Raman studies of nucleic acids. XII. Conformations of oligonucleotides and deuterated polynucleotides

Laser Raman spectra of the trinucleoside diphoshate ApApA and dinucleoside phosphates ApU, UpA, GpC, CpG, and GpU are reported and discussed. Assignments of conformationally sensitive frequencies are-facilitated by comparison with spectra reported here of poly(rA), poly(rC), and poly(rU) in deuterium oxide solutions. The significant spectral differences between ApU and UpA, and between GpC and CpG, reveal that the sequence isomers have nonidentical conformations in aqueous solution. In UpA at low temperature the bases are stacked and the backbone conformation is similar to that found in ordered polynucleotide structures and RNA. In ApU no base stacking can be detected and the backbone conformation differs from that found in UpA, both in the orientation of phosphodiester linkages and in the internal conformation of ribose. At the conditions employed neither ApU nor UpA exhibits base pairing in aqueous solutions. In both GpC and CpG the bases are stacked and the phosphodiester conformations are similar to those encountered for UpA and RNA. However, major differences between spectra of GpC and CpG indicate that the geometries of stacking and ribosyl conformations are different. In GpC the Raman data favor the formation of hydrogen bonded dimers containing GC pairs. Protonation of C in GpC is sufficient to eliminate the ordered conformation detected by Raman spectroscopy. Despite the ordered backbone conformation evident in GpU, this dinucleoside apparently contains neither stacked nor hydrogen bonded bases at the conditions employed here. The Raman data also confirm the stacking interactions in ApApA, poly(rA), and poly(rC) but suggest that the backbone conformation in poly(rC) differs qualitatively from that found in most ordered polynucleotide structures and is thermally more stable. The present results demonstrate the sensitivity of the Raman technique to sequence-related structural differences in oligonucleotides and provide additional spectra–structure correlations for future conformational studies of RNA by laser Raman spectroscopy.     

Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

The DNA bending by acetylaminofluorene residues and by apurinic sites

We have studied the distortions induced in double-stranded oligonucleotides by covalently bound acetylaminofluorene residues and by apurinic sites. Within the acetylaminofluorene-modified oligonucleotide three base-pairs are unpaired as detected by the chemical probes chloroacetaldehyde and osmium tetroxide. These two probes reveal that the bases adjacent to the apurinic site are paired. In both the modified double-stranded oligonucleotides, the backbone on the 5' side of the modification is more reactive with 1,10-phenanthroline copper than the backbone on the 3' side. On polyacrylamide gels, the ligated multimers of acetylaminofluorene or apurinic site-modified oligonucleotides migrate slower than the multimers of the unmodified oligonucleotides. It is suggested that the acetylaminofluorene-modified guanine residues and the apurinic sites behave more as hinge joints than as the centres of directed bends.     

Assembly and characterization of nucleosomal cores on B- vs. Z-form DNA

The ability of right- vs. left-handed alternating purine/pyrimidine copolymers to support the formation of nucleosomes has been examined by using a trout testis assembly factor. The protein, which is thermostable, has a molecular weight of 29000 and will assemble nucleosomes onto both SV40 and calf thymus DNA. This assembly factor has been used to assemble nucleosomes onto the B and Z conformations of poly[d(Gm5C)] and the B conformation of poly[d(GC)]. The isolated B-form particles, which sediment at approximately 11 S in a sucrose density gradient, contain DNA of 140-200 bases in length and the four core histones. The isolated Z-form particles, which also sediment at approximately 11 S, contain the four core histones and DNA of 170-250 bases in length. Physical analysis of the particles by absorbance and circular dichroic spectroscopy indicates that the DNA remains in the original conformation throughout the isolation procedure. Further, the particles reconstituted onto left-handed DNA compete effectively for an anti-Z DNA antibody, while the corresponding right-handed particles do not. Analytical sedimentation velocity determinations indicate that the B-form poly[d(Gm5C)] and poly[d(GC)] particles sediment at 11.2 and 11.1 S, respectively. In contrast, the poly[d(Gm5C)] Z-form particles have an S20,w of 10.6 S. The differences in the sedimentation velocity and the density of the cores, and in the lengths of DNA associated with the particles, suggest that the conformation of the DNA affects the manner in which it associates with the histone octamer.     

Recognition of damaged regions in DNA by oligopeptides and proteins

The binding of various damaged DNAs to the single-strand binding protein coded for by gene 32 from bacteriophage T4, on the one hand, and of oligopeptides containing tryptophan and lysine residues, on the other hand, is described. These molecules exhibit a higher affinity for modified DNA than for native DNA in so far as modification results in a local destabilization of the double-stranded structure of the nucleic acid. Stacking interactions between aromatic amino acids and nucleic acid bases appear to play a crucial role in the recognition of destabilized regions induced by chemical agents (carcinogens and antitumor drugs). These interactions confer to the peptide lysyl-tryptophyl-lysine an endonucleolytic activity specific for apurinic sites. From results obtained with such oligopeptides a model for the active sites of Ap-endonucleases is proposed which could account for the strategy used by the denV endonuclease from phage T4 during the first step of excision repair of pyrimidine dimers in DNA. The effect of the overall conformation of modified DNA on repair efficiency is discussed.     

The structure of triple helical poly(U).poly(A).poly(U) studied by Raman spectroscopy

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U).poly(A).poly(U) triple helix. We compared the Raman spectra of poly(U).poly(A).poly(U) in H2O and D2O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A).poly(U). The presence of a Raman band at 863 cm-1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3' endo; that of the second poly(U) chain may be C2' endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A).poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

Properties of an endonuclease activity in Micrococcus luteus acting on gamma-irradiated DNA and on apurinic DNA

A protein fraction from Micrococcus luteus with endonuclease activity against gamma-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities. The endonuclease activity against gamma-irradiated DNA was stimulated five-fold with 5 mM Mg++, whereas that against apurinic sites was less dependent on the Mg++ concentration. 100 mM KCl inhibited the gamma-ray endonuclease, but not the apurinic endonuclease activity. In gamma-irradiated RNA the protein recognized 1.65 endonuclease sensitive sites per radiation induced single-strand break, among which are 0.45 alkali labile lesions in the nucleotide strand. The affinity of the enzyme for the endonuclease sensitive site was evaluated resulting in a Km-value of 73 nM.     

Conformation of poly(L-arginine). II. Complexes with polyanions

Conformaitons of poly(L -arginine)/polyanion complexes were studies by CD measurements. The polyanions were the homoplolypeptides poly(L -glutamic acid) and poly(L -aspartic acid); the synthetic polyelectrolytes and polyethylenesulfonate; and the polynucleotides were native DNA, denatured DNA, and poly(U). It was found that poly(L -arginine) forms the α-helical conformation by interacting with the acidic homopolypeptides and the synthetic anionic polyelectrolytes. In each complex, poly(L -glutamic acid) is in the α-helical conformation, whereas poly(L -aspartic acid) is mostly in the random structure. The poly(L -glutamic acid) complex changed into the β-sheet structure at the transition temperature about 65°C in 0.01M cacodylate buffer (pH 7). Even in the presence of 5M urea, this complex remained in the α-helical conformation at room temperature. The existence of the stable complex of α-helical poly(L -arginine) and α-helical poly(L -glutamic acid) was successfully supported by the model building study of the complex. The α-helix of poly(L -arginine) induced by binding with polyacrylate was the most stable of the poly(L -arginine)-polyanion complexes examined as evidenced by thermal and urea effects. The lower helical content of the polyethylenesulfonate-complexed poly(L -aginine) was explained in terms of the higher charge density of the polyanion. On the other hand, native DNA, denatured DNA, and poly(U) were not effective in stabilizing the helical structure of poly(L -arginine). This may be due to the rigidity of polyanions and to the steric hindrance of bases. Furthermore, the distinitive structual behavior of poly(L -arginine) and poly(L -lysine) regarding polyanion interaction has been noticed throughout the study.     

Single-strand breakage of DNA in UV-irradiated uvrA, uvrB, and uvrC mutants of Escherichia coli.

We transduced the uvrA6, uvrB5, uvrC34, and uvrC56 markers from the original mutagenized strains into an HF4714 background. Although in the original mutagenized strains uvrA6 cells are more UV sensitive than uvrB5 and uvrC34 cells, in the new background no significant difference in UV sensitivity is observed among uvrA6, uvrB5, and uvrC34 cells. No DNA single-strand breaks are detected in UV-irradiated uvrA6 or uvrB5 cells, whereas in contrast a significant number of single-strand breaks are detected in both UV-irradiated uvrC34 and uvrC56 cells. The number of single-strand breaks in these cells reaches a plateau at 20-J/m2 irradiation. Since these single-strand breaks can be detected by both alkaline sucrose and neutral formamide-sucrose gradient sedimentation, we concluded that the single-strand breaks observed in UV-irradiated uvrC cells are due to phosphodiester bond interruptions in DNA and are not due to apurinic/apyrimidinic sites.     

Proton and phosphorus nmr investigation of the conformational states of acid polyadenylic double helix

Proton and phosphorus nmr have been used to investigate the double-helical structures of polyriboadenylic acid [poly(A)] formed in acidic solutions (pH < 6). The results obtained at low pH (～4.5) are consistent with the model for the acid poly(A) double helix proposed by Rich [Rich, A., Davies, D. R., Crick, F. H. C. & Watson, J. D. (1961) J. Mol. Biol. 3 , 71–86]. Other models that have been proposed are inconsistent with the nmr data. The nmr measurements have also been used to examine the conformation of poly(A) helix in the half-protonated state. Although the base-stacking arrangement of this state is similar to that observed in the more extensively protonated low-pH state, the phosphate backbone conformation is different from that found in either the neutral or low-pH structures.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

The conformation of polycytidylic acid, polyguanylic acid, polyinosinic acid, and their helical complexes in aqueous solution from laser Raman scattering

The Raman spectra of the double helical complexes of poly C–poly G and poly I–poly C at neutral p^H are presented and compared with the spectra of the constituent homopolymers. When a completely double-helical structure is formed in solution a strong sharp band at 810–814 cm^?1 appears which has previously been shown to be due to the A-type conformation of the sugar–phosphate backbone chain. By taking the ratio of the intensity of the 810–814 cm^?1 band to the intensity of the 1090–1100 cm^?1 phosphate vibration, one can obtain an estimate of the fraction of the backbone chain in the A-type conformation for both double-stranded helices and self-stacked single chains. This type of information can apparently only be obtained by Raman spectroscopy. In addition, other significant changes in Raman intensities and frequencies have been observed and tabulated: (1) the Raman intensity of certain of the ring vibrations of guanine and hypoxanthine bases decrease as these bases become increasingly stacked (Raman hypochromism), (2) the Raman band at 1464 cm^?1 in poly I is asigned to the amide II band of the cis-amide group of the hypoxanthine base. It shifts in frequency upon base pairing to 1484 cm^?1, thus permitting the determination of the fraction of I–C pairs formed.     

Effects of polymer structure on the inhibition of cholera toxin by linear polypeptide-based glycopolymers

A variety of important biological events are mediated by the multivalent interaction between relevant oligosaccharides and multiple saccharide receptors on lectins, toxins, and cell surfaces; a variety of glycopolymeric materials have therefore been investigated in studies aimed at manipulating these events. The synthesis of protein- and polypeptide-based glycopolymers via protein engineering and other methods offers opportunities to control both the number and the spacing of saccharides on a scaffold, as well as the conformation of the polymer backbone, and will therefore facilitate the structure-based design of polymers for inhibition of multivalent binding events. In initial studies, we have synthesized a family of galactose-functionalized glycopolymers with a poly(L-glutamic acid) backbone, in which the density and linker length of the pendant carbohydrate moiety were varied. The composition of the glycopolymers was determined via (1)H NMR spectroscopy, and the impact of saccharide density and linker length, as well as the potential for these polypeptide-based glycopolymers to act as high-affinity inhibitors of the cholera toxin, has been indicated via competitive enzyme-linked immunosorbent assay and fluorescence titration experiments. The results of these studies suggest strategies for optimizing the binding of linear glycopolymers to bacterial toxins and will aid in the design of additional protein-based materials for studying the impact of multivalency, spacing, and backbone rigidity in a variety of biologically relevant binding events.     

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA): its conformational and photodynamic properties

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA) polymers with 22 and 35 mol % of azo chromophores in the side chains have been synthesized by condensing 4-methoxy-4'-aminoazobenzene and poly(l-glutamic acid). These polymers have been characterized by NMR, FT-IR, and UV-visible spectroscopic techniques. The conformational features of the polymer backbone chains in the films that were cast from the polymer solutions prepared in different solvents have been investigated by circular dichroism spectroscopy. Experimental data suggested that the thermal cis-trans relaxation and photoinduced birefringence, which are related to the azo chromophores in the side chains of polymer, are not affected by the conformations of polymer backbones. However, the modulations of the surface relief gratings, the result of photoinduced mass transport process, recorded on these polymers are sensitive to polymer main chain conformation, as well as the degree of functionalization.     

A review of the genetic effects of ethyl methanesulfonate

Ethyl methanesulfonate (EMS) is a monofunctional ethylating agent that has been found to be mutagenic in a wide variety of genetic test systems from viruses to mammals. It has also been shown to be carcinogenic in mammals. Alkylation of cellular, nucleophilic sites by EMS occurs via a mixed SN1/SN2 reaction mechanism. While ethylation of DNA occurs principally at nitrogen positions in the bases, because of the partial SN1 character of the reaction, EMS is also able to produce significant levels of alkylation at oxygens such as the O6 of guanine and in the DNA phosphate groups. Genetic data obtained using microorganisms suggest that EMS may produce both GC to AT and AT to GC transition mutations. There is also some evidence that EMS can cause base-pair insertions or deletions as well as more extensive intragenic deletions. In higher organisms, there is clear-cut evidence that EMS is able to break chromosomes, although the mechanisms involved are not well understood. An often cited hypothesis is that DNA bases ethylated by EMS (mostly the N-7 position of guanine) gradually hydrolyze from the deoxyribose on the DNA backbone leaving behind an apurinic (or possibly an apyrimidinic) site that is unstable and can lead to single-strand breakage of the DNA. Data also exist that suggest that ethylation of some chromosomal proteins in mouse spermatids by EMS may be an important factor in causing chromosome breakage.     

Characterization of a depurinated-DNA purine-base-insertion activity from Drosophila.

An activity that binds preferentially to depurinated DNA and inserts purines into those sites was partially purified from Drosophila melanogaster embryos. The protein has a sedimentation coefficient of 4.9 S and is devoid of AP (apurinic/apyrimidinic) endonuclease activity. Upon incorporation of purines into apurinic DNA, the number of alkali-labile sites decreases, thus establishing the conversion of depurinated sites into normal nucleotides. The activity requires K+, and is totally inhibited by caffeine or EDTA. Guanine is specifically incorporated into partially depurinated poly(dG-dC) and adenine is specifically incorporated into poly(dA-dT), thus demonstrating the apparent template specificity of the enzyme.     

Structure of branched poly-alpha-amino acids in dimethylformamide. II. Viscosity, sedimentation, and diffusion

The intrinsic viscosity, sedimentation and diffusion of a series of branched, multichain poly-α-amino acids having a poly(L -lysine) backbone and poly(γ-benzyl L -glutamate) and poly (β-benzyl L -aspartate) side chains was studied at room temperature in dimethylformamide. The molecules were found to be extremely compact structures in which the molecular backbone is either lying along the major axis in a slightly twisted configuration (the longer the side chain the smaller the twist) or is coiled up in the form of a disk with backbone and side chains coplanar. Heat treatment (to 70°C.) introduces only small changes in the hydrodynamic parameters showing that the heat-labile aggregates detected by light scattering are reversibly broken up during the hydrodynamic measurements. The above structural information concerns the initial metastable conformation of the molecules which is irreversibly destroyed by heat treatment.     

Apurinic acid endonuclease activity from mouse epidermal cells.

An endonuclease activity making single-strand breaks into depurinated and alkylated DNA has been purified 500-fold from carcinogen-transformed mouse epidermal cells. The enzyme was active only at apurinic/apyrimidinic sites, regardless of whether they were produced by heating at an acidic pH or by alkylation with the ultimate carcinogen MeSO2OMe. The enzyme did not act on native DNA nor on ultraviolet-induced pyrimidine-dimers nor on steric distortions caused by modification of DNA with the carcinogen (Ac)2ONFln. The enzyme was active in the presence of 1 mM EDTA; however, at pH 7.4 optimal conditions were: 6mM MgCl2 and 40--120 mM KCl or 10--40 mM potassium phosphate. The enzyme eluted from hydroxyapatite, phosphocellulose and heparin-cellulose between 100--250 mM potassium phosphate but did not bind to DEAE-cellulose. Using four chromatographic steps the endonuclease was obtained free of exonuclease, demethylase and DNA glycosylase activity specific for DNA bases methylated with MeSO2OMe or MeNOUr. The molecular weight was 31 000 +/- 3000 as calculated from the diffusion coefficient (8.2 x 10-7 cm2/s) and the sedimentation value (2.7 S).     

Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins

Amino acids in peptides and proteins display distinct preferences for alpha-helical, beta-strand, and other conformational states. Various physicochemical reasons for these preferences have been suggested: conformational entropy, steric factors, hydrophobic effect, and backbone electrostatics; however, the issue remains controversial. It has been proposed recently that the side-chain-dependent solvent screening of the local and non-local backbone electrostatic interactions primarily determines the preferences not only for the alpha-helical but also for all other main-chain conformational states. Side-chains modulate the electrostatic screening of backbone interactions by excluding the solvent from the vicinity of main-chain polar atoms. The deficiency of this electrostatic screening model of amino acid preferences is that the relationships between the main-chain electrostatics and the amino acid preferences have been demonstrated for a limited set of six non-polar amino acid types in proteins only. Here, these relationships are determined for all amino acid types in tripeptides, dekapeptides, and proteins. The solvation free energies of polar backbone atoms are approximated by the electrostatic contributions calculated by the finite difference Poisson-Boltzmann and the Langevin dipoles methods. The results show that the average solvation free energy of main-chain polar atoms depends strongly on backbone conformation, shape of side-chains, and exposure to solvent. The equilibrium between the low-energy beta-strand conformation of an amino acid (anti-parallel alignment of backbone dipole moments) and the high-energy alpha conformation (parallel alignment of backbone dipole moments) is strongly influenced by the solvation of backbone polar atoms. The free energy cost of reaching the alpha conformation is by approximately 1.5 kcal/mol smaller for residues with short side-chains than it is for the large beta-branched amino acid residues. This free energy difference is comparable to those obtained experimentally by mutation studies and is thus large enough to account for the distinct preferences of amino acid residues. The screening coefficients gamma(local)(r) and gamma(non-local)(r) correlate with the solvation effects for 19 amino acid types with the coefficients between 0.698 to 0.851, depending on the type of calculation and on the set of point atomic charges used. The screening coefficients gamma(local)(r) increase with the level of burial of amino acids in proteins, converging to 1.0 for the completely buried amino acid residues. The backbone solvation free energies of amino acid residues involved in strong hydrogen bonding (for example: in the middle of an alpha-helix) are small. The hydrogen bonded backbone is thus more hydrophobic than the peptide groups in random coil. The alpha-helix forming preference of alanine is attributed to the relatively small free energy cost of reaching the high-energy alpha-helix conformation. These results confirm that the side-chain-dependent solvent screening of the backbone electrostatic interactions is the dominant factor in determining amino acid conformational preferences.