Photophysical and calorimetric investigation on the structural reorganization of poly(A) by phenothiazinium dyes azure A and azure B.

Poly(A) has significant relevance to mRNA stability, protein synthesis and cancer biology. The ability of two phenothiazinium dyes azure A (AA) and azure B (AB) to bind single-stranded poly(A) was studied by spectroscopic and calorimetric techniques. Strong binding of the dyes and the higher affinity of AA over AB were ascertained from absorbance and fluorescence experiments. Significant perturbation of the circular dichroism spectrum of poly(A) in the presence of these molecules with formation of induced CD bands in the 300-700 nm region was observed. Strong emission polarization of the bound dyes and strong energy transfer from the adenine base pairs of poly(A) suggested intercalative binding to poly(A). Intercalative binding was confirmed from fluorescence quenching experiments and was predominantly entropy driven as evidenced from isothermal titration calorimetry data. The negative values of heat capacity indicated involvement of hydrophobic forces and enthalpy-entropy compensation suggested noncovalent interactions in the complexation for both the dyes. Poly(A) formed a self-assembled structure on the binding of both the dyes that was more favored under higher salt conditions. New insights in terms of spectroscopic and thermodynamic aspects into the self-structure formation of poly(A) by two new phenothiazinium dyes that may lead to structural and functional damage of mRNA are revealed from these studies.

Antibodies to oxidative DNA damage: characterization of antibodies to 8-oxopurines.

The 8-oxo-7,8-dihydropurines (8-oxopurines) are important cellular premutagenic lesions produced in DNA by free radicals. Specific antibodies were prepared to detect these lesions. For antigens, 8-oxo-7,8-dihydroadenosine (8-oxoAdo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) were synthesized from the bromonucleosides, and the immunogens were produced by conjugating these to either bovine serum albumin or rabbit serum albumin by the periodate method. Polyclonal antibodies specific for the haptens were elicited from rabbits immunized with the BSA conjugates. The antibodies to 8-oxoAdo (anti-8-oxoAdo) and 8-oxoGuo (anti-8-oxoGuo) precipitated the homologous antigens in an Ouchterlony gel diffusion assay and no cross-reactivity was observed toward the normal nucleosides or to the heterologous 8-oxopurine. Specificity was also examined by hapten inhibition of antibody reactivity with the homologous conjugates using ELISA. For anti-8-oxoAdo, the IC50 for 8-oxoAdo was 8 mumol/L and 8-bromoadenosine, guanosine, and inosine did not inhibit, even at concentrations of 1.25 mmol/L. Similarly, the IC50 for anti-8-oxoGuo for 8-oxoGuo was 0.1 mumol/L. 8-Methoxyguanosine also inhibited the reaction but was about 500-fold less effective than the eliciting hapten. Other nucleosides tested did not inhibit at concentrations up to 100 mumol/L. Both antibodies could easily detect the corresponding damage in x-irradiated f1 DNA at a dose of 7.5 Gy and both antibodies recognized the corresponding lesion in duplex DNA; however, with anti-8-oxoGuo the signal was reduced about 50% compared to single-stranded DNA. In order to determine the exact amount of each lesion produced in irradiated DNA, and to standardize the ELISA signal, both products were measured after alkaline phosphatase digestion of x-irradiated calf thymus DNA using high-pressure liquid chromatography (HPLC) coupled to an electrochemical detector. Anti-8-oxoGuo could detect ten 8-oxoG residues and anti-8-oxoAdo could detect two 8-oxoA residues per 10,000 nucleotides. Thus, these antibodies should be useful for the detection and measurement of 8-oxopurines in cellular DNA.

Laser-induced modification of DNA and Poly[A,G] at guanine moiety using acetone as photosensitizer.

The interactions of triplet acetone with polyadenylic acid (Poly[A]), polyguanylic acid (Poly[G]), polyadenylic-guanylic acid (Poly[A,G]) and single-stranded DNA (ssDNA) were investigated in neutral aqueous solution using KrF (248 nm) laser flash photolysis. The transient absorption spectra and kinetics of DNA and polynucleotides obtained under acetone sensitization demonstrated that the predominant transient species was guanine radical. These novel findings have offered time-resolved evidence for photochemical modification of DNA and Poly[A,G] at guanine moiety.

Electron paramagnetic resonance investigation of X-irradiated poly(U), poly(A) and poly(A):poly(U): influence of hydration, packing and conformation on radical yield at 4 K.

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.

Base release from DNA and polynucleotides upon 193 nm laser excitation.

Release of bases form calf thymus DNA and three polynucleotides, induced by 20 ns excitation at 193 nm in aqueous solution at pH 7, was detected by HPLC. The quantum yields of formation of free bases (phi B) from double-stranded DNA (0.4 mM) are independent of intensity, indicating a one-quantum mechanism of N-glycosidic bond cleavage. The phi B values increase in the order guanine, thymine, adenine, cytosine, the latter being phi C approximately 7 x 10(-4) for double-stranded DNA under Ar and O2. The larger phi B values in N2O-saturated solution, e.g., phi C = 1.2 x 10(-3), are ascribed to additional base release via OH-adduct radicals. The phi B values of homopolynucleotides increase in the order poly(G), poly(A) and poly(C), e.g. phi C = 7 x 10(-3) under Ar, as do the efficiencies for base release per radical cation (eta B). A comparison of the eta B values with the efficiencies of single-strand breakage for poly(C), poly(A) and DNA shows a similar trend; both are markedly larger for pyrimidines than for purines. Pathways to undamaged bases, initiated from base radical cations, are proposed.

Coronavirus mRNA transcription: UV light transcriptional mapping studies suggest an early requirement for a genomic-length template.

Mouse hepatitis virus (MHV) synthesizes seven to eight mRNAs, each of which contains a leader RNA derived from the 5' end of the genome. To understand the mechanism of synthesis of these mRNAs, we studied how the synthesis of each mRNA was affected by UV irradiation at different time points after infection. When MHV-infected cells were UV irradiated at a late time in infection (5 h postinfection), the syntheses of the various mRNAs were inhibited to different extents in proportion to the sizes of the mRNAs. Analysis of the UV inactivation kinetics revealed that the UV target size of each mRNA was equivalent to its own physical size. In contrast, when cells were irradiated at 2.5 or 3 h postinfection, there appeared to be two different kinetics of inhibition of mRNA synthesis: the synthesis of every mRNA was inhibited to the same extent by a small UV dose, but the remaining mRNA synthesis was inhibited by additional UV doses at different rates for different mRNAs in proportion to RNA size. The analysis of the UV inactivation kinetics indicated that the UV target sizes for the majority of mRNAs were equivalent to that of the genomic-size RNA early in the infection. These results suggest that MHV mRNA synthesis requires the presence of a genomic-length RNA template at least early in the infection. In contrast, later in the infection, the sizes of the templates used for mRNA synthesis were equivalent to the physical sizes of each mRNA. The possibility that the genomic-length RNA required early in the infection was used only for the synthesis of a polymerase rather than as a template for mRNA synthesis was ruled out by examining the UV sensitivity of a defective interfering (DI) RNA. We found that the UV target size for the DI RNA early in infection was much smaller than that for mRNAs 6 and 7, which are approximately equal to or smaller in size than the DI RNA. This result indicates that even though DI RNA and viral mRNAs are synthesized by the same polymerase, mRNAs are synthesized from a larger (genomic-length) template. We conclude that a genomic-length RNA template is required for MHV subgenomic mRNA synthesis at least early in infection. Several transcription models are proposed.

Irradiation-induced expression of O6-methylguanine-DNA methyltransferase in mammalian cells.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein which plays an important role in chemotherapy, mutagenesis, and carcinogenesis. The specific activity of MGMT in female rat liver can be induced by approximately 20-fold by treatment of the rats with gamma-irradiation. Maximum response occurred 48 h after 15 Gy irradiation. MGMT levels in male rats were induced by only 3-fold. MGMT activity was also induced by irradiation of rat hepatoma H4IIE cells with a 3-fold increase noted after treatment with 3 Gy. Northern analysis and nuclear run-on assays indicated that the induction of MGMT was regulated at the transcriptional level. The radiation-mediated increase in MGMT was blocked by H7, a protein kinase inhibitor, but not by H89, an inhibitor of protein kinase A. Hydroxyl radicals may play a role in the induction mechanism since dimethyl sulfoxide, a radical scavenger, blocked the radiation-mediated increase in MGMT. MGMT activity was also increased by treatment of the cells with H2O2, in accordance with the involvement of activated oxygen species in the induction of MGMT. Finally, the addition of cycloheximide, an inhibitor of protein synthesis, prior to but not after irradiation, abolished the increase in MGMT activity.

Strand breakage in poly(C), poly(A), single- and double-stranded DNA induced by nanosecond laser excitation at 193 nm.

Single- and double-stranded calf thymus DNA and two polynucleotides (0.4 mM) were studied in aqueous solution at pH approximately 7 using pulsed, 20 ns laser excitation at 193 nm. Monophotonic ionization of the nucleic acids is suggested from the linear dependences of the concentration of ejected electrons and the number of single- and double-strand breaks (ssb, dsb, respectively) on laser intensity (IL) in the range (0.2-3) x 10(6) W cm-2. The quantum yields of formation of hydrated electrons (phi e-) and ssb and dsb (phi ssb and phi dsb) are therefore independent of IL. In contrast, under 248 nm excitation these quantum yields increase linearly with IL under otherwise comparable conditions. Nevertheless, several effects and mechanistic implications are analogous using lambda exc = 193 and 248 nm. For polycytidylic acid, poly(C), in Ar-saturated solution for example, the efficiency of ssb per radical cation (eta RC = phi ssb/phi e-) is similar to the efficiency of ssb per OH radical (eta OH). For polyadenylic acid, poly(A), and single- and double-stranded DNA eta RC (lambda exc = 193 nm) is significantly smaller than eta OH. The ratio phi ssb (N2O)/phi ssb (Ar) is approximately 2 for poly(C), approximately 4 for poly(A) approximately 10 for DNA; the conversion of hydrated electrons into OH radicals in N2O-saturated solution and smaller eta RC than eta OH values in the case of DNA account for these results. For double-stranded DNA phi dsb does not depend on IL but increases linearly with the dose, indicating an accumulative effect of two ssb to generate one dsb. The critical distance for this event is 60-85 phosphoric acid diester bonds.

The kinetics of radiation-induced strand breakage in polynucleotides in the presence of oxygen: a time-resolved light-scattering study.

The time-resolved light-scattering changes of aqueous, aerated solutions of poly-C, poly-U and poly-A at pH 7.8, following pulse irradiation, have been studied as indices of strand break formation. With doses of 4-24 Gy/pulse a number of kinetically distinct components have been detected. For the poly-pyrimidines an immediate and fast reduction (tau 1/2 less than or equal to 50 microseconds) in light-scattering intensity (LSI), accounting for approximately 20% of the total LSI change, is followed by a much slower loss (k1 approximately 1.6 s-1) which constitutes their major LSI change. For poly-A a similar fast component is observed, present to an extent equivalent to the one noted with poly-C; it constitutes, however, over 50% of the purine polynucleotide's total response, with the remainder of the change being a slower loss (tau 1/2 approximately 0.09 s). Optical pulse radiolysis studies of poly-C and poly-U, in support of the LSI investigations, show that transient absorbances in a region assigned to base peroxyl radicals decay in a complex fashion, with some at a rate equivalent to that for the slow (major) component of LSI loss. These observations support a proposal that the rate-limiting step of major strand breakage for these polynucleotides, in the presence of oxygen, is a base peroxyl radical-mediated abstraction of a H-atom from an adjacent sugar moiety (Bothe et al. 1986), with the resulting sugar peroxyl radicals then leading to strand break formation at a rate equivalent to that for loss of the initial, fast LSI components. These latter processes are attributed to strand breaks arising from the direct interaction of .OH with the polynucleotide sugar phosphate backbone.

Formation of alpha-deoxyadenosine in polydeoxynucleotides exposed to ionizing radiation under anoxic conditions.

When poly(dA), poly(dA-dT), and salmon testis DNA were gamma-irradiated under nitrogen, the major deoxyadenosine damage product (excluding liberated adenine) was identified as the alpha-anomer of deoxyadenosine. The yields of alpha-deoxyadenosine from poly(dA), poly(dA-dT), and salmon testis DNA irradiated with a dose of 500 Gy under anoxic conditions were 1.5, 1.3, and 1.3%, respectively. No alpha-deoxyadenosine was detected after irradiation under oxic conditions. The presence of nucleotides with the alpha-configuration at the anomeric carbon atom in the DNA chain may have a significant effect on its tertiary structure and possibly modify its biological activity.

[Indices of RNA metabolism in the brain stem of rats under superlethal exposure to ionizing radiation]. / Nekotorye pokazateli metabolizma RNK v stvole golovnogo mozga krys pri sverkhletal'nom vozdeistvii ioniziruiushchei radiatsii.

A study was made of the content of hnRNA, nuclear poly(A) RNA and biosynthesis of rlnRNA in truncus cerebri of rats divided into 3 groups by the forced swimming test 6-8 min and 60 min after a short-term exposure to sparsely ionizing radiation of 100 Gy. The observed changes in the nuclear RNA metabolism can subsequently lead to the impairment of the synthesis of proteins required for normal functioning of CNS, and to the development of CNS syndrome.

Extent of formation of a dimeric adenine photoproduct in polynucleotides and DNA.

Quantum yields are reported for the formation of a dimeric adenine photoproduct, A = A, in adenine homopolymers and DNA irradiated at 254 nm. The A = A content of irradiated samples was assayed by using reversed-phase HPLC to isolate the 4,6-diamino-5-guanidinopyrimidine (DGPY) which is produced from A = A on acid hydrolysis. Acid hydrolysates derived from DNA radiolabelled with [14C] 2'-deoxyadenosine were spiked with unlabelled DGPY before fractionation on HPLC and the recovered material was further purified by chromatography on Sephadex G-10 followed by co-crystallization with DGPY sulphate. Although A = A is formed with a relatively high quantum yield of 1.6 X 10(-3) mol einstein-1 in single-stranded poly(dA) the photoaddition reaction is strongly quenched in base-paired poly(dA).poly(dT) and undetectable in poly(rA).poly(dT). Respective quantum yields of 6 X 10(-5) and 9 X 10(-6) were estimated for the formation of A = A in single- and double-stranded E. coli DNA implying that the photoproduct has very limited biological significance. From studies with d(ApG), d(GpA), ApG, GpA, d(A)20 and d(A4G)4 it is concluded that adjacent guanine and adenine bases do not form a photoadduct analogous to A = A and also that guanine residues have no local or long-range quenching effect on photodimerization within A-A doublets.

Gamma-radiolysis of poly(A) in aqueous solution: efficiency of strand break formation by primary water radicals.

gamma-Radiation-induced single-strand break formation (ssb) in polyadenylic acid (poly(A] has been determined in Ar and N2O-saturated aqueous solution in the presence of different concentrations of t-butanol. Strand breaks were monitored by a low-angle laser light-scattering technique. The efficiencies for strand breakage caused by solvated electrons, hydrogen atoms and OH radicals have been found to be 0.25, 0.20 and 7.8 per cent, respectively. The efficiency of OH radicals depends only slightly on pH (pH 5.0, 7.5 and 9.0) and is independent of the presence of salt (0.01 mol dm-3 NaC1O4) and of the irradiation temperature (20 degrees C and 70 degrees C). The efficiency of OH for ssb formation obtained in this work with poly(A) is much smaller than that of poly(dA). This is explained by the different molecular conformations of the sugar moiety of poly(A) (3'-endo) and poly(dA) (2'-endo). With increasing t-butanol concentration more strand breaks are formed than expected from simple homogeneous competition kinetics of poly(A) and t-butanol for OH radicals. This result is considered to be due to nonhomogeneous reaction kinetics in the above-mentioned competition. The rate constants for the reaction of OH and H with poly(A) have been determined.

Intramolecular cyclization in irradiated nucleic acids: correlation between high-performance liquid chromatography and an immunochemical assay for 8,5'-cycloadenosine in irradiated poly(A).

A correlation between high-performance liquid chromatography (HPLC) analysis and an in situ enzyme-linked immunosorbent assay (ELISA) for 8,5'-cycloadenosine formation in irradiated poly(A) has been established. The correlation shows that the ELISA precisely reflects changes in the combined yield of R- and S-8,5'-cycloadenosine but that a correction factor must be applied to the ELISA values for accuracy. The HPLC analysis reveals that the intramolecular cyclization proceeds stereoselectively in irradiated poly(A) to preferentially produce the R isomer at pH 7.0 which is similar to the result for irradiated adenosine but in contrast to the result for 5'-AMP where the S isomer predominates at neutral pH. The HPLC analysis shows that two events originating in hydroxyl radical attack at the sugar phosphate backbone in poly(A); that is, adenine release and 8,5'-cycloadenosine formation have somewhat different dose-yield responses. The formation of 8-hydroxyadenosine was detected in the HPLC chromatograms of poly(A) irradiated under N2O at neutral pH, and the yield of this compound was similar to the yield observed in 5'-AMP or adenosine irradiated under similar conditions.

Interaction of nitroaromatic radiosensitizers with irradiated polyadenylic acid as measured by an indirect immunochemical assay with specificity for the 8,5'-cycloadenosine moiety.

The relative reactivity of a series of nitroaromatic radiosensitizers toward the C(5') radical intermediate leading to 8,5'-cycloadenosine formation in deoxygenated solutions of irradiated polyadenylic acid (poly A) was assessed using standard competition kinetic analysis. Formation of 8,5'-cycloadenosine was assayed by an indirect, competitive, enzyme-linked immunosorbent assay (ELISA) described in an earlier report. In the absence of oxygen, the nitroaromatics inhibit 8,5'-cyclonucleoside formation in a way which generally increases with radiosensitizer electron affinity. Although hydroxyl radical scavenging by the nitroaromatics may account for a relatively small decrease in 8,5'-cyclonucleoside formation, the data suggest that oxidation of the C(5') radical intermediate is the more plausible explanation for the decreased yield of the 8,5'-cyclonucleoside with increasing nitroaromatic electron affinity.

The isolation and characterisation of a new type of dimeric adenine photoproduct in UV-irradiated deoxyadenylates.

A new type of dimeric adenine photoproduct has been isolated from d(ApA) irradiated at 254 nm in neutral aqueous solution. It is formed in comparable amounts to another, quite distinct, adenine photoproduct first described by Pörschke (J. Am. Chem. Soc. (1973), 95, 8440-8446). Results from high resolution mass spectrometry and 1H NMR indicate that the new photoproduct comprises a mixture of two stereoisomers whose formation involves covalent coupling of the adenine bases in d(ApA) and concomitant incorporation of the elements of one molecule of water. The photoproduct is degraded specifically by acid to 4,6-diamino-5-guanidinopyrimidine (DGPY) whose identity has been confirmed by independent chemical synthesis. Formation of the new photoproduct in UV-irradiated d(pA)2 and poly(dA), but not poly(rA), has been demonstrated by assaying their acid hydrolysates for the presence of DGPY. The properties of the photoproduct are consistent with it being generated by the hydrolytic fission of an azetidine photoadduct in which the N(7) and C(8) atoms of the 5'-adenine in d(ApA) are linked respectively to the C(6) and C(5) positions of the 3'-adenine.