Geometry and mechanics of DNA superhelicity

This paper analyzes the elastic equilibrium conformations of duplex DNA constrained by the constancy of its molecular linking number, Lk. The DNA is regarded as having the mechanical properties of a homogeneous, linearly elastic substance with symmetric cross section. Integral representations of the writhing number Wr and of Lk are developed, in terms of which the equilibria are given as solutions to an isoperimetric problem. It is shown that the Euler angles defining equilibrium conformations must obey equations identical to those governing unconstrained equilibria. A scaling law is developed stating that molecules supercoiled the same amount ΔLk will have geometrically similar elastic equilibria regardless of their length. Thus, comparisons among molecules of properties related to their large-scale tertiary structure should be referred to differences in ΔLk rather than to their superhelix densities. Specific conditions on the elastic equilibrium conformations are developed that are necessary for ring closure. The equilibrium superhelical conformations accessible to closed-ring molecules are shown to approximate toroidal helices. Questions relating to the stability and nonuniqueness of equilibria are treated briefly. A comparison is made between these toroidal conformations and interwound configurations, which are shown to be stable, although they are not equilibria in the present sense. It is suggested that entropic factors are responsible for favouring the toroidal conformation in solution.     

Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA

We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.     

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.     

The translation of DNA primary base sequence into three-dimensional structure

A procedure is outlined to obtain a reliable computer-generatedrepresentation of the DNA duplex from its primary sequence ofbase pairs. The calculations are based on the potential energiesof interaction of adjacent side groups. The methods are, however,completely general and can be adapted to any set of base sequencedependent conformational rules. Static representations of theDNA are compared with the distributions of conformations obtainedfrom Monte Carlo simulation studies. Direct matrix generatorcalculations of the average (equilibrium) extension and orientationof various sequences and numerical estimates of the flexibilityof the chains as a whole are also reported. The methods areapplied to three short fragments of kinetoplast DNA from Crithidiafasciculata which exhibit dramatically different behavior onnon-denaturing poly-acrylamide gels. Received on August 17, 1987; accepted on December 19, 1987     

Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA·ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA·ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA·ligand complexes.     

Reversible decatenation of kinetoplast DNA by a DNA topoisomerase from trypanosomatids.

DNA topoisomerase activity detected in cell extracts of the trypanosomatid Crithidia fasciculata interlocks kinetoplast DNA duplex minicircles into huge catenane forms resembling the natural kinetoplast DNA networks found in trypanosomes. Catenation of duplex DNA circles is reversible and equilibrium is affected by ionic strength, and by spermidine. The reaction requires magnesium, is ATP dependent and is inhibited by high concentrations of novobiocin. Extensive homology between duplex DNA rings was not required for catenane formation since DNA circles with unrelated sequences could be interlocked into mixed network forms. Covalently sealed catenaned DNA circles are specifically used as substrates for decatenation. No such preference for covalently sealed duplex DNA rings was observed for catenate formation. Its catalytic properties and DNA substrate preference, suggest a potential role for this eukaryotic topoisomerase activity in the replication of kinetoplast DNA.     

Effects of HU binding on the equilibrium cyclization of mismatched, curved, and normal DNA

The effects of HU, the histone-like protein from Escherichia coli, on the equilibrium cyclization of duplex DNAs have been observed as a function of protein concentration and DNA sequence. The results indicate that the presence of HU significantly enhances the extent of cyclization and increases the melting temperature, T(m), of the cyclized form of the DNA by >10 K. The stabilization of equilibrium cyclization by HU binding is at least -1.2 kcal/mol. The results are consistent with two HU homotypic dimers binding to each of the three 29-mer duplexes studied. One of the 29-mer duplexes contains a central dA tract, one contains mismatched sites, and one a conventional sequence. Stepwise or microscopic association constants, determined from the fluorescence data, range from 1.5 to 0.6 micro M(-1). The binding affinity of the HU dimer is strongest for the mismatched duplex and lowest for the dA tract, consistent with HU dimers having a preference for flexible DNA substrates. These results demonstrate the utility of the equilibrium cyclization approach to monitor DNA-protein interactions. These results have been considered along with those previously obtained to refine a model for the interaction of HU with duplex DNA.     

Escherichia coli DNA helicases: mechanisms of DNA unwinding

DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair. These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown. Although it is clear that not all helicases unwind duplex DNA in an identical way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action. For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric. The oligomeric nature of helicases provides them with multiple DNA-binding sites, allowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either single-stranded and duplex DNA simultaneously or two strands of single-stranded DNA. Modulation of the relative affinities of these binding sites for single-stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases. The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oligomeric structures, with emphasis on the E. coli Rep, RecBCD and phage T7 gene 4 helicases.     

Structural polymorphism exhibited by a quasipalindrome present in the locus control region (LCR) of the human beta-globin gene cluster

Structural polymorphism of DNA is a widely accepted property. A simple addition to this perception has been our recent finding, where a single nucleotide polymorphism (SNP) site present in a quasipalindromic sequence of β-globin LCR exhibited a hairpin-duplex equilibrium. Our current studies explore that secondary structures adopted by individual complementary strands compete with formation of a perfect duplex. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, circular dichroism (CD) techniques, we have demonstrated the structural transitions within a perfect duplex containing 11 bp quasipalindromic stretch (TGGGG(G/C)CCCCA), to hairpins and bulge duplex forms. The extended version of the 11 bp duplex, flanked by 5 bp on both sides also demonstrated conformational equilibrium between duplex and hairpin species. Gel-electrophoresis confirms that the duplex coexists with hairpin and bulge duplex/cruciform species. Further, in CD spectra of duplexes, presence of two overlapping positive peaks at 265 and 285 nm suggest the features of A- as well as B-type DNA conformation and show oligomer concentration dependence, manifested in A → B transition. This indicates the possibility of an architectural switching at quasipalindromic region between linear duplex to a cruciform structure. Such DNA structural variations are likely to be found in the mechanics of molecular recognition and manipulation by proteins.     

Modulating DNA configuration by interfacial traction: an elastic rod model to characterize DNA folding and unfolding

As a continuum model of DNA, a thin elastic rod subjected to interfacial interactions is used to investigate the equilibrium configuration of DNA in intracellular solution. The interfacial traction between the rod and the solution environment is derived in detail. Kirchhoff’s theory of elastic rods is used to analyze the equilibrium configuration of a DNA segment under the action of the interfacial traction. The influences of the interfacial energy factor and bending stiffness on the toroidal spool formation of the DNA segment are discussed. The results show that the equilibrium configuration of DNA is mainly determined by competition between the interfacial energy and elastic strain energy of the DNA itself, and the interfacial traction is one of the forces that drives DNA folding and unfolding.     

Recombination apparatus of T4 phage

The substantial process of general DNA recombination consists of production of ssDNA, exchange of the ssDNA and its homologous strand in a duplex, and cleavage of branched DNA to maturate recombination intermediates. Ten genes of T4 phage are involved in general recombination and apparently encode all of the proteins required for its own recombination. Several proteins among them interact with each other in a highly specific manner based on a protein-protein affinity and constitute a multicomponent protein machine to create an ssDNA gap essential for production of recombinogenic ssDNA, a machine to supply recombinogenic ssDNA which has a free end, or a machine to transfer the recombinogenic single strand into a homologous duplex.     

An elastic rod model to evaluate effects of ionic concentration on equilibrium configuration of DNA in salt solution

As a coarse-gained model, a super-thin elastic rod subjected to interfacial interactions is used to investigate the condensation of DNA in a multivalent salt solution. The interfacial traction between the rod and the solution environment is determined in terms of the Young–Laplace equation. Kirchhoff’s theory of elastic rod is used to analyze the equilibrium configuration of a DNA chain under the action of the interfacial traction. Two models are established to characterize the change of the interfacial traction and elastic modulus of DNA with the ionic concentration of the salt solution, respectively. The influences of the ionic concentration on the equilibrium configuration of DNA are discussed. The results show that the condensation of DNA is mainly determined by competition between the interfacial energy and elastic strain energy of the DNA itself, and the interfacial traction is one of forces that drive DNA condensation. With the change of concentration, the DNA segments will undergo a series of alteration from the original configuration to the condensed configuration, and the spiral-shape appearing in the condensed configuration of DNA is independent of the original configuration.     

Enthalpy distribution functions for the unwinding of a short DNA duplex

In this article we use the published heat capacity data of Dragan et al. (J Mol Biol 2003, 327, 293-411) for a short DNA duplex to calculate the enthalpy probability distribution for this species as a function of temperature. Our approach is based on a procedure that we developed (Poland, D. J Chem Phys 2000, 112, 6554) whereby one obtains moments of the enthalpy distribution from the temperature dependence of the heat capacity. One then uses the maximum-entropy method to construct the enthalpy probability distribution from the set of enthalpy moments. For the DNA duplex treated here the heat capacity goes through a maximum as a function of temperature reflecting the unwinding of the duplex structure. In the neighborhood of the heat capacity maximum, the enthalpy distribution functions show a clear bimodal structure, indicating the coexistence of two distinct states, the duplex and the single-strand state. The probabilities of theses two states can be estimated from the enthalpy distribution functions and can be used to calculate the temperature dependence of the equilibrium constant for the unwinding of the DNA duplex. This example illustrates that the temperature dependence of the heat capacity can be used to give a detailed picture of conformational transitions in biological macromolecules. In particular, the structure of the enthalpy distribution in this case allows one to see the temperature evolution of the two-state distribution in detail.     

The herpes simplex virus type 1 DNA polymerase processivity factor, UL42, does not alter the catalytic activity of the UL9 origin-binding protein but facilitates its loading onto DNA

The herpes simplex virus type 1 UL42 DNA polymerase processivity factor interacts physically with UL9 and enhances its ability to unwind short, partially duplex DNA. In this report, ATP hydrolysis during translocation of UL9 on single-stranded (ss) or partially duplex DNA was examined in the presence and absence of UL42 to determine the effect of UL42 on the catalytic function of UL9. Our studies reveal that a homodimer of UL9 is sufficient for DNA translocation coupled to ATP hydrolysis, and the steady-state ATPase catalytic rate was greater on partially duplex DNA than on ss DNA in the presence or absence of UL42. Although UL42 protein increased the steady-state rate for ATP hydrolysis by UL9 during translocation on either partially duplex or ss DNA, UL42 had no significant effect on the intrinsic ATPase activity of UL9. UL42 also had no effect on the catalytic rate of ATP hydrolysis when UL9 was not limiting but enhanced the steady-state ATPase rate at only subsaturating UL9 concentrations. At subsaturating UL9 to DNA ratios, stoichiometric concentrations of UL42 were shown to increase the amount of UL9 bound to ss DNA at equilibrium. These data support a model whereby UL42 increases the ability of UL9 to load onto DNA, thus increasing its ability to assemble into a functional complex capable of unwinding duplex DNA.     

Enhanced recA protein binding to Z DNA represents a kinetic perturbation of a general duplex DNA binding pathway

recA protein binding to duplex DNA is enhanced when a B form DNA substrate is replaced with a left-handed Z form helix. This represents a kinetic rather than an equilibrium effect. Binding to Z DNA is much faster than binding to B DNA. In other respects, binding to the two DNA forms is quite similar. recA protein binds to B or Z DNA with a stoichiometry of 1 monomer/4 base pairs. The final protein filament exhibits a right-handed helical structure when either B or Z form DNAs are bound. There are only two evident differences: the kcat for ATP hydrolysis is reduced 3-4-fold when Z DNA is bound, and recA binding at equilibrium is less stable on Z DNA than on B DNA. At steady state, the binding favors B DNA in competition experiments. The results indicate that Z DNA binding by recA protein follows the same pathway as for recA binding to B DNA, but that the nucleation step is faster on the Z form helix.     

DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair

Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the k(on) and k(off) to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein-protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase alpha but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein-protein interactions.     

Energetics of B-Z junction formation in a sixteen base-pair duplex DNA

We report analysis of the NaCl-induced B-Z transition in a 16 base-pair duplex DNA with sequence designed such that when NaCl is increased the left half of the molecule undergoes the B-Z transition while the right half remains in the B-form. An equilibrium thermodynamic model based on the body of available published experimental data and the recent theoretical work of Soumpasis, which indicate, in the salt range above approximately 0.9 M-NaCl, the transition free-energy of B-Z conversion in DNA is a linear function of the NaCl concentration, is employed. Analysis of the B-Z transition of the junction-containing molecule indicates the B-Z junction formed in this 16 base-pair DNA is composed of approximately three base-pairs and has a free energy of formation of approximately 4.7 kcal/mol junction. These values for the junction are in excellent agreement with published estimates of B-Z junction size and energy derived from much longer DNA pieces.     

DNA structural changes as the basis for a nanomolecular device.

There is currently great interest in the design of nanodevices that are capable of performing movements. Protein molecular machines are abundant in biology but it has recently been proposed that nucleic acids could also act as nanomolecular machines in model systems. Several types of movements have been described with DNA machines: rotation, extension-contraction and "scissor-like" opening and closing. Here we analyze the properties of a simple and robust device composed of a single 21-base-long oligonucleotide which relies on a duplex/quadruplex equilibrium fueled by the sequential addition of DNA single-strands, generating a DNA duplex as a by-product. The interconversion between two well-defined topological states induces a five nanometer two-stroke, linear motor type movement, which is detected by FRET spectroscopy.