Statistical mechanical theory of melting transition in supercoiled DNA

The melting curve for a covalently closed circular DNA has been analyzed on the basis of an expression for the supercoiling energy derived in terms of the elastic parameters of the macromolecule, treated as a homopolymer. The result obtained by applying the usual methods of statistical mechanics indicate close agreement with the available experimental data. It is found that the elevation of the melting temperature, as compared to that of the nicked circular or linear DNA, is a natural consequence of the fact that the supercoiled molecule is constrained by an invariant linking number. The flattening of the melting curve, on the other hand, arises as the closed circular duplex melts into a loose helix rather than random coils.     

Is DNA really a double helix?

Do the two chains of the DNA molecule coil round one another plectonemically ? If so, what is the approximate value of Lk (the linking number) for any closed, circular DNA molecule? Experiments using gel electrophoresis have shown that supercoiled DNA molecules usually migrate in a series of discrete bands. The only tenable explanation for this quantized behavior is that the molecules in one band all have the same value of Lk and that this value differs by unity from that of the adjacent bands. Various experiments in which circular DNA is unwound by known amounts show that (given this assumption) Lk for relaxed DNA is very roughly equal to $\text{N}\text{10}$ (where N is the number of base-pairs), as expected from the classical double helix.The original model for the double helix was right-handed. The experimental evidence for this feature is suggestive but not yet completely compelling.     

Inverted repeat sequences can influence the melting transitions of linear DNAs

The influence of inverted repeat sequences on the melting transitions of linear DNAs has been examined. Derivative melting curves (DMC) of a 514 base pair (bp) DNA, seven subfragments of this DNA, and four other DNAs have been compared to predictions of DNA melting theory. The 514-bp DNA contains three inverted repeat sequences that can form cruciform structures in supercoiled DNA. We refer to these sequences as c-inverted repeats. Previous work showed that the DMC of this DNA, unlike a number of other DNAs, is not accurately predicted by DNA melting theory. Since the theoretical model does not include hairpin-like structures, it was suggested that hairpin or cruciform formation in these inverted repeats may be responsible for this discrepancy. Our results support this hypothesis. Predicted DMCs are in good agreement with DNAs with no inverted repeats, or inverted repeats not evident in supercoiled DNA. Differences between the theoretical and experimental Tm's are less than or equal to 0.3 degrees C. DNA molecules that contain one or more of the three c-inverted repeats are not as accurately predicted. Experimental Tm values are lower than predicted values by 0.7-3.8 degrees C. It is concluded that some inverted repeat sequences can form hairpin-like structures during the melting of linear DNAs. These structures appear to lower overall DNA stability.     

Melting characteristics of highly supercoiled DNA

The effect of high supercoil densities on the melting characteristics of a supercoiled DNA has been studied. It is found that although the melting temperature increases abruptly on converting a linear DNA merely into the relaxed circular form, it falls back substantially at high supercoil densities. It is further predicted, in such cases, that the number of melted base pairs should be significantly enhanced even at the physiological temperature, which may facilitate the binding of other molecules to the highly supercoiled DNA.     

Visualization of drug-nucleic acid interactions at atomic resolution. III. Unifying structural concepts in understanding drug-DNA interactions and their broader implications in understanding protein-DNA interactions.

Structural information afforded by the X-ray crystallographic studies of ethidium-dinucleoside monophosphate crystalline complexes described in the preceding two papers has led to a detailed model for ethidium-DNA binding. Features of ethidium-DNA binding, in turn, have led to unifying structural concepts in understanding a wide range of drug-DNA interactions. It is possible that these concepts have still broader implications in understanding the nature of protein-DNA interactions.This paper begins by summarizing the stereochemical aspects of ethidium-DNA, actinomycin-DNA and irehdiamine-DNA binding, molecules that use intercalative and kinked-type geometries in binding to DNA. It then describes superhelical DNA structures formed by kinking DNA periodically varying numbers of base-pairs apart. κ-kinked B DNA, a structure formed by kinking DNA every ten base-pairs, is a left-handed superhelical structure that may be utilized in the organization of DNA within the nucleosome in chromatin. β-kinked B DNA is a right-handed superhelical structure formed by kinking DNA every two base-pairs. It is possible that premelting conformational changes occur in DNA which utilize elements of this structure. This would expose base-pairs to solvent denaturation, and could lower the activation energy necessary for strand separation during DNA denaturation. RNA polymerase and other DNA melting proteins could capitalize on this type of premelting conformational change when binding to DNA.The concept that conformational flexibility exists in DNA structure (and that drug intercalation is a phenomenon that reflects this flexibility) can, in addition, explain a wide variety of physicochemical data about DNA. In this paper we discuss the nature of these data in detail.     

Letter: Electron microscopic visualization of the folded chromosome of Escherichia coli

The folded Escherichia coli chromosome has been analyzed in the electron microscope following the Kleinschmidt spreading technique. In all cases, the DNA molecule appears intact and supercoiled. In addition, the membrane-associated form of the chromosome shows DNA fibers attached to one or two membrane patches. The DNA molecule is intact, as evidenced by the absence of free ends. Two types of DNA coiling are generally observed: (1) spirals with about four to seven turns per spiral, and (2) stretched plectonemic supercoils, more abundant on the outside of the complex where the spreading has been more extensive.The released folded chromosome appears to be less compact; it extends over a larger area and frequently breaks with the spreading. The intact molecules which can be found show the DNA concentrated in few nueleation areas, with one or a few interconnecting DNA fibers. The folded DNA has very few or no single strand nicks, as evidenced by its extensive supertwisting at high ethidium bromide concentrations.     

Comparison of partial denaturation maps with the known sequence of simian virus 40 and phi X174 replicative form DNA.

Electron microscope partial denaturation maps of two viral DNAs, simian virus 40 and φX174 replicative form, have been obtained. A simple computer program has been developed to predict denaturation maps from any given DNA sequence, based on the percentage of A · T base-pairs along the molecule. Maps constructed from the SV40 DNA and φX174 replicative form DNA base sequence show a good correlation with the experimental maps. The results show that the regions of a DNA molecule that denature first are, in fact, those regions with the highest content of adenine and thymine base-pairs.     

Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA

We report that two types of stable protein-DNA complexes, or transpososomes, are generated in vitro during the Mu DNA strand transfer reaction. The Type 1 complex is an intermediate in the reaction. Its formation requires a supercoiled mini-Mu donor plasmid, Mu A and HU protein, and Mg2+. In the Type 1 complex the two ends of Mu are held together, creating a figure eight-shaped molecule with two independent topological domains; the Mu sequences remain supercoiled while the vector DNA is relaxed because of nicking. In the presence of Mu B protein, ATP, target DNA, and Mg2+, the Type 1 complex is converted into the protein-associated product of the strand transfer reaction. In this Type 2 complex, the target DNA has been joined to the Mu DNA ends held in the synaptic complex at the center of the figure eight. Supercoils are not required for the latter reaction.     

Effects of 5 cytosine methylation on the B-Z transition in DNA restriction fragments and recombinant plasmids

Alternating (dC-dG)n regions in DNA restriction fragments and recombinant plasmids were methylated at the 5 position of the cytosine residues by the HhaI methylase. Methylation lowers the concentration of NaCl or MgCl2 necessary to cause the B-Z conformational transition in these sequences. Ionic strengths higher than physiological conditions are required to form the Z conformation when the methylated (dC-dG)n tract is contiguous with regions that do not form Z structures, in contrast to the results with the DNA polymer poly(m5dC-dG) . poly(m5dC-dG). In supercoiled plasmids containing (dC-dG)n sequences, methylation reduces the number of negative supercoils necessary to stabilize the Z conformation. Calculations of the observed free energy contributions of the B-Z junction and cytosine methylation suggest that two junctions offset the favorable effect of methylation on the Z conformation in (dC-dG)n sequences (about 29 base-pairs in length). Studies with individual methylated topoisomers demonstrate that increasing Na+ concentration up to approximately 0.2 M inhibits the formation of the Z conformation in the (m5dC-dG)n region of supercoiled plasmids. The results suggest that methylation may serve as a triggering mechanism for Z DNA formation in supercoiled DNAs.     

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.     

Mode of replication of the conjugative R-plasmid RSF1040 in Escherichia coli.

Replicating deoxyribonucleic acid (DNA) molecules of plasmid RSF1040, a deletion mutant of the conjugative R plasmid R6K, appear in the electron microscope as partially supercoiled structures with two open circular branches of equal size, although open structures with three branches, two branching points and no supercoiled regions (theta structures) were also found at a lower frequency. The partially supercoiled molecules sediment more rapidly than native covalently closed circular DNA in neutral sucrose gradients and band at a position intermediate between covalently closed circular and open circular DNA in CsClethidium bromide gradients. Electron microscope measurements of the linear EcoRI-treated replicative intermediates indicate that replication can be initiated at two sites (origins) on the plasmid DNA molecule located at about 23% (alpha) and 39% (beta) of the total genome length from an EcoRI end designated arbitrarily as the "left-hand" end of the molecule. The overall replication of RSF1040 is asymmetrically bidirectional. Replication from the alpha origin proceeds first to the "right" to a unique termination site located some 55% of the total genome length from the left-hand end of the molecule. At this point replication proceeds from the alpha origin to the "left" (i.e., opposite to the original direction of replication) until replication of the molecule is completed. Replication also proceeds from the beta origin asymmetrically to the unique terminus site.     

Large-scale opening of A + T rich regions within supercoiled DNA molecules is suppressed by salt.

Large-scale cooperative helix opening has been previously observed in A + T rich sequences contained in supercoiled DNA molecules at elevated temperatures. Since it is well known that helix melting of linear DNA is suppressed by addition of salt, we have investigated the effects of added salts on opening transitions in negatively supercoiled DNA circles. We have found that localised large-scale stable melting in supercoiled DNA is strongly suppressed by modest elevation of salt concentration, in the range 10 to 30 mM sodium. This has been shown in a number of independent ways: 1. The temperature required to promote cruciform extrusion by the pathway that proceeds via the coordinate large-scale opening of an A + T rich region surrounding the inverted repeat (the C-type pathway, first observed in the extrusion of the ColE1 inverted repeat) is elevated by addition of salt. The temperature required for extrusion was increased by about 4 deg for an addition of 10 mM NaCl. 2. A + T rich regions in supercoiled DNA exhibit hyperreactivity towards osmium tetroxide as the temperature is raised; this reactivity is strongly suppressed by the addition of salt. At low salt concentrations of added NaCl (10 mM) we observe that there is an approximate equivalence between reducing the salt concentration, and the elevation of temperature. Above 30 mM NaCl the reactivity of the ColE1 sequences is completely supressed at normal temperatures. 3. Stable helix opening transitions in A + T rich sequences may be observed with elevated temperature, using two-dimensional gel electrophoresis; these transitions become progressively harder to demonstrate with the addition of salt. With the addition of low concentrations of salt, the onset of opening transitions shifts to higher superhelix density, and by 30 mM NaCl or more, no transitions are visible up to a temperature of 50 degrees C. Statistical mechanical simulation of the data indicate that the cooperativity free energy for the transition is unaltered by addition of salt, but that the free energy cost for opening each basepair is increased. These results demonstrate that addition of even relatively low concentrations of salt strongly suppress the large-scale helix opening of A + T rich regions, even at high levels of negative supercoiling. While the opening at low salt concentrations may reveal a propensity for such transitions, spontaneous opening is very unlikely under physiological conditions of salt, temperature and superhelicity, and we conclude that proteins will therefore be required to facilitate opening transitions in cellular DNA.     

DNA supercoiling promotes formation of a bent repression loop in lac DNA

Titration experiments on supercoiled lac DNA show that one repressor tetramer can bind simultaneously to the primary lac operator and to the very weak lac pseudo-operator, located 93 base-pairs apart. The formation of this complex is accompanied by the appearance of an extreme hypersensitive site in a five base-pair sequence located approximately midway between the operators. This remote sequence is hypersensitive to attack by two different chemical probes, dimethyl sulfate and potassium permanganate, the latter of which is a new probe for distorted DNA. We interpret these results in terms of a complex in which lac repressor holds two remote operators together in a DNA loop. The formation of this bent DNA loop requires negative DNA supercoiling. In vivo, both lac operators bind repressor even though the presence of multiple operator copies has forced the two operators to compete for a limited amount of repressor. This suggests that the operator and pseudo-operator have similar affinities for repressor in vivo. Such similar affinities were observed in vitro only when DNA supercoiling forced formation of a repression loop.     

Features of the geometry of a supercoiled DNA molecule

The features of the geometry of a supercoiled DNA molecule are considered. A model of supercoiled structure of DNA taking into account its natural helical structure has been constructed. Force factors arising in DNA has been calculated depending on winding angle.     

The conformational variability of an adenosine.inosine base-pair in a synthetic DNA dodecamer.

A crystal structure analysis of the synthetic deoxydodecamer d(CGCAAATTIGCG) which contains two adenosine.inosine (A.I) mispairs has revealed that, in this sequence, the A.I base-pairs adopt a A(anti).I(syn) configuration. The refinement converged at R = 0.158 for 2004 reflections with F greater than or equal to 2 sigma(F) in the range 7.0-2.5A for a model consisting of the DNA duplex and 71 water molecules. A notable feature of the structure is the presence of an almost complete spine of hydration spanning the minor groove of the whole of the (AAATTI)2 core region of the duplex. pH-dependent ultraviolet melting studies have suggested that the base-pair observed in the crystal structure is, in fact, a protonated AH+ (anti).I(syn) species and that the A.I base-pairs in the sequence studied display the same conformational variability as A.G mispairs in the sequence d(CGCAAATTGGCG). The AH+(anti).I(syn) base-pair predominates below pH 6.5 and an A(anti).I(anti) mispair is the major species present between pH 6.5 and 8.0. The protonated base-pairs are held together by two hydrogen bonds one between N6(A) and O6(I) and the other between N1(A) and N7(I). This second hydrogen bond is a direct result of the protonation of the N1 of adenosine. The ultraviolet melting studies indicate that the A(anti).I(anti) base-pair is more stable than the A(anti).G(anti) base-pair but that the AH+(anti).I(syn) base pair is less stable than its AH+(anti).G(syn) analogue. Possible reasons for this observation are discussed.     

Melting of Cross-Linked DNA IV. Methods for Computer Modeling of Total Influence on DNA Melting of Monofunctional Adducts,Intrastrand and Interstrand Cross-Links Formed by Molecules of an Antitumor Drug

Abstract

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.     

Molecular mechanics model of supercoiled DNA

We describe a pseudo-atomic model of supercoiled DNA. Each base-pair of the DNA is represented in the model by three particles placed in a plane. The particle triplets are stacked to model stacked base-pairs in double-helical DNA, and closed circular conformations are generated to investigate supercoiling. This model is less detailed than all-atom models, which are too computationally demanding to be used to study supercoiling. On the other hand, this model contains details at the base-pair level and is therefore more elaborate than elastomechanical models. A potential energy function is written in terms of a set of internal co-ordinates defined to resemble a limited number of helical parameters. The modeled helical parameters, helical twist, base-roll, tilt and rise, are the most important parameters of the global shape of DNA. Experimentally measured mechanical properties of DNA are used to define the forces holding the particles together. We then use a procedure incorporating energy minimization and molecular dynamics to locate low energy conformations of the model DNA. The model was found to behave very much like rubber-tubing and elastomechanical models. The conformations and the effects of supercoiling pressure (a number proportional to the degree to which the total twist of the DNA has been altered from its natural value) on these conformations are all very similar to those observed in the latter two models. We also used this model to examine the effects of supercoiling pressure, base-sequence and mechanical properties on the conformations and energies of five sequences. The sequences studied include models of naturally straight DNA and DNA with static or natural bends.     

Melting of cross-linked DNA IV. Methods for computer modeling of total influence on DNA melting of monofunctional adducts, intrastrand and interstrand cross-links formed by molecules of an antitumor drug

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.