Structures of poly(dA-dT, ip5dU) containing various small amounts of the antiherpetic 5-isopropyl-2'-deoxyuridine.

Three different concentrations of the antiherpetic agent 5-isopropyl-2'-deoxyuridine (ip5dU) were introduced into the synthetic DNA poly(dA-dT) to analyze resulting copolymers by electron microscopy, UV absorption and CD spectroscopy. The poly(dA-dT, ip5dU) containing 1.3 and 4.3% ip5dU did not much differ from the parent poly(dA-dT) but poly (dA-dT, ip5dU) with 7.1% ip5dU behaved in an unusual way. Results are explained by the notion that if bulky isopropyls occur sufficiently close to each other then stable hairpins protruding from the double helix are formed, presumably to accommodate the ip5dU-s into the loops.     

Modified polynucleotides. VII. Impaired integrity of a synthetic DNA containing the antiherpetic agent 5-isopropyl-2''-deoxyuridine.

5-Isopropyl-2'-deoxyuridine (ip5dU) was recently recognized as a clinically useful antiherpetic (HSV-1) agent. An ip5dU-containing polynucleotide, poly (dA-dT, ip5dU) was prepared to study how physical and bio-organic properties of the synthetic DNA model poly (dA-dT) would change upon partial substitution of thymidine. Synthesis was carried out with DNA polymerase enzyme and the polymers contained 7-9% of ip5dU. It proved to be less thermostable than poly (dA-dT) and the transition width was highly increased. Although it was a very efficient template for DNA polymerase enzyme, template activity for RNA synthesis was strongly reduced by the presence of ip5dU. Diminished stability against enzymic degradation, especially against single-strand-specific Nuclease S1 was also observed.     

Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase

Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG were 55-65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C≈U/G>U/T≈U/AP≈U/->U/U≈U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases.     

Evidence for a near UV-induced photoproduct of 5-hydroxymethylcytosine in bacteriophage T4 that can be recognized by endonuclease V

Summary Non-photoreactivable endonuclease V-sensitive sites have been detected in the DNA of wild type bacteriophage T4 irradiated with near UV light (320 nm). Such sites were not detected in the DNA of (a) wild type T4 irradiated with far UV (254 nm) or (b) in T4 mutants in which non-glucosylated 5-hydroxy-methylcytosine (5HMC) or cytosine replaces glucosylated 5HMC normally present in T4, irradiated with 320 nm or 254 nm light. Although the non-photoreactivable sites accounted for 50% of the endonuclease V-sensitive sites in the DNA of glucosylated T4 irradiated with near UV, there was very little difference in the sensitivities of T4 containing glucosylated 5HMC, non-glucosylated 5HMC and cytosine to near UV (313 nm). We propose that the photoproduct responsible for the non-photoreactivable, but endonuclease V-sensitive, sites in glucosylated DNA is formed from glucosylated 5HMC and that a similar photoproduct is formed from non-glucosylated 5HMC or cytosine in the appropriate phage strains. We further propose that the glucosylated 5HMC photoproduct is non-photoreactivable whereas the cytosine and non-glucosylated 5HMC photoproducts are photoreactivable and are therefore possibly cyclobutane dimers.AECL Refence No. 6370Communicated by B.A. Bridges     

Binding of ethidium to DNA measured using a 2D diffusion-modulated gradient COSY NMR experiment

The binding of ethidium bromide to a DNA hairpin (dU(5)-hairpin) was investigated using a novel 2D diffusion-modulated gradient correlation spectroscopy (DMG-COSY) experiment to evaluate the applicability of this technique for studying the binding of drugs to DNA. The DMG-COSY experiment includes a preparation period during which coherent magnetization is attenuated due to molecular self-diffusion. Magnetization then evolves due to scalar coupling during an evolution delay, and is detected using gradient pulses for coherence selection. The time-domain data are processed in an analogous manner as for gradient-selected COSY experiments. The diffusion coefficient for uridine in DMSO solution was determined from the H5-H6 crosspeak intensities for a series of 2D DMG-COSY experiments that differed in the magnitude of the gradient pulses applied during the preparation period of the DMG-COSY experiment. The diffusion coefficient for uridine calculated from the DMG-COSY experiments was identical (within experimental error) to that determined from 1D diffusion experiments (5.24x10(-6) cm(2)/s at 26 degrees C). The diffusion coefficients for ethidium bromide and for the dU(5)-hairpin were first measured separately using the DMG-COSY experiment, and then measured in the putative complex. The diffusion coefficient for free ethidium bromide (4.15x10(-6) cm(2)/s at 26 degrees C) was considerably larger than for the dU(5)-hairpin (1. 60x10(-6) cm(2)/s at 26 degrees C), as expected for the smaller molecule. The diffusion coefficient for ethidium was markedly decreased upon addition of the dU(5)-hairpin, consistent with complex formation (1.22x10(-6) cm(2)/s at 26 degrees C). Complex formation of 1:1 stoichiometry between ethidium and the stem of the dU(5)-hairpin was verified independently by fluorescence spectroscopy. These results demonstrate the utility of the DMG-COSY experiment for investigating the binding of drugs to DNA in aqueous solution.     

A dual-app nucleoside probe reports G-quadruplex formation and ligand binding in the long terminal repeat of HIV-1 proviral genome

We have developed a dual-app nucleoside analog, 5-selenophene-modified 2′-deoxyuridine (^SedU), to probe the structure and ligand-binding properties of a G-rich segment present in the long terminal repeat (LTR) of the HIV-1 proviral DNA promoter region. The nucleoside probe is made of an environment-responsive fluorophore and X-ray crystallography phasing label (Se atom). ^SedU incorporated into LTR-IV sequence, fluorescently reports the formation of G-quadruplex (GQ) structure without affecting the native fold. Further, using the environment sensitivity of the probe, a fluorescence assay was designed to estimate the binding affinity of small molecule ligands to the GQ motif. An added feature of this probe system is that it would enable direct correlation of structure and recognition properties in solution and atomic level by using a combination of fluorescence and X-ray crystallography techniques.     

Determining the origin of the stabilization of DNA by 5-aminopropynylation of pyrimidines

DNA duplexes are stabilized by aminopropynyl modification of pyrimidines at the 5 position. A combination of thermodynamic analyses as a function of ionic strength, NMR, and molecular modeling has been applied to determine the origin of the stabilization. UV melting studies of a dodecamer bearing one, two, or three nonadjacent modified dU and dC and of a single dU(8) in the Dickerson-Drew dodecamer revealed that the modifications are essentially additive in terms of T(m), DeltaG, and DeltaH, and there is little difference between dU and dC. The free energy change was parsed into electrostatic and nonelectrostatic components, which showed a significant contribution from charge interactions at physiological ionic strength but also a nonelectrostatic contribution that arises in part from hydration. NMR spectroscopy of the modified Dickerson-Drew dodecamer revealed that the conformation of the duplexes is not significantly altered by the modifications, though (31)P NMR shows that the positive charge may affect ionic interactions with the oxygen atoms of the neighboring phosphates. The modified duplex showed significant hydration in both major and minor grooves. The single strands were also analyzed by NMR, which showed evidence of significant stacking interactions in the modified oligonucleotide. Parsing the energy contribution has shown that electrostatics and hydration can produce substantial increases in thermodynamic stability without significant changes in the conformation of the duplex state. These considerations have significance for the design of oligonucleotides used for hybridization.     

DNA duplexes containing photoactive derivatives of 2'-deoxyuridine as photocrosslinking probes for EcoRII DNA methyltransferase-substrate interaction

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.     

The stabilizing contribution of thymine in duplexes of (dA)24 with (dU)24, (dT)24, (dU12-dT12), (dU-dT)12, (dU2-dT2)6, or (dU3-dT3)4: nearest neighbor and next-nearest neighbor effects

Ultraviolet melting curves are used to determine the effect of the pyrimidine 5-methyl group on the stability of duplexes of (dA)(24) with (dU)(24), (dT)(24), (dU(12)-dT(12)), (dU-dT)(12), (dU(2)-dT(2))(6), and (dU(3)-dT(3))(4). Substitution of a T for a U results in an increase in stability, which is attributed to an increase in strength of dipole-induced dipole and dispersion (van der Waals) interactions. Significant additional enhancement occurs when two T residues are adjacent. A further increase in the number of adjacent T's has a relatively slight effect on T(m). The sequence effect appears to be largely attributable to an increment in dispersion forces.The CD spectra of the duplexes are all closely similar except in the region between 260 and 290 nm. A band near 272 nm associated with the presence of U in the spectrum of (dA)(24).(dU)(24) decreases in intensity when T's are incorporated in the pyrimidine strand. The band is completely replaced in the spectrum of (dA)(24).(dT)(24) with a new maximum at 282 nm and a minimum at 268 nm, both of lower magnitude. The emergence of the two new bands is correlated with the presence of adjacent T's once more, and only two adjacent T's appear necessary for a major part of the change to occur. The degree of cation release on thermal dissociation of the oligomer dimers ranges from Deltai = 0.14 to 0.16, about the same or slightly less than values reported for polynucleotide duplexes and less than predicted from theoretical calculations.     

C5-(1-propynyl)-2'-deoxy-pyrimidines enhance mismatch penalties of DNA:RNA duplex formation

UV melting experiments show that C5-(1-propynyl)ation of seven pyrimidines to give a fully propynylated oligodeoxynucleotide (PrODN) heptamer increases the thermodynamic stability of six Watson-Crick paired DNA:RNA duplexes by 8.2 kcal/mol, on average, at 37 degrees C. About 2.5 kcal/mol of this enhancement is due to long-range cooperativity between the propynylated pyrimidines, Y(p)'s. On average, penalties for dU(p):rG, dC(p):rA, dU(p):rC, and dC(p):rC mismatches are enhanced by 2.9 kcal/mol in PrODN:RNA duplexes over those in unmodified duplexes. This results in penalties as large as 10 kcal/mol for a single mismatch. Removing a single propyne two base pairs away from a mismatch in a PrODN:RNA duplex eliminates the enhancement in specificity. Evidently, enhanced specificity is directly linked to long-range cooperativity between Y(p)'s. In most cases, the enhanced specificity is larger for internal than for terminal mismatches. PrODN:RNA duplexes are destabilized by full phosphorothioate backbone substitution to give S-PrODN:RNA duplexes. The S-PrODN:RNA duplexes retain enhanced mismatch penalties, however. These results provide insight for utilizing long-range cooperativity and enhanced specificity to improve nucleic acid based probe and drug design.     

UV-induced pyrimidine hydrates in DNA are repaired by bacterial and mammalian DNA glycosylase activities

Escherichia coli endonuclease III and mammalian repair enzymes cleave UV-irradiated DNA at AP sites formed by the removal of cytosine photoproducts by the DNA glycosylase activity of these enzymes. Poly(dG-[3H]dC) was UV irradiated and incubated with purified endonuclease III. 3H-Containing material was released in a fashion consistent with Michaelis-Menten kinetics. This 3H material was determined to be cytosine by chromatography in two independent systems and microderivatization. 3H-Containing material was not released from nonirradiated copolymer. When poly(dA-[3H]dU) was UV irradiated, endonuclease III released 3H-containing material that coeluted with uracil hydrate (6-hydroxy-5,6-dihydrouracil). Similar results are obtained by using extracts of HeLa cells. There results indicate that the modified cytosine residue recognized by endonuclease III and the mammalian enzyme is cytosine hydrate (6-hydroxy-5,6-dihydrocytosine). Once released from DNA through DNA-glycosylase action, the compound eliminates water, reverting to cytosine. This is consistent with the known instability of cytosine hydrate. The repairability of cytosine hydrate in DNA suggests that it is stable in DNA and potentially genotoxic.     

Mismatch recognition and uracil excision provide complementary paths to both Ig switching and the A/T-focused phase of somatic mutation

AID-mediated deamination of dC residues within the immunoglobulin locus generates dU:dG lesions whose resolution leads to class-switch recombination and somatic hypermutation. The dU:dG pair is a mismatch and comprises a base foreign to DNA and is, thus, recognized by proteins from both base excision (uracil-DNA glycosylase, UNG) and mismatch recognition (MSH2/MSH6) pathways. Strikingly, while antibody diversification is perturbed by single deficiency in either UNG or MSH2, combined UNG/MSH2 deficiency leads to a total ablation both of switch recombination and of IgV hypermutation at dA:dT pairs. The initiating dU:dG lesions appear not to be recognized and are simply replicated over. The results indicate that the major pathway for switch recombination occurs through uracil excision with mismatch recognition of dU:dG providing a backup; the second phase of hypermutation (essentially introducing mutations solely at dA:dT pairs) is triggered by mismatch recognition of the dU:dG lesion with uracil excision providing a backup.     

The role of 5-methylcytidine in the anticodon arm of yeast tRNA(Phe): site-specific Mg2+ binding and coupled conformational transition in DNA analogs.

The tDNA(Phe)AC, d(CCAGACTGAAGAU13m5C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+)-induced structural transition that was not exhibited by an unmodified tDNA(Phe)AC d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., & Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(Phe)AC molecules having m5C14, tDNA(Phe)AC d(U13m5C14U15), d(U13m5C14T15), and d(T13,5C14U15), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(Phe)AC, d(T13C14T15), d(U13C14U15), and d(A7;U13m5C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+)-induced structural transitions and only monophasic thermal transitions (Tm of approximately 52 degrees C). The tDNA(Phe)AC d(U13m5C14U15) had a single, strong Mg2+ binding site with a Kd of 1.09 x 10(-6) M and a delta G of -7.75 kcal/mol associated with the Mg(2+)-induced structural transition. In thermal denaturation of tDNA(Phe)AC d(U13m5C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe).(ABSTRACT TRUNCATED AT 250 WORDS)     

The degradation of 5-iododeoxyuridine and 5-bromodeoxyuridine by serum from different sources and its consequences for the use of the compounds for incorporation into DNA

Enzymes are present in sera that convert 5-iododeoxyuridine (IdU) to deoxyuridine (dU) and 5-iodouracil (IU). Although in the presence of serum 5-bromodeoxyuridine (BrdU) is not subject to extensive debromination it is converted to 5-bromouracil (BrU) at approx. 50% of the rate for IdU. These conversions are likely brought about by the enzymes thymidylate synthetase and thymidine phosphorylase. In vivo and in culture the dU enters DNA as thymidine 5'-monophosphate (dTMP) via the de novo pathway. Deoxyuridine is often found as a contaminant of [3H]IdU and [3H]BrdU. For these reasons, complications can arise in the interpretation of experimental work using these radioactive compounds. The problems may be overcome by purifying the compounds by high performance liquid chromatography (HPLC) before use together with identification of the DNA components with which the 3H is associated by chromatographic analysis.     

Optically detected zero field magnetic resonance characterization of a bromine atom-containing polynucleotide, poly(dA-br5dU)

Phosphorescence and optically detected zero field magnetic resonance ( ODMR ) spectra are reported for a bromine atom-containing polynucleotide, poly(dA- br5dU ). The triplet state luminescence of poly(dA- br5dU ) is dominated by the phosphorescence of the bromouracil base which possesses sub-millisecond triplet lifetimes. Characteristic multiple slow passage ODMR transitions, which are observed in both br5dUrd and poly(dA- br5dU ), are assigned to the triplet state of bromouracil. In addition, an abnormally-perturbed adenine triplet state, which is not apparent in the phosphorescence spectrum of poly(dA- br5dU ), is detected and identified by its slow passage ODMR and amplitude-modulated phosphorescence microwave double resonance spectra. It is proposed that the perturbed adenine is a minor component of the polynucleotide structure which is present in regions of altered stacking induced by the high polarizability of the Br atom.     

Preapoptotic chromatin changes induced by ultraviolet B irradiation in human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.     

Pyrimidine 5-methyl groups influence the magnitude of DNA curvature

DNA containing short sequences of the form (dA)n.(dT)n can exhibit pronounced degrees of stable curvature of the helix axis, provided that these homooligomeric stretches are approximately in phase with the helix repeat. However, the precise origin of this effect is unknown. We have observed that pyrimidine 5-methyl groups can have a significant effect on the degree of curvature, depending on their locations within the homooligomeric sequences. Such effects are observed in both (dA)n.(dT/dU)n and (dI)n.(dC/d5meC)n sequence motifs, arguing for a general structural perturbation due to the methyl group. The current observations suggest that pyrimidine methyl groups could influence protein-DNA interactions not only through direct protein-methyl group contacts but also by methyl group induced alterations in local DNA structure.     

USER friendly DNA engineering and cloning method by uracil excision

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10nt away from the 5′-end of each PCR primer. 5′ of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3′ ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly.