Search for an adenine photoproduct in DNA.

Poly(d[14C]A), p(dA)2, and [14C]adenosine-labeled DNA were irradiated at 254 nm with fluences up to 50 J/m2, and then following formic acid hydrolysis at 170 degrees C WERE SUBJECTED TO PAPER CHROMAtography using a butanol:water:acetic acid (80:30:12) solvent system. For poly(dA), up to 25% of the radioactivity appeared as fluorescent material located in the Rf 0.21-0.29 region. The hydrolysate of the purified photoproduct, p(dA)2, isolated from irradiated p(dA)2 by DEAE chromatography also had an Rf of 0.29 as well as an absorbance maximum at 310 nm. In all cases studied, however, the photoproduct yield in the Rf 0.29 region for native DNA was less than 2%. Denaturation of the DNA appeared to enhance the yield slightly, although no pronounced peak in this region of the chromatogram was discerned. Mechanistic studies indicate that the yield of the adenine photoproduct in poly(dA) is favored by base stacking, has a singlet excimer as a precursor, and is quenched by hydrogen bonding to a pyrimidine. It is concluded that the yield of the adenine photoproduct in both native and denatured DNA is considerably less than in poly (dA) and in all probability does not represent a biologically significant product.     

Resonance Raman spectroscopy of complexes of the helix destabilizing proteins GP32 and GP5 with poly(rA) and poly(dA)

The bacteriophage T4 helix destabilizing protein (hdp) gp32 and its complexes with poly(rA) and poly(dA) were studied with ultra-violet resonant Raman spectroscopy. The UV-resonant Raman (UV-RR) spectrum of the complex of gp5, the coat protein of bacteriophage M13, with poly(dA) was also measured and is compared with the spectrum of the gp 32/poly(dA) complex. The excitation wavelength was 245.1 nm. This is on the far UV-side of the first absorption bands of adenine and near a "window" in the protein absorption spectrum. The overlap of fluorescence due to chromophores present in the protein and resonance Raman scattering was prevented by this choice of wavelength. The spectra of the protein/polynucleotide complexes are compared with the native nucleotide spectra measured at varying temperatures. The hyperchromicity which is expected when a nucleotide changes from a stacked to an unstacked conformation was not observed for poly(rA), neither upon temperature increase nor on protein binding. In both cases poly(dA) revealed a clear hyperchromicity. This different behavior of poly(rA) and poly(dA) is probably a consequence of their different conformations. The contributions of the proteins to the spectra is weak except for two bands, at 1550 and 1610 cm-1 due to tryptophan (in case of gp32) and one band near 1610 cm-1 due to tyrosine and phenylalanine.     

The poly dA strand of poly dA.poly dT adopts an A-form in solution: a UV resonance Raman study.

The study by resonance Raman spectroscopy with a 257 nm excitation wave-length of adenine in two single-stranded polynucleotides, poly rA and poly dA, and in three double-stranded polynucleotides, poly dA.poly dT, poly(dA-dT).poly(dA-dT) and poly rA.poly rU, allows one to characterize the A-genus conformation of polynucleotides containing adenine and thymine bases. The characteristic spectrum of the A-form of the adenine strand is observed, except small differences, for poly rA, poly rA.poly rU and poly dA.poly dT. Our results prove that it is the adenine strand which adopts the A-family conformation in poly dA.poly dT.     

Circular dichroism of adenine and thymine containing synthetic polynucleotides

Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs.     

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.     

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.     

The photoreactivity of T-A sequences in oligodeoxyribonucleotides and DNA.

Photoaddition between adjacent adenine and thymine bases occurs, with a quantum yield of approximately 5 X 10(-4) mol einstein-1, when d(T-A), dT-A, d(pT-A), d(T-A-T), d(T-A-T-A) and poly(dA-dT) are irradiated, at 254 nm, in aqueous solution. The photoadduct thus formed is specifically degraded by acid to the fluorescent heterocyclic base 6-methylimidazo[4,5-b]pyridin-5-one (6-MIP) with retention of C(8) of adenine and the methyl group of thymine. This reaction, coupled with either spectrofluorimetric or radiochemical assay of 6-MIP isolated by high voltage paper electrophoresis, has been used to demonstrate formation of the adenine-thymine photoadduct on UV irradiation of poly(dA-dT).poly(dA-dT) and both native and denatured DNA from calf thymus and E. coli. Estimated quantum yields for this new type of photoreaction in DNA show that it is substantially quenched by base pairing. Possible biological implications of the photoreaction are discussed.     

A UV Resonant Raman Spectroscopic Study of the Interaction of Metallic Derivatives of Tetrakis(4-N-methylpyridyl)porphine with Polynucleotides

Abstract

Resonance Raman spectra excited at 257 nm are reported for the complexes of the Nickel, Cobalt and Zinc derivatives of Tetrakis(4-N-methylpyridyl)porphine with poly(dA.dT)₂, poly(dA)poly(dT), poly(dG.dC)₂ and poly(dG).poly(dC). These spectra are interpreted as evidence of multiple outside binding modes with poly(dA).poly(dT), and of evidence for an outside binding mode with Poly(dG.dC)₂. Some results obtained for the zinc derivative with poly(dA).poly(dT) suggest a binding mode peculiar to this derivative.     

UV Light-Induced Crosslinking of the Strands of Poly(dA-dT) and Related Alternating Purine-Pyrimidine DNAs

Abstract

Alkaline agarose gel electrophoresis was used to detect UV-induced crosslinking of the strands of poly(dA-dT) and related alternating purine-pyrimidine DNAs in solutions stabilizing various polynucleotide conformations. Strands of the B-form and A-form of poly(dA-dT) were not crosslinked but a UV dose-dependent retarded species appeared in the denaturing gels in parallel with the polynucleotide isomerization into the unusual X-form. Most other polynucleotides adopting the X-form were crosslinked as well. The exceptions include the X-forms of poly(dA-butyl⁵dU) and poly(dA-pentyl⁵dU) whose strands do not crosslink because the long exocyclic substituents attached to uracil make the photodimerization impossible. Strands of poly (amino²dA-dT) and poly(dA, amino ²dA-dT), the latter polynucleotide containing roughly equal amounts of amino ²adenine and adenine, also do not crosslink upon UV irradiation because they isomerize into an A-like conformation which is different from the X- form of poly (dA-dT). In contrast, strands of the mixed copolymers of poly(dA, amino ²dA-dT) containing low amino ²adenine contents are crosslinked upon UV irradiation, in accordance with the observation that they isomerize into the X-form.     

fd gene 5 protein binds to double-stranded polydeoxyribonucleotides poly(dA.dT) and poly[d(A-T).d(A-T)]

Circular dichroism (CD) data indicated that fd gene 5 protein (G5P) formed complexes with double-stranded poly(dA.dT) and poly[d(A-T).d(A-T)]. CD spectra of both polymers at wavelengths above 255 nm were altered upon protein binding. These spectral changes differed from those caused by strand separation. In addition, the tyrosyl 228-nm CD band of G5P decreased more than 65% upon binding of the protein to these double-stranded polymers. This reduction was significantly greater than that observed for binding to single-stranded poly(dA), poly(dT), and poly[d(A-T)] but was similar to that observed for binding of the protein to double-stranded RNA [Gray, C.W., Page, G.A., & Gray, D.M. (1984) J. Mol. Biol. 175, 553-559]. The decrease in melting temperature caused by the protein was twice as great for poly[d(A-T).d(A-T)] as for poly(dA.dT) in 5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7. Upon heat denaturation of the poly(dA.dT)-G5P complex, CD spectra showed that single-stranded poly(dA) and poly(dT) formed complexes with the protein. The binding of gene 5 protein lowered the melting temperature of poly(dA.dT) by 10 degrees C in 5 mM Tris-HCl, pH 7, but after reducing the binding to the double-stranded form of the polymer by the addition of 0.1 M Na+, the melting temperature was lowered by approximately 30 degrees C. Since increasing the salt concentration decreases the affinity of G5P for the poly(dA) and poly(dT) single strands and increases the stability of the double-stranded polymer, the ability of the gene 5 protein to destabilize poly(dA.dT) appeared to be significantly affected by its binding to the double-stranded form of the polymer.     

Extensive photodimerization of non-adjacent pyrimidines

In a prior study we found that non-adjacent thymidyl residues in the single-stranded alternating copolymer poly[d(G-T)] are subject to photodimerization by germicidal lamp irradiation (lambda max 254 nm). The maximum yield of this photoproduct was 1% of the total thymine of poly[d(G-T)]. We now report that dimer formation in this polymer is increased to 10 to 40% thymine as dimer between non-adjacent pyrimidines, using near-ultraviolet irradiation (lambda max 310 nm) with or without acetone triplet-sensitization. As previously observed for 254 nm irradiation, dimer formation was nearly absent in double-stranded poly[d(G-T).d(C-A)]. These observations extend prior findings by demonstrating high-yield dimerization between non-adjacent pyrimidines via direct irradiation at environmentally relevant wavelengths (greater than or equal to 280 nm), and are potentially relevant to the mechanism of the ultraviolet light-induced targeted -1 frameshift mutation.     

Ultraviolet resonance Raman spectroscopy of distamycin complexes with poly(dA)-poly(dT) and poly(dA-dT): role of H-bonding

Raman spectra are reported for distamycin, excited at 320 nm, in resonance with the first strong absorption band of the chromophore. Qualitative band assignments to pyrrole ring and amide modes are made on the basis of frequency shifts observed in D2O. When distamycin is dissolved in dimethyl sulfoxide or dimethylformamide, large (30 cm-1) upshifts are seen for the band assigned to amide I, while amides II and III shift down appreciably. Similar but smaller shifts are seen when distamycin is bound to poly(dA-dT) and poly(dA)-poly(dT). Examination of literature data for N-methylacetamide in various solvents shows that the amide I frequencies correlate well with solvent acceptor number but poorly with solvent donor number. This behavior implies that acceptor interactions with the C = O group are more important than donor interactions with the N-H group in polarizing the amide bond and stabilizing the zwitterionic resonance form. The resonance Raman spectra therefore imply that the distamycin C = O groups, despite being exposed to solvent, are less strongly H-bonded in the polynucleotide complexes than in aqueous distamycin, perhaps because of orienting influences of the nearby backbone phosphate groups. In this respect, the poly(dA-dT) and poly(dA)-poly(dT) complexes are the same, showing the same RR frequencies. Resonance Raman spectra were also obtained at 200-nm excitation, where modes of the DNA residues are enhanced. The spectra were essentially the same with and without distamycin, except for a perceptable narrowing of the adenine modes of poly(dA-dT), suggesting a reduction in conformational flexibility of the polymer upon drug binding.     

Excited states and energy transfer among DNA bases in double helices.

The study of excited states and energy transfer in DNA double helices has recently gained new interest connected to the development of computational techniques and that of femtosecond spectroscopy. The present article points out contentious questions regarding the nature of the excited states and the occurrence of energy transfer and shows how they are currently approached. Using as example the polymer poly(dA) . poly(dT), composed of about 2000 adenine-thymine pairs, a model is proposed on the basis of time-resolved measurements (fluorescence decays, fluorescence anisotropy decays and fluorescence spectra, obtained with femtosecond resolution), associated to steady-state spectra. According to this qualitative model, excitation at 267 nm populates excited states that are delocalized over a few bases (excitons). Ultrafast internal conversion directs the excited state population to the lower part of the exciton band giving rise to fluorescence. Questions needing further investigations, both theoretical and experimental, are underlined with particular emphasis on delicate points related to the complexity and the plasticity of these systems.     

Experimental and theoretical study of the interaction of single-stranded DNA homopolymers and a monomethine cyanine dye: nature of specific binding.

The photophysical properties of an intercalating unsymmetrical monomethine cyanine dye and single-stranded DNA homopolymers show strong association for poly(dA) and poly(dG), but not for poly(dC) and poly(dT), as determined by several spectroscopic techniques and molecular dynamics calculations. While poly(dA) and poly(dG) appear to bind the dye as a monomer (with dramatic increase in fluorescence), poly(dC) and poly(dT) bind only very weakly, and seem to promote dye aggregation. Only in the case of poly(dA) there seems to be a unique, well defined form of intercalation, that molecular dynamics calculations suggest involve the quinoline ring between two bases, in an arrangement that should favor pi-stacking; consistently with this, the decay of the fluorescence shows a single exponential, the absorption spectrum shows a shift in the dye maximum, the fluorescence is strong, and the induced circular dichroism follows a simple pattern.     

Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA.

Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs.     

A Raman spectroscopic study of the interaction between nucleotides and the DNA binding protein gp32 of bacteriophage T4

Raman spectra of the bacteriophage T4 denaturing protein gp32, its complex with the polynucleotides poly(rA), poly(dA), poly(dT), poly(rU), and poly(rC), and with the oligonucleotides (dA)₈ and (dA)₂, were recorded and interpreted. According to an analysis of the gp32 spectra with the reference intensity profiles of Alix and co-workers [M. Berjot, L. Marx, and A. J. P. Alix (1985) J. Ramanspectrosc., submitted; A. J. P. Alix, M. Berjot, and J. Marx (1985) in Spectroscopy of Biological Molecules, A. J. P. Alix, L. Bernard, and M. Manfait, Eds., pp. 149–154], 1 gp32 contains ≈ 45% helix, ≈ 40% β-sheet, and 15% undefined structure. Aggregation of gp32 at concentrations higher than 40 mg/mL leads to a coordination of the phenolic OH groups of 4–6 tyrosines and of all the sulfhydryl (SH) groups present in the protein with the COO^? groups of protein. The latter coordination persists even at concentrations as low as 1 mg/mL. In polynucleotide–protein complexes the nucleotide shields the 4–6 tyrosine residues from coordination by the COO^? groups even at high protein concentration. The presence of the nucleotide causes no shielding of the SH groups. With Raman difference spectroscopy it is shown that binding of the protein to a single-stranded nucleotide involves both tyrosine and trytophan residues. A change in the secondary structure of the protein upon binding is observed. In the complex, gp32 contains more β-sheet structure than when uncomplexed. A comparison of the spectra of complexed poly(rA) and poly(dA) with the spectra of their solution conformations at 15°C reveals that in both polynucleotides the phosphodiester vibration changes upon complex formation in the same way as upon a transition from a regular to a more disordered conformation. Distortion of the phosphate–sugar–base conformation occurs upon complex formation, so that the spectra of poly(rA) and poly(dA) are more alike in the complex than they are in the free polynucleotides. The decrease in intensity of the Raman bands at 1304 cm^?1 in poly(rA), at 1230 cm^?1 in poly(rU), and at 1240 and 1378 cm^?1 of poly(dT) may be indicative of increased stacking interactions in the complex. No influence of the nucleotide chain length upon the Raman spectrum of gp32² in the complex was detected. Both the nucleotide lines and the protein lines in the spectrum of a complex are identical in poly(dA) and (dA)₈.     

Binding of ethidium bromide to a DNA triple helix. Evidence for intercalation

The interaction of ethidium, a DNA intercalator, with the poly(dA).poly(dT) duplex and the poly (dA).2poly(dT) triplex has been investigated by a variety of spectrophotometric and hydrodynamic techniques. The fluorescence of ethidium is increased when either the duplex or triplex form is present. Binding constants, determined from absorbance measurements, indicate that binding to the triple helical form is substantially stronger than to the duplex, with a larger binding site size (2.8 base triplets compared to 2.4 base pairs). Furthermore, while binding to poly(dA).poly(dT) shows strong positive cooperativity, binding to the triplex is noncooperative. Thermal denaturation experiments demonstrate that ethidium stabilizes the triple helix. Binding to either form induces a weak circular dichroism band in the visible wavelength region, while in the region around 310 nm, there is a band that is strongly dependent on the degree of saturation of the duplex, and which is positive for the duplex but negative for the triplex. Both fluorescence energy transfer and quenching studies provide evidence of intercalation of ethidium in both duplex and triplex complexes. Binding of ethidium leads to an initial decrease in viscosity for both the duplex and triplex structures, followed by an increase, which is greater for the duplex. Taken together, these results strongly suggest that ethidium binds to the poly (dA).2poly(dT) triple helix via an intercalative mechanism.     

Molecular states in single-stranded adenylate chains by relaxation analysis

The single-strand helix-coil transition in various oligo- and polyadenylates is characterized by means of an improved cable temperature-jump technique. In all the polymers studied {poly(rA), poly(dA), poly[A(m^2′)] and poly[A(e^2′)]} helix-coil relaxation is observed in the time range from 30 to 1000 nsec. Relaxation-time constants observed at wavelengths λ<280 nm (τ_α) are different from those found at λ >280 nm (τβ), indicating the presence of more than two conformational states. The time constants τ_α increase in the series poly(dA), poly[A(m^2′)], constants τ_β/τ_α is approximately 2.5, except in poly(dA) where τ_β/τ_α ≈ 9. Relaxation measurements with r(A)_n- oligomers show a decrease in conformational mobility with increasing chain length. The relaxation curves also demonstrate that “internal” residues have lower reaction rates than residues at the ends of the oligomer chain. Measurement in D₂O reveal a solvent isotope effect for τ_α of +87% for poly(rA), and of +53% for poly(dA), whereas no isotope effect is found in τ_β. The absence of “slow” relaxation processes in the model compound 9,9′ -trimethylenebisadenine shows that the relatively low rate of the single-strand helix-coil transitions is due to the coupling of base stacking with the folding of the sugar–phosphate chain. The absence of a seprate relaxation process (corresponding to τ_β) in 9,9′-trimethylenebisadenine, as well as in the dinucleotides ApC and CpA, suggests that this relaxation process is dependent upon the presence of both the sugar–phosphate chain and of adjacent adenine bases. The experimental data provide evidence that there is more than one ordered conformation in various single-stranded oligo- and polyadenylates and that the transition between these conformations is influenced by the sugar conformation.     

The photochemistry of some main chain liquid crystalline 4,4'-stilbene dicarboxylate polyesters.

Several aspects of the photochemistry and photophysics of four main chain liquid crystalline polyesters with a rigid trans-stilbene 4,4'-dicarboxylate mesogen as chromophore and flexible spacer groups are reported. The three polymers with the longest 'spacer' groups are liquid crystalline at room temperature, two have smectic phases. Chromophore aggregation has a dramatic effect on the photophysics and photochemistry of these polymers. Each of the polymers in poor solvents or as films has greatly perturbed UV-Vis absorption and fluorescence spectra due to aggregation of the stilbene chromophore. These effects are more pronounced upon annealing above the glass transition temperature, T(g), and in the mesophase. Film fluorescence is excitation wavelength dependent and is suppressed at elevated temperatures. The stilbene 'environment' in both films and solution is clearly heterogeneous and energy transfer processes relatively slow. The dominant photochemical reaction upon direct excitation above 300 nm is 2 + 2 photocycloaddition rendering polymer films insoluble. No significant trans-to-cis photoisomerization can be detected upon initial irradiation of the polymer films. There is evidence for the formation of aldehyde and carboxylate functionality upon irradiation in the presence of air. Loss of the aggregate UV-Vis absorption and fluorescence occurs during irradiation. Difference UV-Vis spectra of irradiated films suggest preferential initial consumption of dimeric aggregates. Loss of stilbene UV-Vis absorption upon irradiation above 300 nm can be partly photoreversed upon subsequent 254 nm irradiation. The rate of stilbene chromophore loss from films increased significantly above Tg and in the smectic phase above room temperature.