Preferential interactions of glycine betaine and of urea with DNA: implications for DNA hydration and for effects of these solutes on DNA stability

Interactions of the solutes glycine betaine (GB) and urea with mononucleosomal calf thymus DNA in aqueous salt solutions are characterized by vapor pressure osmometry (VPO). Analysis of osmolality as a function of solute and DNA concentration yields the effect of the solute on the chemical potential, mu(2), of the DNA. Although both GB and urea generally are nucleic acid denaturants and therefore must interact favorably with the nucleic acid surface exposed upon melting, VPO demonstrates that neither interacts favorably with duplex DNA. Addition of GB greatly increases mu(2) of DNA, indicating that the average local concentration of GB in the vicinity of the double helix is much less than its bulk concentration. By contrast, addition of urea has almost no effect on mu(2) of duplex DNA, indicating that the average local concentration of urea in the vicinity of duplex DNA is almost the same as in bulk solution. Qualitatively, we conclude that the nonuniform distribution of GB occurs primarily because duplex DNA and GB prefer to interact with water rather than with each other. Comparison with thermodynamic data for the interaction of GB with various protein surfaces (Felitsky et al., Biochemistry, 43, 14732-14743) shows that GB is excluded primarily from anionic DNA surface and that the hydration of anionic DNA phosphate oxygen surface (>or approximately 17 H(2)O per nucleotide or >or approximately 0.22 H(2)O A(-)(2)) involves at least two layers of water. From analysis of literature data for effects of urea and of GB on DNA melting, we propose that urea is an effective nonspecific nucleic acid denaturant because of its favorable interactions with the polar amide-like surface of G, C, and especially T or U bases exposed in denaturation, whereas GB is a specific GC denaturant because of its favorable interaction with G and/or C surface in the single-stranded state.     

Large-scale isolation of plasmid DNA and purification of lambda phage DNA using hydroxylapatite chromatography

A rapid and relatively simple procedure for purifying large quantities of plasmid DNA is described. Plasmid thus purified contains no detectable chromosomal DNA and little RNA or protein. The procedure combines alkaline denaturation and hydroxylapatite chromatography and utilizes an improved method of separating DNA from RNA. It was observed that the phosphate concentrations at which previously bound DNA as well as RNA elute from hydroxylapatite changed markedly as a function of urea concentration. In the presence of urea concentrations higher than 4 M, the ranges of phosphate concentration over which DNA and RNA elute show no overlap. This permits efficient washing of hydroxylapatite-bound DNA under conditions which should remove all bound RNA. lambda Phage DNA is also easily eluted from hydroxylapatite under the conditions used.     

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

The impact of urea on viscosity of hydroxethyl cellulose and observed mobility of deoxyribonucleic acids

The influence of urea on the viscosity of hydroxyethyl cellulose (HEC), and the state and separation of double-stranded DNA, was studied by viscometry, fluorometry, and capillary electrophoresis. The results show that double logarithm plots of specific viscosity against the volume fraction of HEC in very dilute polymer solutions are linear, the slopes of which decrease from 0.96 in 0M to 0.29 in 7M urea. The linear regression plots converge at 0.0029 g/mL, the entanglement threshold of HEC. The inclusion of urea in HEC solution thus provides an accurate method of determining its entanglement threshold from such plots. Above the entanglement threshold of HEC, urea has no effect on the specific viscosity of HEC. Results also show that urea has no effect on double-stranded DNA. No change in fluorescence was observed when increasing amounts of urea were added to a fixed concentration of DNA. To examine the influence of urea on the migration of DNA in HEC, the separation of DNA was carried out by polymer-solution capillary electrophoresis in HEC solutions containing 0 or 7M urea using unmodified capillary. Observed mobilities were used in data reduction. It was found that a parallel relationship exists between the observed mobilities and the true mobilities. In buffers containing no urea, the pseudo-free solution mobility appears to be independent of the DNA size. It was also observed to be independent of the electric field below 300 V/cm, but relates exponentially to it in 7M urea. The pseudo-retardation constants obtained by Ferguson-like plots were observed to be positive for smaller DNA molecules below 300 V/cm and increasing linearly with electric field in 0M urea, but nearly constant in 7M urea.     

Conformation and thermostability of double-stranded nucleic acids in aqueous urea solutions

The conformation and thermostability of DNA and double-helical synthetic RNA in aqueous solutions with 0-10 M urea have been investigated. A weak dependence of DNA conformation, realized in the presence of urea, on the GC-content has been found. The increase of urea concentration leads to destabilization of DNA and synthetic RNA. The character of changes in the spectra of RNA circular dichroism at the increase of urea concentration testifies that a conformational transition (different from A----A' transition) takes place. Urea stimulates the B----Z transition in poly(dG-dC).poly(dG-dC) molecules upon NaCl addition.     

Ionic and nonionic interactions in adenoviral nucleoprotein complexes.

Ionic and nonionic interactions between the adenoviral histone-like proteins and DNA were examined by determining effects of ionic strength and urea concentration on disruption of viral nucleoprotein. The viral proteins were as susceptible to dissociation by salt in the presence of urea as histones. Nonionic interactions between viral proteins appeared more extensive than those between histones.     

Multiple restraints to the unfolding of spermidine nucleoids from Escherichia coli

Bacterial DNA is largely localized in compact bodies known as nucleoids. The structure of the bacterial nucleoid and the forces that maintain its DNA in a highly compact yet accessible form are largely unknown. In the present study, we used urea to cause controlled unfolding of spermidine nucleoids isolated from Escherichia coli to determine factors that are involved in nucleoid compaction. Isolated nucleoids unfolded at approximately 3.2 M urea. Addition of pancreatic RNase reduced the urea concentration for unfolding to approximately 1.8 M urea, indicating a role of RNA in nucleoid compaction. The transitions at approximately 3.2 and approximately 1.8 M urea reflected a RNase-sensitive and a RNase-resistant restraint to unfolding, respectively. Removal of the RNase-sensitive restraint allowed us to test for roles of proteins and supercoiling in nucleoid compaction and structure. The remaining (RNase-resistant) restraints were removed by low NaCl concentrations as well as by urea. To determine if stability would be altered by treatments that caused morphological changes in the nucleoids, transitions were also measured on nucleoids from cells exposed to chloramphenicol; the RNase-sensitive restraint in such nucleoids was stabilized to much higher urea concentrations than that in nucleoids from untreated cells, whereas the RNase-resistant transition appeared unchanged.     

Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro.

Thymine glycols were produced in M13 DNA in a concentration dependent manner by treating the DNA with osmium tetroxide (OsO4). For the formation of urea-containing M13 DNA, OsO4-oxidized DNA was hydrolyzed in alkali (pH 12) to convert the thymine glycols to urea residues. With both thymine glycol- and urea-containing M13 DNA, DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment was decreased in proportion to the number of damages present in the template DNA. Sequencing gel analysis of the products synthesized by E. coli DNA polymerase I and T4 DNA polymerase showed that DNA synthesis terminated opposite the putative thymine glycol site and at one nucleotide before the putative urea site. Substitution of manganese for magnesium in the reaction mix resulted in increased processivity of DNA synthesis so that a base was incorporated opposite urea. With thymine glycol-containing DNA, processivity in the presence of manganese was strongly dependent on the presence of a pyrimidine 5' to the thymine glycol in the template.     

Stability of the primary organization of nucleosome core particles upon some conformational transitions.

The sequential arrangement of histones along DNA in nucleosome core particles was determined between 0.5 and 600 mM salt and from 0 to 8 M urea. These concentrations of salt and urea up to 6 M had no significant effect on the linear order of histones along DNA but 8 M urea caused the rearrangement of histones. Conformational changes in cores have been identified within these ranges of conditions by several laboratories 8-21. Also, abrupt structural changes in the cores, apparently their unfolding, were found by gel electrophoresis to occur at urea concentration, between 4 and 5 M. 600 mM salt and 6 M urea were shown to relax the binding of histones to DNA in cores but do not however release histones or some part of their molecules from DNA. It appears therefore that nucleosomal cores can undergo some conformational transitions and unfolding whereas their primary organization remains essentially unaffected. These results are consistent with a model of the core particles in which the histone octamer forms something like a helical "rim" along the superhelical DNA and histone-histone interactions beyond the "rim" are rather weak in comparison with those within the "rim".     

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.     

Composition and DNA-binding properties of the nuclear matrix proteins from mammalian cell nuclei

A rapidly sedimenting DNA-protein complex was isolated from nuclear lysates in 2 M NaCl and characterized with regard to its polypeptide composition and the DNA-binding properties of the purified proteins. The complex consists of the nuclear matrix with attached DNA. Electrophoresis in SDS-polyacrylamide gels revealed two major and five minor polypeptide bands, mainly in the 60 to 75 kDa molecular weight region. The DNA-matrix complex dissociated into free DNA and proteins in the presence of 2 M NaCl and 5 M urea. The proteins could be purified by chromatography on hydroxyapatite and showed a strong tendency to reassociate at 0.15 M NaCl concentration in the absence of urea. DNA was bound to the reassociated proteins at 0.15 M NaCl concentration. Part of the DNA-protein complex was stable at 1 M NaCl concentration. The binding appeared to be random with regard to the DNA sequence.     

Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea

Nucleus pulposus intervertebral disc cells are routinely confronted with high osmolality in their microenvironment and respond to this stress in vitro by regulating cell cycle progression and by activating a DNA repair machinery in order to counteract its genotoxic effect. In the present study, we attempted to identify the origin of this osmo-regulatory response, by using an ionic NaCl/KCl solution, the compatible osmolyte sorbitol, and the readily permeant urea. High salt and sorbitol were found to activate similar molecular pathways, including the p38 MAPK and the p53-p21(WAF1)-pRb axis, that were not stimulated by high urea. On the other hand, only high urea led to the phosphorylation of ERKs and JNKs. Furthermore, salt- and sorbitol-treated cells were able to phosphorylate histone H2A.X on Ser139, in contrast to cells exposed to urea, indicating a common mechanism for DNA repair, which was achieved by a p53-dependent activation of the G1 checkpoint by both solutes. DNA repair, as directly measured by a host cell reactivation assay, occurred under conditions of hyperosmolar salt and sorbitol, although to a lesser extent in sorbitol-treated cells than in cells exposed to high salinity. Taken as a whole, our findings suggest that the hyperosmolality-provoked DNA damage and the responses of nucleus pulposus cells induced by this genotoxic stress most probably originate from cell volume alterations mediated by hypertonicity and not from increased intracellular ionic concentration.     

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non-Watson-Crick base pair in SECIS element plays an important role in the selenocysteine expression by UGA codon.     

Urea Facilitates the Translocation of Single-Stranded DNA and RNA Through the α-Hemolysin Nanopore

The staphylococcal α-hemolysin (αHL) protein nanopore is under investigation as a fast, cheap detector for nucleic acid analysis and sequencing. Although discrimination of all four bases of DNA by the αHL pore has been demonstrated, analysis of single-stranded DNAs and RNAs containing secondary structure mediated by basepairing is prevented because these nucleic acids cannot be translocated through the pore. Here, we show that a structured 95-nucleotide single-stranded DNA and its RNA equivalent are translocated through the αHL pore in the presence of 4 M urea, a concentration that denatures the secondary structure of the polynucleotides. The αHL pore is functional even in 7 M urea, and therefore it is easily stable enough for analyses of challenging DNA and RNA species.     

Urea effect on Cu(2+)-induced DNA structural transitions in solution

Cu(2+) ion interaction with DNA in aqueous solutions containing urea (0-5 M) was studied by IR spectroscopy. It was shown that upon the Cu(2+) ion binding DNA transition into a compact form occurs. This transition is of positive cooperativity. We suppose that the mechanism of Cu(2+)-induced DNA compaction in solutions containing urea is not completely electrostatic. Urea addition to the DNA solution decreases the Cu(2+) ion concentration required to induce DNA compaction. As the urea content in solution rises, the binding constant of Cu(2+) ions interacting with DNA increases, going through the maximum in the case of 2 M solution; further increase of the urea content in solutions leads to decrease of the binding constant. DNA transition into the compact form under the Cu(2+) ion action is determined not only by the effects of the solution dielectric permeability but by the solvation effects; when changes of the dielectric permeability are small the solvation effects may prevail. Urea addition to the DNA solution also decreases cooperativity of the DNA compaction process. Perhaps, cooperativity of the DNA transition into the compact state depends on the ordered spatial structure of water adjacent to the macromolecule and decreases on the structure destruction.     

Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea.

This paper reports the results of a systematic study of the effects of formamide and urea on the thermal stability and renaturation kinetics of DNA. Increasing concentrations of urea in the range 0 to 8 molar lower the Tm by 2.25 degrees C per molar, and decreases the renaturation rate by approximately 8 percent per molar. Increasing concentrations of formamide in the range from 0 to 50 percent lowers the Tm by 0.60 degrees C per percent formamide for sodium chloride concentrations ranging from 0.035M to 0.88M. At higher salt concentrations the dependence of Tm on percent formamide was found to be slightly greater. Increasing formamide concentration decreases the renaturation rate linearly by 1.1% per percent formamide such that the optimal rate in 50% formamide is 0.45 the optimal rate in an identical solution with no formamide. The effects of urea and formamide on the renaturation rates of DNA are explained by consideration of the viscosities of the solutions at the renaturation temperatures.     

Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) modified oligonucleotides containing neutral urea linkages: effect of charge deletions on binding and fidelity

A solid-phase synthesis for a DNA analogue with a mixed guanidinium and urea backbone is reported. This material is nearly identical in structure to deoxynucleic guanidine (DNG) but the neutral urea internucleoside linkages can be used to attenuate the overall positive charge on the oligomer. The opposite charge attraction between urea containing DNG oligomers (DNGUs) and complimentary DNA can be controlled so that the affinity of DNG for DNA does not overwhelm the base-pairing discrimination necessary for specific binding. Octameric DNGU containing between 1 and 3 urea substitutions covered the range between very tight and very weak bonding. Each deletion of a positive charge reduced the thermal denaturation temperature (Tm) by approximately 5 degrees C. Mismatches in the DNA oligomers reduced the Tm values by 3 to 5 degrees C for each of the DNGU oligomers. DNGUs were found to bind in a 2:1 fashion to complimentary DNA in the same manner as DNG.     

Stepwise unfolding of chromatin by urea. A flow linear dichroism and photoaffinity labeling study

The unfolding of chromatin by urea (0-7 M) was studied by means of flow linear dichroism, photoaffinity labeling and nuclease digestion. The linear dichroism results indicate that the unfolding of the DNA is accomplished through two distinct transitions at 1-2 M urea and 6-8 M urea, respectively. The photoaffinity labeling studies indicate that an opening of the nucleosome histone core occurs above 2 M urea, accompanied by general loosening of the structure. Based on the results a model for the unfolding of chromatin fibers by urea is proposed, which includes a stretching of the linker DNA (0-2 M urea) followed by a "loosening" of the nucleosome core, possibly to a one-loop DNA conformation (2-6 M urea), and finally resulting in an almost total stretching of the DNA (greater than 6 M urea).     

Alteration of the groove width of DNA induced by the multimodal hydrogen bonding of denaturants with DNA bases in its grooves affects their stability

BackgroundDenaturants, namely, urea and guanidinium chloride (GdmCl) affect the stability as well as structure of DNA. Critical assessment of the role of hydrogen bonding of these denaturants with the different regions of DNA is essential in terms of its stability and structural aspect. However, the understanding of the mechanistic aspects of structural change of DNA induced by the denaturants is not yet well understood.MethodsIn this study, various spectroscopic along with molecular dynamics (MD) simulation techniques were employed to understand the role of hydrogen bonding of these denaturants with DNA bases in their stability and structural change.Results and conclusionIt has been found that both, GdmCl and urea intrude into groove region of DNA by striping surrounding water. The hydrogen bonding pattern of Gdm⁺ and urea with DNA bases in its groove region is multimodal and distinctly different from each other. The interaction of GdmCl with DNA is stabilized by electrostatic interaction whereas electrostatic and Lennard-Jones interactions both contribute for urea. Gdm⁺ forms direct hydrogen bond with the bases in the minor groove of DNA whereas direct and water assisted hydrogen bond takes place with urea. The hydrogen bond formed between Gdm⁺ with bases in the groove region of DNA is stronger than urea due to strong electrostatic interaction along with less self-aggregation of Gdm⁺ than urea. The distinct hydrogen bonding capability of Gdm⁺ and urea with DNA bases in its groove region affects its width differently. The interaction of Gdm⁺ decreases the width of the minor and major groove which probably increases the strength of hydrogen bond between the Watson-Crick base pairs of DNA leading to its stability. In contrast, the interaction of urea does not affect much to the width of the grooves except the marginal increase in the minor groove width which probably decreases the strength of hydrogen bond between Watson Crick base pairs leading to the destabilization of DNA.General significanceOur study clearly depicts the role of hydrogen bonding between DNA bases and denaturants in their stability and structural change which can be used further for designing of the guanidinium based drug molecules.