tracks in H(2)O is taken into account, we show that the probability for electron damage to heterogeneous oligonucleotides is enhanced by a factor of 2.5-3 for electron energies below 20 eV for both sensitized and unsensitized strands.

Radiation-induced electron migration in nucleic acids.

Radiation-induced electron migration along DNA is a mechanism by which randomly produced stochastic energy deposition events can lead to non-random types of damage along DNA manifested distal to the sites of the initial energy deposition. Radiation-induced electron migration in nucleic acids has been examined using oligonucleotides containing 5-bromouracil (5-BrU). Interaction of 5-BrU with solvated electrons results in release of bromide ions and formation or uracil-5-yl radicals. Monitoring either bromide ion release or uracil formation provides an opportunity to study electron migration processes in model nucleic acid systems. Using this approach we have discovered that electron migration along oligonucleotides is significantly influenced by the base sequence and strandedness. Migration along 7 base pairs in oligonucleotides containing guanine bases was observed for oligonucleotides irradiated in solution, which compares with mean migration distances of 6-10 bp for Escherichia coli DNA irradiated in solution and 5.5 bp for E. coli DNA irradiated in cells. Evidence also suggests that electron migration can occur preferentially in the 5' to 3' direction along a double-stranded oligonucleotide containing a region of purine bases adjacent to the 5-BrU moiety. Our continued efforts will provide information regarding the contribution of electron transfer along DNA to formation of locally multiply damaged sites created in DNA by exposure to ionizing radiation.

Ring opening photoreactions of 5-bromouracil and 5-bromo-2'-deoxyuridine with selected alkylamines.

Several studies in the literature indicate that histones (lysine rich proteins found associated with DNA in eukaryotic chromatin), as well as poly-L-lysine, can be photocross-linked by ultraviolet (UV) light to DNA in which 5-bromo-2'-deoxyuridine has been substituted for thymidine. To gain some insight into the possible nature of this cross-linking, we have studied the photoreactions occurring in deoxygenated aqueous solutions containing 5-bromouracil (I) (BrUra) or 5-bromo-2'-deoxyuridine (III) (BrdUrd) and ethylamine, a lysine side chain analog. In the case of I this reaction produced the ring opened compound N-(N'-ethylcarbamoyl)-3-amino-2-bromoacrylamide (Ia). A small amount of N-(N'-ethylcarbamoyl)-3-ethylamino-2-bromoacrylamide (Ic) was also isolated. It was found that purified Ia, standing in the presence of ethylamine, was gradually converted to Ic in a dark reaction. The beta and alpha anomers of N-(N'-ethylcarbamoyl)-3-(2'deoxyribofuranos-1'-yl) amino-2-bromoacrylamide (IIIa and IIIb respectively) were isolated as products in the photoreaction of III with ethylamine; the alpha anomer was produced in a dark reaction from the beta anomer. The identity of these anomers was established by comparison of their proton NMR spectra with those of the four corresponding alpha and beta furanosyl and pyranosyl isomeric nucleosides of thymine, which are presented in the Appendix. A study was also made of the reaction of I with methylamine; a ring opened product analogous to Ia, viz. N-(N'-methylcarbamoyl)-3-amino-2-bromoacrylamide (IIa) was formed. A similar study with 5-bromo-1-methyluracil produced N-(N'-methylcarbamoyl)-3-methylamino-2-bromoacrylamide (IIc) as a product. Likewise, the reaction of 5-chlorouracil with ethylamine was studied and N-(N'-ethylcarbamoyl)-3-amino-2-chloroacrylamide (Ie), which is analogous in structure to Ia, was found to be produced. Structural identifications were made through use of UV spectroscopy, high resolution 1H-NMR spectroscopy, mass spectrometry and, in the case of Ia and IIa, 13C-NMR spectroscopy. In the BrUra and BrdUrd reaction systems, described above, dehalogenation reactions accounted for a major portion of the products. The yields of ring opened products, determined at pH 10, ranged from a high of 10.3% in the BrUra-ethylamine system to a low of 1.7% in the MeBrUra-methylamine system.(ABSTRACT TRUNCATED AT 400 WORDS)

Photochemical reduction of 5-bromouracil by cysteine derivatives and coupling of 5-bromouracil to cystine derivatives.

Irradiation of pH 7, aqueous solutions of 5-bromouracil (BU) in the presence of cysteine peptide-like derivatives at 308 nm using a XeCl excimer laser yielded initial formation of only uracil (U) and the corresponding cystine derivative. Continued irradiation yielded an S-uracilylcysteinyl adduct as well as additional U and cystine derivative. Similar irradiation of a solution of BU and a cystine derivative yielded initial formation of U and the S-uracilylcysteinyl adduct. Formation of these products as well as secondary products of uracil photochemistry was observed upon irradiation of the respective solutions with 254 nm light. With 308 nm laser excitation, U-Cys adduct formation and reduction of BU to U are proposed to occur via initial electron transfer from the disulfide of the cystine derivative to triplet BU. The quantum yield of BU destruction with 308 nm excitation in the presence of cystine derivative is 1.1 X 10(-3). Reaction of triplet BU with the cysteine derivative does not yield U-Cys adduct but U and cystine derivative. A possible byproduct of reduction of triplet BU to U by a cysteinyl residue in a protein BU-DNA complex is a sulphenyl bromide which might yield a protein-DNA crosslink via nucleophilic substitution on sulfur by a nucleophilic site in DNA.

Photochemical cross-linking of lac repressor to nonoperator 5-bromouracil-substituted DNA.

Ultraviolet irradiation of lac repressor bound to 5-bromouracil-substituted nonoperator DNA leads to the formation of cross-links between the protein and the nucleic acid. The cross-links are formed between the DNA and the 1-51 N-terminal peptide of the repressor, the "headpiece". The tetrameric core (4 X 60-360 amino acids) was never found to be cross-linked to the DNA. With isolated headpieces, which are able to bind DNA, no cross-link was detected. These results are discussed considering the fundamental role of the core in keeping the headpieces in an adequate geometry for the DNA-repressor interaction. It has been possible to cross-link two DNA molecules to one repressor molecule, thus showing the existence of at least two binding sites for nonoperator DNA on the repressor. The attached peptides were analyzed after extensive proteolytic cleavage, and the most abundant peptide found was peptide 23-33. Histidine-29 seems to be the photo-cross-linked amino acid. Analysis of the results required a computation method discussed in the Appendix.

Spin trapping of reactive uracilyl radicals produced by ionizing radiation in aqueous solutions.

The DNA base analogue 5-bromouracil (BU) reacts with hydrated electrons to produce a highly reactive uracil-5-yl (uracilyl) radical. The uracilyl radical was spin trapped by 5,5-dimethylpyrroline-1-oxide (DMPO) and t-nitrosobutane (tNB). DMPO solutions (0.01 M at pH = 7, N2 or N2O saturated) without BU were irradiated with 60Co gamma-rays and the resulting spin adducts produced by e-aq, H., and OH. were identified. The irradiation of alkaline (pH 12, N2 saturated) solutions of DMPO alone simplified the e.p.r. spectrum of spin adducts in that only the H. adduct was observed. Irradiated DMPO solutions (pH 12, N2 saturated) containing BU yielded a composite of two six-line e.p.r. spectra, attributed to uracilyl spin adducts that have two distinct steric configurations, and a background spectrum of H.-DMPO adducts which was removed by computer-subtraction. Uracilyl-tNB spin adducts were detected by first trapping uracilyl radicals in irradiated photobleached 10 M NaOH glasses at 77 K and then dissolving these glasses in pH-adjusted tNB solutions.

Solid-state radiation chemistry of DNA and its constituents.

The molecular structure as well as the mechanisms of formation and decay of free radicals produced in DNA and its constituents by ionizing radiation is reviewed. Starting with the description of the spectral parameters for cations and anions in natural nucleic acid bases, emphasis is given to the comparable species formed in the group of the 5-halogen substituted uracil derivatives. The consequences of the attachment of a ribose or ribosephosphate group to the bases is discussed in terms of the distribution of primary radicals which, again, is shown to be different from those of the subunits in DNA itself. The quantitative aspects of radical formation are discussed in terms of G values and their dependence on the temperature of irradiation. Finally, a schematic presentation of the major modes of radical reactions is given occurring upon warming of the primary species in the DNA subunits and in DNA itself.

Radiation-induced free radicals in solid 5-halouracil derivatives: single crystals of 1-methyl-5-bromouracil.

Single crystals of the 5-halogen substituted uracil derivative 1-methyl-5-bromouracil have been irradiated at 77 and 295 K and studied by electron spin resonance spectroscopy. At 295 K a hydrogen abstraction radical (I) from the methyl group was observed, together with a modified 5-yl radical (II). The latter species seem to be formed by addition of a non-interacting residue (probably CH3) to C6. At 77 K, radical I is observed together with two bromine-centered species. One of these (III) exhibits only bromine hyperfine interaction (maximum splitting of 515 G) and is assigned to a complexed cation radical. The third 77 K resonance could be due to a beta-bromine radical R'-C5HBr-C6H-R'. A computer program able to calculate powder spectra of radicals with dominant halogen interaction is described. The results are discussed in relation to radiation damage processes in the isomorphous crystals of 1-methylthymine.

gamma-Ray-induced demethylation of thymine-(methyl-14C) in aqueous solution.

gamma-Ray-induced demethylation of thymine in deaerated aqueous solution was observed and quantitatively measured for the first time using thymine-(methyl-14C). An airfree 5 x 10(-5) M aqueous solution was irradiated with doses of 2 x 10(3)-2 x 10(4) Gy. Radio-gaschromatographic analyses provided proof for the formation of methanol (G - 7.6 x 10(-3)), and methane (G = 2.5 x 10(-5)).

Mutagenesis of lambda phage: weigle mutagenesis is induced by coincident lesions in the double helical DNA of the host cell genome.

We have studied the increase in mutation in mutagenized lambda phage when the host cells are also irradiated with ultraviolet light, "Weigle mutagenesis." The increase in mutation is induced mainly on coincidences between a radiation-produced lesion in one strand of the host cell DNA and a second lesion in the complementary strand. This conclusion is based on experiments in which incorporation of the base analog bromouracil sensitized the host cells to ultraviolet light. For the same number of bromouracil incorporated per cell, uniform substitution gave a higher level of Weigle mutagenesis than did substitution in only one strand of the DNA double helix. The data also show some induction of Weigle mutagenesis by processes linear in ultraviolet fluence; possibility include: lesions involving both complementary strands such as crosslinks, lesions in one strand opposite pre-existing discontinuities in the complementary strand, and very small contributions to induction from lesions in one strand only of the DNA.

E.s.r. studies of halogenated pyrimidines in gamma-irradiated alkaline glasses.

The reactions of mobile electrons (em-) and oxygen radical anions (O--) with halogenated bases and nucleosides have been studies in gamma-irradiated alkaline glasses by e.s.r. and specific halogen-ion electrode techniques. It is shown that electrons react with halogenated uracil bases (XUr where X = Cl, Br. I but not F) by dissociative electron attachment to form uracil-5-yl radicals (U-) and halogen anions. The relative rates of reaction of em- with XUr decrease in the sequence BrUr greater than ClUr greater than FUr greater than IUr. Thermal annealing studies carried out on U- in H2O and D2O matrices support the hypothesis that U- in H2O hydrates across the 5-6 double bond in the temperature region 135 degrees-155 degrees K, and deuterates to a much smaller extent in D2O at temperatures above 155 degrees K. Studies on bromouridine and bromodeoxyurinde suggest that em- reacts with the base moieties to form U- type radicals which abstract H- from the sugar moieties of adjacent nucleosides.

Thermoluminescent properties of crystalline halogen substituted uracils.

The thermoluminescent properties of crystalline uracil (U), and its halogen-substituted derivatives, 5 bromo-uracil (5BrU), 5 fluorouracil (5FU) and 5 iodo-uracil (5IU), have been investigated. The X-ray-induced thermoluminescent output increases in the order U less than 5 FU less than 5BrU less than 5IU. However, a reversed relationship is found for the U.V.-stimulated thermoluminescence. The thermoluminescence-emission spectra suggest that the same de-excitation process operates in all the halogen-substituted uracils, but the glow curves show that the charge-trapping characteristics are different for each material.