Double-stranded DNA Helix Research Articles

On the simultaneous binding of eukaryotic DNA topoisomerase II to a pair of double-stranded DNA helices

Stabilization of crossings of pairs of DNA helices by binding of eukaryotic DNA topoisomerase II was studied by two types of experiments. In one, mixtures of yeast DNA topoisomerase II and supercoiled DNA were incubated with vaccinia virus topoisomerase, and the linking numbers of the DNA products were measured to quantitate supercoils that were constrained by the stoichiometrically bound yeast enzyme molecules; in parallel, the same yeast enzyme-supercoiled DNA mixtures were incubated with a nonhydrolyzable ATP analog AMPPNP (adenosine 5'-(beta, gamma-imido)triphosphate) instead of the vaccinia enzyme, and DNA linking number changes following the addition of AMPPNP were measured to monitor DNA transport mediated by the yeast enzyme and AMPPNP. In the second type of experiments, formation of knotted DNA rings by the addition of AMPPNP to mixtures of yeast DNA topoisomerase II and different topological forms of DNA rings was studied. These experiments indicate that binding of yeast DNA topoisomerase II to DNA crossings is significant, especially in low salt media containing Mg(II), and that this mode of binding strongly affects DNA knotting. It appears, however, that stabilization of DNA crossovers by the eukaryotic type II enzyme is not directly related to its DNA transport activity.

Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.

Direct measurement of temperature-dependent solvation forces between DNA double helices

The assembly of double stranded DNA helices with divalent manganese ion is favored by increasing temperature. Direct force measurements, obtained from the osmotic stress technique coupled with x-ray diffraction, show that the force characteristics of spontaneously precipitated Mn(2+)-DNA closely resemble those observed previously by us for other counterion condensed DNA assemblies. At temperatures below the critical one for spontaneous assembly, we have quantitated the changes in entropy and manganese ion binding associated with the transition from repulsive to attractive interactions between helices mediated by osmotic stress. The release of structured water surrounding the DNA helix to the bulk solution is the most probable source of increased entropy after assembly. Increasing the water entropy of the bulk solution by changing the manganese salt anion from CI- to ClO4- predictably and quantitatively increases the transition entropy. This is further evidence for the dominating role of water in the close interaction of polar surfaces.

Evidence for structural deformation of the DNA helix by a psoralen diadduct but not by a monoadduct.

We have investigated the structural change in a double-stranded DNA helix caused by covalent addition of a psoralen. A synthetic double-stranded DNA was constructed to contain either a psoralen furan-side monoadduct or an interstrand diadduct at a specific site. When the unmodified and psoralen modified DNAs were examined by electron microscopy in the presence of distamycin, which stiffens the DNA helix, the DNA containing the psoralen interstrand diadduct appeared bent (or kinked), whereas the furan-side monoadducted DNA appeared similar to the unmodified DNA. RecA protein from E. coli has been shown to preferentially bind UV (ultra violet) irradiated DNA presumably due to alterations in the normal DNA helical structure. Using a nitrocellulose filter binding assay, we have found that the psoralen interstrand diadduct enhances the binding of recA protein to the double-stranded DNA, whereas a furan-side monoadduct has little effect. Thus both the recA protein binding and the electron microscopic data suggest that a psoralen diadduct causes deformation of a DNA helix, most likely by kinking the helix, and that a monoadduct has little effect on the DNA helix structure.

DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction

The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant ( k cat K m ), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein- nucleic acid interactions observed for the EcoRI restriction endonuclease.

Raman spectrum and secondary structure of the phage λ operator site O L1: evidence for multiple nucleoside conformations in an aqueous B-form DNA

The laser Raman spectrum of O L1 has been obtained in the region 600–850 cm −1 and the data have been interpreted in terms of different nucleoside conformations within the 17 base pair operator site. The O L1 sequence, which is one of the tightest binding sites for the cI and Cro repressors of bacteriophage λ, displays several Raman conformation markers indicative of more than one backbone geometry for the same double-stranded DNA helix. Specific assignments for the Raman conformation markers are suggested by analogy with spectra of DNA single crystals and DNA fibers of known structure. Two Raman bands diagnostic of B-DNA backbone geometry are observed at 825 ± 3 and 838 ± 3 cm −1, and may be due, respectively, to inequivalent conformations of GC and AT pairs. In addition, a weak band at 706 cm −1 and a shoulder near 807 cm −1 are consistent with a minor contribution from residues which assume the A-DNA backbone geometry or a structurally related configuration. The complex bandshape in the 650–700 cm −1 interval, which is resolved into four peaks by Fourier deconvolution, is also consistent with the presence of multiple nucleoside conformers in O L1 in physiological conditions.

Unwinding of double-stranded DNA helix by dehydration.

Conformation changes of the double-stranded DNA helix in response to dehydration were investigated by monitoring, by agarose gel electrophoresis, the linking number of covalently closed circular DNA generated by ligation of linear DNA in the presence of different organic solvents or different temperatures. It was found that: (i) The DNA helix unwinds upon addition of certain organic solvents or elevation of temperature. (ii) The conformational change observed under the experimental conditions is a continuous process in response to the organic solvent concentration. (iii) The delta H of unwinding one linking of the DNA helix is constant at approximately 12.2 +/- 0.4 kcal/mol (1 kcal = 4.184 kJ); the corresponding delta S and d(delta S)/dn are 2nkR and 2kR, in which n is the relative equivalent linking number (referred to the state of delta S = 0 for unwinding) of the DNA, R is the gas constant, and k is equal to 1117/number of base pairs. The delta H, delta S, and d(delta S)/dn for unwinding i linkings are i X 12.2 kcal/mol, 2inkR, and 2ikR, respectively. (iv) d(delta S)/dn, like k, is inversely proportional to the number of base pairs in DNA. (v) Double-stranded DNAs of different chain lengths have average delta S = 35 cal/mol.K for unwinding one linking under the experimental conditions; this corresponds to 127 +/- 14 base pairs per "relative linking."

Repairable damage in DNA: overview.

Due to the central functional role of DNA and the fact that each cell contains only one or at the most a few copies of each chromosome, damage to DNA has more severe implications for the functional integrity of the cell than does damage to most other cellular components. The chemical makeup and the large target size of chromosomal DNA make it particularly susceptible to attack by exogenous chemical and physical agents. Most, if not all, reactions of exogenous agents with the sugar residues of the DNA backbone result in strand breakage. While the continuity of the sugar-phosphate backbone usually remains intact for reactions involving the heterocyclic bases, such reactions may cause local distortion of the DNA conformation and of the native structure of eukaryotic chromatin. The preservation of the unique three-dimensional structure of the double-stranded DNA helix appears to be a prerequisite for its unimpaired biological activity. DNA base damage and concomitant helix distortion may lead to inhibition of replication and transcription and to a deterioration of the fidelity and a breakdown of the regulation of these processes. Restoration of the structural integrity of the DNA by repair processes is, therefore, a vital function of every cell.

On the possibility of intercalation of aromatic amino acid residues into double-stranded DNA helix

The relative sedimentation rates of linear and covalently closed λ DNA in sucrose gradients containing varying amounts of tryptamine, tyramine, histamine and phenethylamine were measured. The presence of the aromatic amines does not significantly change the sedimentation coefficient of the closed DNA, indicating that the DNA helix rotation angle is not significantly altered. This lack of angular alteration is also supported by sedimentation measurements of λ DNA which had been covalently closed by ligase in the presence of tryptamine. These results suggest that intercalation of aromatic amino acid residues into DNA is unlikely, contrary to the conclusion drawn from other measurements.

Explanation of Ionic Sequences in Various Phenomena. V. Determination of the Structure of DNA and RNA

Abstract The Watson-Crick model for DNA has been modified to account for observed physical phenomena associated with DNA. The viscosity data obtained on both partially denatured DNA and on undenatured DNA in various aqueous salt solutions give rise to acidic-type cationic sequences. These results can only be explained if there exist additional bonds other than the hydrogen bonds between base pairs. Considering the experimental data, the only possible type of bond that could exist is an ionic bond between the negatively charged phosphate group and the polar or positively charged nitrogen atom attached to the C'-1 position of the deoxyribose unit. The formation of such an ionic bond produces a natural twist in the DNA strand. The model is also applicable to RNA, although steric factors affect the stability and structure of such an RNA. Values of ΔH for the formation of the DNA and RNA double helix substantiate the proposed models. The most plausible structure for the double-stranded DNA helix is a model where the base pairs are on the surface rather than on the interior of the helix. Hydrophobic bonds do not exist except possibly in the altered structure produced during preparation of the DNA for X-ray analyses. The viscosity results on undenatured DNA in various salt solutions are caused by two factors: a destruction of the ionic bond and a reversal of charge effect on the DNA. The results show that the charge on DNA must change from a negative to a positive charge with addition of salt. The proposed model is applied to replication of DNA and formation of RNA from DNA and associated phenomena.

Localized strand separations within deoxyribonucleic acid during selective transcription.

MOLECULES of deoxyribonucleic acid isolated under gentle conditions are found to possess a double-stranded helical structure, in which the bases of each DNA strand are exactly paired within the interior of the helix with complementary bases of the opposite strand1. During gene transcription in vivo or under gentle conditions in vitro, only one of the two DNA strands serves as a template for new RNA synthesis2. Furthermore, within a well-differentiated tissue, only a fraction of loci within the total genome are transcribed in vivo, each such selection of loci being characteristic of the particular tissue3. It has recently been demonstrated that stable localized strand separations are induced within the double-stranded DNA helix during selective gene transcription within higher organisms4,5. Within such localized separation loops, one DNA strand appears to function as a template for RNA synthesis5. The complementary DNA strand of each separation loop appears to function as a binding site for specific de-repressor RNA molecules5 in an epigenetic control mechanism which selectively restricts gene transcription to only such strand-separated portions of the genome5,6.

BIOLOGICAL INFORMATION IN A SINGLE STRAND OF DEOXYRIBONUCLEIC ACID.

INVESTIGATIONS with several virus systems have indicated clearly that only one strand of the double-stranded DNA helix carries the coding for protein synthesis1–4. To distinguish this strand it might be called the ‘coding’ strand since in addition to priming the formation of complementary DNA, its complementary RNA copy contains the information for protein synthesis. The second strand might be called the ‘replicating’ strand, meaning that it serves only to prime the formation of complementary DNA with DNA polymerase.