Effect of intermolecular triplex formation on the yield of cyclobutane photodimers in DNA.

We have studied the effect of intermolecular triplexes formation on the yield of cyclobutane photodimers in DNA. DNA duplex within the pyrimidine-purine-pyrimidine triplex d(TC)nd(GA)nd(CT)n is protected from the formation of cyclobutane photodimers in the case of the stabilization of this triplex by acid pH, and in the case of supplementary stabilization by Mg2+ or Zn2+. We have studied pH-independent pyrimidine-purine-purine triplexes stabilized by bivalent cations. In such triplexes, the protection from the formation of [6-4] photodimers is observed, whereas the protection from cyclobutane dimer formation does not take place. The formation of the d(TC)nd(GA)nd(GA)n triplex leads to an inversion of the intensities of cyclobutane CT and TC photodimers. We observed a sharp decrease in photoreactivity with respect to cyclobutane dimers in the duplex tract d(C)18d(G)18 in the presence of Ba2+, Cd2+, Co2+, Mn2+, Zn2+ and Ni2+. The formation of the d(C)nd(G)nd(G)n triplex leads to 'antifootprinting', i.e. an increase in the yield of cyclobutane photodimers.     

Enzymatic analysis of oligonucleotides containing cyclobutane pyrimidine photodimers with a cleaved intradimer phosphodiester linkage.

Our recent studies indicate that enzymatic hydrolysis of the intradimer phosphodiester linkage constitutes an early reaction in processing UV light-induced cis-syn-cyclobutane pyrimidine dimers in cultured human fibroblasts. Before characterizing the resultant modified dimer sites in cellular DNA, it is necessary to establish experimental conditions that can distinguish backbone-nicked from intact dimers. We thus constructed a model substrate, i.e. p(dT) 10 <> p(dT)10 containing a dimer with a ruptured sugar-phosphate bond, and determined the products of its reaction with snake venom phosphodiesterase and alkaline phosphatase, an enzymatic digestion mixture known to release dimers from UV-treated poly(dA).poly(dT) within trinucleotides with the photoproduct intact at the 3'-end (d-TpTT). The model substrate was prepared by (i) end labeling p(dT)9 using terminal deoxynucleotidyltransferase and [3H]thymine-labeled TTP; and (ii) annealing the chromatographically purified p(dT)10 oligomers to poly(dA) followed by UV (290 nm)-induced ligation. Photoligated 20-mers with one radioactive and modified internal dimer were isolated and enzymatically digested. High performance liquid chromatographic analysis of the reaction products revealed a novel trithymidylate with its backbone severed at the 3'-terminus (d-TpT<>dT), demonstrating that this procedure could discriminate between intact and modified dimers. The procedure was then exploited to show that (i) Escherichia coli DNA photolyase can monomerize, albeit inefficiently, backbone-ruptured dimers; and (ii) phage T4 polynucleotide kinase can catalyze the phosphorylation of d-TpT<>dT, thus facilitating the development of a sensitive postlabeling assay suitable for modified dimer detection under biologically relevant conditions.     

Substituent effects on the puckering mode of the cyclobutane ring and the glycosyl bond of cis–syn photodimers

The cyclobutane ring (CB) puckering of a cis–syn DNA photodimer (cis–syn d-T[p]T) differs from that of a cis–syn RNA photodimer (cis–syn r-U [p] U) [J.-K. Kim and J. L. Alderfer (1992) Journal of Biomolecular Structure and Dynamics, Vol. 9 , p. 1705]. In cis–syn d-T [p] T, interconversion of the CB ring between CB⁺ and CB^? is observed, while in cis–syn r-U [p] U only CB^? is observed. In the CB⁺ conformation, the two thymine rings of the dimer are twisted in a right-handed fashion, as are the bases in B-form DNA. In case of CB^? they are twisted in a left-handed fashion. The C5 (base) and/or C2′ (sugar) substituents apparently affect the CB ring flexibility in cis–syn d-T [p] T and cis–syn r-U [p] U. To study the effects of the C5 substituent on CB ring flexibility, two-dimensional nuclear Overhauser effect (NOE) and ³¹P-nmr experiments were performed on cis–syn d-T [p] U, cis–syn d-U [p] T, and cis–syn d-U [p] U photodimers to investigate the CB puckering mode and overall molecular conformation and dynamics. The NOE results indicate the 5-methyl group in the photodimer induces conformational flexibility of the CB ring. In cis–syn d-T [p] U and cis–syn d-U [p] T, both CB⁺ and CB^? puckering modes are observed. This indicates interconversion between two modes takes place as observed in cis–syn d-T [p] T. In the case of cis–syn d-U [p] U, only the puckering CB^? mode is observed. All three DNA-type dimers—cis–syn d-T [p] U, cis–syn d-U [p] T, cis–syn d-U [p] U—show a characteristic flexibility of glycosidic bonds at the 5′ residue; cis–syn d-T [p] T demonstrates syn–anti interconversion for both the 3′ and 5′ sides, while the others are exclusively anti on the 3′ side. In contrast, the ribophotodimer, cis–syn r-U [p] U, lacking the C5 methyls and having a C2′-OH, demonstrates no conformational flexibility in the CB ring or in either of the glycosidic bonds. Differential flexibility of the three DNA-type dimers (cis–syn d-T [p] U, cis–syn d-U [p] T, cis–syn d-U [p] U) and the RNA dimer (cis–syn r-U [p] U) in the sugar-phosphate backbone region is also apparent from the temperature dependence of the ³¹P chemical shifts of these photodimers compared to their normal dimer analogues. Over the temperature range 18-63°C, the chemical shift change is reduced 22–42% in three DNA-type dimers, while it is reduced 71% in cis–syn r-U [p] U, suggesting the RNA-type dimer is more rigid. © 1993 John Wiley & Sons, Inc.     

Conformations of deoxydinucleoside phosphates containing pyrimidine-pyrimidine photodimers

Conformational analysis of deoxydinucleoside monophosphates with the sequences TpT and CpC have been carried out with the incorporation of both cyclobutane type pyrimidine dimers and 6-4 photoadducts using the methods of molecular mechanics energy minimization. The effect of flexibility with respect to sugar geometries and glycosidic torsions have been studied and the relative energies of a large variety of structures have been compared. The salient features obtained from these calculations have been compared with the crystallographic and spectroscopic data on pyrimidine dimer incorporated deoxydinucleoside monophosphates. Effects of "inserting" the energetically favourable conformations of such structures into B-DNA helices have been discussed in terms of the distortions in helical structures.     

Investigation of the cyclobutane pyrimidine dimer (CPD) photolyase DNA recognition mechanism by NMR analyses

The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from (31)P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.     

Solid-state NMR studies of amyloids

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NMR studies of prebiotic polypeptides

Several polypeptides prepared by means of pyrocondensation have been the subject of structural investigations. Attention has been focused on the constitutional characterization of homo-and co-polymers containing Asp and Glu residues, whose role is essential for the formation of the so-called proteinoids. Contrary to the literature data based on chemical degradation, nmr studies show conclusively that in thermal poly-aspartic acid only β-peptide linkages are present. This result casts serious doubt on the role thermal condensation might have played in prebiotic polypeptide syntheses.     

NMR studies of protein interactions

Interactions of proteins with other macromolecules or small molecules play important roles in most biological processes. Often, such interactions are weak and transient, and the complexes do not easily crystallize. NMR spectroscopy has the unique ability to retrieve information about these interactions and is increasingly used. Recent methodological developments have helped characterize weak protein interactions, and have in particular been applied to the study of proteins that are mostly unfolded alone but form well-defined complexes upon interaction. In addition, NMR methods have been applied to the identification and characterization of small chemicals that inhibit protein function, a primary objective of rational drug design.     

NMR studies of retinal proteins

A review is given of the use of nuclear magnetic resonance (NMR) spectroscopy to study bacteriorhodopsin and bovine rhodopsin. Solution and solid-state approaches are included. The studies of the bacterial proton pump examine the chromophore, the peptide backbone, and the protein side chains. The studies of the bovine visual pigment are limited to the chromophore. Various forms of each pigment are considered. Both structural and dynamic features are addressed.     

NMR studies of water-gas interactions

Analysis of published data concerning the solubility of different gases in water as dependent on temperature was carried out. These dependences could be described by mono-or bi-exponential functions. Solubilities of nitrogen and oxygen are additive and depend on their percentage in the atmosphere over the liquid. The temperature dependence of oxygen and nitrogen dissolved in water corresponds to that in the atmospheric air. Measurements of water spin-lattice relaxation times, changing with the concentration of dissolved paramagnetic oxygen, showed that oxygen could be substantially but not completely eliminated by saturation with any gas. The best method is the contact with a water-immiscible liquid of gas capacity higher than water. However, all this leads to an unstable state of a gas-water system, converging to equilibrium.     

Fluorine NMR studies of fluorocinnamoylchymotrypsins

Three monofluorocinnamoylchymotrypsins have been examined at pH 4 by fluorine NMR spectroscopy. Protein-induced fluorine chemical shifts are quite large (~7 ppm) when fluorine is present at the para position but nearly zero for ortho fluorine. The shifts roughly parallel those observed in complexes formed between the enzyme and the analogous N-acetylfluorophenylalanines, suggesting a similarity in molecular environment for the aromatic ring in both systems. Little correlation is found, however, between the shifts for the acylenzymes and those of the corresponding enzyme-cinnamate complexes, indicating that the environment for the aromatic ring in the complexes is dissimilar from that experienced by the aromatic group in the acylated enzyme. Solvent isotope effects ($\text{H}{}_2\text{O}\text{D}{}_2\text{O}$) on the fluorine chemical shifts for the fluorocinnamoylchymotrypsins are small and downfield. Fluorine NMR observations suggest that the presence of the fluorocinnamoyl group greatly stabilizes the enzyme toward denaturation in 8 m urea.     

Synthesis of cyclobutane nucleosides

2-(6-Chloropurinyl)-3-benzoyloxymethylcyclobutanone can be prepared by reaction of 6-chloropurine with 3-benzoyloxymethyl-2-bromocyclobutanone. The N-alkylation gave both N-9 and N-7 regioisomers. Both regioisomers upon hydride reduction followed by aminolysis gave the corresponding adenine nucleoside analogues. However, the N-7 series led to the hypoxanthine analogues as byproducts.     

Deuterium NMR studies of cerebroside-phospholipid bilayers

²H-NMR was used to probe the interaction of non-hydroxy fatty acid cerebroside and 2-hydroxy fatty acid cerebroside with the polar head group and with the acyl chains of dipalmitoylphosphatidylcholine in unsonicated bilayers. It is shown that the interior of the bilayer exhibits uniformly increasing orientational order as the concentration of both types of cerebroside increases, whereas the surface of the bilayer, as reflected by the head group motion, becomes disordered. The extent of the disorder at the surface is dependent upon the type and concentration of the cerebroside. These results are discussed in terms of hydrogen-bonding interactions.     

NMR studies of fluorophenylalanine-containing carbonic anhydrase

Rabbits ingesting 4-fluorophenylalanine are known to incorporate small amounts of this fluorinated amino acid into their proteins. Carbonic anhydrase I isolated from the erythrocytes of animals so maintained exhibits a well-resolved fluorine NMR signal for each phenylalanine in the sequence. The chemical shifts of most of these signals respond to the binding of inhibitors, suggesting that most if not all of the tertiary structure of the enzyme adjusts to the presence of inhibitors at the active site.