DNA sequencing and helix–coil transition. III. DNA sequencing

We show that the fine oscillatory structure of the DNA melting curve can be used to determine explicitly the nucleotide composition and the order of certain domains within the DNA. If DNA is specifically fragmented, the order of fragments can be learned directly from a comparison of the differential melting curves of the nonfragmented and fragmented DNA. The indicated information may complement exact methods of DNA sequencing. The proposed analysis is applied to bacteriophage ?X-174, whose melting curve is known. Compared to the known ?X-174 DNA sequence, the results of the analysis are found to be very accurate.     

An A + T-rich satelitte DNA in a monocotyledonous plant, Cymbidium.

The DNA of aseptically grown protocorms of a Cymbidium hybrid and in vitro developed leaves, as well as DNA of leaves and flower buds of Cymbidium ceres from the greenhouse, was analysed by analytical ultracentrifugation and thermal denaturation. Upon ultracentrifugation a satellite DNA with a buoyant density of 1.682 g/cm-3 appears as a shoulder on the main band (density 1.694 g/cm-3). Thermal denaturation reveals an inhomogeneous main peak with the major component melting at 84 degrees C and a separate peak melting at 75 degrees C. This is the first demonstration of a satellite DNA in a monocot, and one of the rare examples of a major A + T-rich DNA fraction in a plant.     

Analysis of heat-denatured DNA using native agarose gel electrophoresis.

The use of native or neutral gels to resolve denatured DNA affords a rapid and convenient analytical method for assessing the consequences of a number of procedures employed in molecular biology research. We demonstrate that this method can be used to analyze transition melting temperature (Tm) and strand breakage in heat-denatured duplex DNA. This shows that some commonly recommended denaturation procedures can result in significant degradation of DNA and that reannealing or aggregation can occur when samples are concentrated or ionic conditions altered.     

DNA sequencing and helix-coil transition. I. Theory of DNA melting

An explicit analytic formula accurately describing the melting of a natural DNA is derived. For phage ?X-174 and virus SV-40, the nucleotide sequences of which are known, the formula fits experimental data for the differential melting curve almost within the experimental accuracy.     

Polymer- and salt-induced toroids of hexagonal DNA.

A model is proposed for polymer- and salt-induced toroidal condensates of DNA, based on a recent theory of the undulation enhancement of the electrostatic interaction in the bulk hexagonal phase of semiflexible polyions. In a continuum approximation, the thermodynamic potential of a monomolecular toroid may be split up in bulk, surface, and curvature contributions. With the help of an approximate analytical minimization procedure, the optimal torus dimensions are calculated as a function of the concentrations of inert polymer and added salt. The stability of the torus is analyzed in terms of its surface tension and a bulk melting criterion. The theory should be applicable to psi-toroids that are not too thick.     

Determination of DNA cooperativity factor.

The paper presents measurements of the difference in the melting temperature of a colE1 DNA region when it is located inside the DNA helix and at its end. A direct comparison of calculations based on the rigorous theory of helix-coil transition with experimental data for .2 M Na+ (the conditions for fully reversible melting) yielded the value of 2.5-5x10(-5) for the cooperatively factor sigma. We discuss the reversibility of DNA melting and the possibility of applying the "all-or-nothing" concept to the melting of DNA regions.     

A study of DNA denaturation in the ultracentrifuge

The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable molecules melt early and are found at heavier densities in the melting region. An idealized ultracentrifuge melting transition is described. The melting transition of singly nicked PM-2 DNA resembles the idealized curve. The DNA profile is a Gaussian band at all points in the melt. DNA's from mouse, D. Melanogaster, M. lysodeikticus, T4, and T7 also show equilibrium bands at partially denatured densities, some of which are highly asymmetric. Simple sequence satellite DNA shows an all-or-none transition with no equilibrium bands at partially denatured densities. The temperature at which a DNA denatures is an increasing function of the (G + C) content of the DNA. The T_m does not show a molecular-weight dependence in the range 1.2 × 10⁶–1.5 × 10⁷ daltons (single strand) for mouse, M. lysodeikticus, or T4 DNA. The mouse DNA partially denatured bands do not change shape as a function of molecular weight. The T4 DNA intermediate band develops a late-melting tail at low molecular weight. M. lysodeikticus DNA bands at partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components at each point in the melting transition.     

Preparation and melting of single strand circular DNA loops.

A method for preparation of single strand DNA circles of almost arbitrary sequence is described. By ligating two sticky ended hairpins together a linear duplex is formed, closed at both ends by single stranded loops. The melting characteristics of such loops are investigated using optical absorbance and NMR. It is shown by comparison with the corresponding linear sequence (closed circle minus the end loops) that the effects of end fraying and the strand concentration dependence of the melting temperature are eliminated in the circular form. Over the concentration range examined (0.5 to 2.0 micromolar strands), the circular DNA has a monophasic melting curve, while the linear duplex is biphasic, probably due to hairpin formation. Since effects of duplex to single strands dissociation do not contribute to melting of the circular molecules (dumbells), these DNAs present a realistic experimental model for examining local thermal stability in DNA.     

Melting fine structure of filamentous fungus nuclear DNA.

Melting fine structure of the nuclear DNA isolated from the filamentous fungus Fusarium graminearum Schwabe is presented. Optical melting profiles of nuclear DNA were analyzed by using a combination of curve fitting and derivative techniques. The "melting components" were obtained from the derivative curve by a simple decomposition technique. Differential optical melting curves of unsheared nuclear DNA indicate the presence of 15 "melting components" in filamentous fungus nuclear genome. It should be emphasized that the "melting components" observed here are different from the "thermalites" which can be observed in bacteriophage DNA. The "melting components" reported here represent the separately melting of large "blocks" of fungus nuclear DNA.     

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA structure.     

Hyperfine structure in melting profile of bacteriophage lambda DNA.

A high-resolution plotter of differential melting profiles of DNA, RNA, or related biopolymers with an on-line mini-computer is described. With this device, more than 15 transition steps were identified in the thermal melting profile of DNA from bacteriophage lambda. These fine structures were found to be reproducible, and some of them disappear in the deletion mutant. To Examine the melting profile, computer simulations for several hypothetical polynucleotide sequences were performed, and compared with experimental data. The sharp peaks that appeared in the differential melting profile of λ DNA may come from some homogeneous sequences of 500 bases or longer.     

Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis.

The melting behavior of a DNA fragment carrying the mouse beta maj-globin promoter was investigated as a means of establishing procedures for separating DNA fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lerman (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp, to a 135 bp DNA fragment carrying the mouse beta-globin promoter significantly alters the pattern of DNA melting within the promoter. When the promoter is attached to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agreement with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels.     

Force-induced melting of the DNA double helix 1. Thermodynamic analysis

The highly cooperative elongation of a single B-DNA molecule to almost twice its contour length upon application of a stretching force is interpreted as force-induced DNA melting. This interpretation is based on the similarity between experimental and calculated stretching profiles, when the force-dependent free energy of melting is obtained directly from the experimental force versus extension curves of double- and single-stranded DNA. The high cooperativity of the overstretching transition is consistent with a melting interpretation. The ability of nicked DNA to withstand forces greater than that at the transition midpoint is explained as a result of the one-dimensional nature of the melting transition, which leads to alternating zones of melted and unmelted DNA even substantially above the melting midpoint. We discuss the relationship between force-induced melting and the B-to-S transition suggested by other authors. The recently measured effect on T7 DNA polymerase activity of the force applied to a ssDNA template is interpreted in terms of preferential stabilization of dsDNA by weak forces approximately equal to 7 pN.     

A study of the reversibility of helix-coil transition in DNA.

The reversibility of DNA melting has been thoroughly investigated at different ionic strengths. We concentrated on those stages of the process that do not involve a complete separation of the strands of the double helix. The differential melting curves of pBR 322 DNA and a fragment of T7 phage DNA in a buffer containing 0.02M Na+ have been shown to differ substantially from the differential curves of renaturation. Electron-microscopic mapping of pBR 322 DNA at different degrees of unwinding (by a previously elaborated technique) has shown that the irreversibility of melting under real experimental conditions is connected with the stage of forming new helical regions during renaturation. In a buffer containing 0.2M Na+ the melting curves of the DNAs used (pBR322, a fragment of T7 phage DNA, a fragment of phage Lambda DNA, a fragment of phiX174 phage DNA) coincide with the renaturation curves, i.e. the process is equilibrium. We have carried out a semi-quantitative analysis of the emergence of irreversibility in the melting of a double helix. The problem of comparing theoretical and experimental melting curves is discussed.     

Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting

The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, have determined that AT-rich sequences have a lower binding energy than GC-rich sequences. In cells, however, the double-stranded backbone of DNA is destabilized by negative supercoiling, and not by temperature. To investigate what the effect of GC content is on DNA melting induced by negative supercoiling, we studied DNA molecules with a GC content ranging from 38% to 77%, using single-molecule magnetic tweezer measurements in which the length of a single DNA molecule is measured as a function of applied stretching force and supercoiling density. At low force (<0.5pN), supercoiling results into twisting of the dsDNA backbone and loop formation (plectonemes), without inducing any DNA melting. This process was not influenced by the DNA sequence. When negative supercoiling is introduced at increasing force, local melting of DNA is introduced. We measured for the different DNA molecules a characteristic force F _char, at which negative supercoiling induces local melting of the dsDNA. Surprisingly, GC-rich sequences melt at lower forces than AT-rich sequences: F _char = 0.56pN for 77% GC but 0.73pN for 38% GC. An explanation for this counterintuitive effect is provided by the realization that supercoiling densities of a few percent only induce melting of a few percent of the base pairs. As a consequence, denaturation bubbles occur in local AT-rich regions and the sequence-dependent effect arises from an increased DNA bending/torsional energy associated with the plectonemes. This new insight indicates that an increased GC-content adjacent to AT-rich DNA regions will enhance local opening of the double-stranded DNA helix.     

Melting of Cross-Linked DNA: I. Model and Theoretical Methods

Abstract

Covalent and strong coordination binding to DNA of a large number of antitumour drugs and other compounds leads to interstrand cross-link formation. To investigate cross-link influence on double helix stability, two methods are developed for the calculation of melting curves. The first method is based on Poland's approach. It requires computer time proportional to u-N, where u is the average distance (in base pairs) between neighboring crosslinks, and N is the number of base pairs in the DNA chain. The method is more suitable when u is not large, and small loops formed by interstrand cross-links in melted regions strongly affect DNA melting. The computer time for the second method, based on the Fixman-Freire approach, does not depend on the number of cross-links and is proportional to I N (I is the number of exponential functions used for a decomposition of the loop entropy factor). It is more appropriate when N and u are large, and therefore particular values of the entropy factors of small loops do not influence DNA melting behavior.     

Equilibrium melting of plasmid ColE1 DNA: electron-microscopic visualization.

The fine structure of the melting curve for the linear colE1 DNA has been obtained. To find the ColE1 DNA regions corresponding to peaks in the melting curve's fine structure, we fixed the melted DNA regions with glyoxal /12/. Electron-microscopic denaturation maps were obtained for nine temperature points within the melting range. Thereby the whole process of colE1 DNA melting was reconstructed in detail. Spectrophotometric and electron microscopic data were used for mapping the distribution of Gc-pairs over the DNA molecule. The most AT-rich DNA regions (28 and 37% of GC-pairs), 380 and 660 bp long resp., are located on both sides of the site of ColE1 DNA's cleavage by EcoR1 endonuclease. The equilibrium denaturation maps are compared with maps obtained by the method of Inman /20/ for eight points of the kinetic curve of ColE1 DNA unwinding by formaldehyde.     

Predicting sequence-dependent melting stability of short duplex DNA oligomers

Many important applications of DNA sequence-dependent hybridization reactions have recently emerged. This has sparked a renewed interest in analytical calculations of sequence-dependent melting stability of duplex DNA. In particular, for many applications it is often desirable to accurately predict the transition temperature, or t_m, of short duplex DNA oligomers (∼ 20 base pairs or less) from their sequence and concentration. The thermodynamic analytical method underlying these predictive calculations is based on the nearest-neighbor model. At least 11 sets of nearest-neighbor sequence-dependent thermodynamic parameters for DNA have been published. These sets are compared. Use of the nearest-neighbor sets in predicting t_m from the DNA sequence is demonstrated, and the ability of the nearest-neighbor parameters to provide accurate predictions of experimental t_m's of short duplex DNA oligomers is assessed. © 1998 John Wiley & Sons, Inc. Biopoly 44: 217–239, 1997