Competition between supercoils and toroids in single molecule DNA condensation

The condensation of free DNA into toroidal structures in the presence of multivalent ions and polypeptides is well known. Recent single molecule experiments have shown that condensation into toroids occurs even when the DNA molecule is subjected to tensile forces. Here we show that the combined tension and torsion of DNA in the presence of condensing agents dramatically modifies this picture by introducing supercoiled DNA as a competing structure in addition to toroids. We combine a fluctuating elastic rod model of DNA with phenomenological models for DNA interaction in the presence of condensing agents to compute the minimum energy configuration for given tension and end-rotations. We show that for each tension there is a critical number of end-rotations above which the supercoiled solution is preferred and below which toroids are the preferred state. Our results closely match recent extension rotation experiments on DNA in the presence of spermine and other condensing agents. Motivated by this, we construct a phase diagram for the preferred DNA states as a function of tension and applied end-rotations and identify a region where new experiments or simulations are needed to determine the preferred state.     

Potential-Energy surface for double-helical DNA with Watson-Crick Pairs. I. Complete scanning in the space of torsion angles

Complete scanning of correct double-helical DNA structures was performed in the space of torsion angles using rapid analytical procedures for generation of regular double helices with Watson-Crick base pairs. Potential energy of structures was calculated by pair-wise potential functions and used as necessary condition to single out physically meaningful forms. We found 12 discrete potential wells containing these physically meaningful forms, with only one well containing all classical forms (A, B, etc.). On the basis of the new structures listed, alternative models for DNA and DNA-like polymers and appropriate conformations for fluctuations leading to DNA bending and kink formation are suggested.     

Effect of Bending Strain on the Torsion Elastic Constant of DNA

The torsion constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the torsion constant increased by a factor of 1.42, the intrinsic binding constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between torsion constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4°C for eight months, the torsion constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the torsion constant and intrinsic binding constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that torsion constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.     

Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation

The mechanisms of RecA-mediated three-strand homologous recombination are investigated at the single-molecule level, using magnetic tweezers. Probing the mechanical response of DNA molecules and nucleoprotein filaments in tension and in torsion allows a monitoring of the progression of the exchange in real time, both from the point of view of the RecA-bound single-stranded DNA and from that of the naked double-stranded DNA (dsDNA). We show that strand exchange is able to generate torsion even along a molecule with freely rotating ends. RecA readily depolymerizes during the reaction, a process presenting numerous advantages for the cell's 'protein economy' and for the management of topological constraints. Invasion of an untwisted dsDNA by a nucleoprotein filament leads to an exchanged duplex that remains topologically linked to the exchanged single strand, suggesting multiple initiations of strand exchange on the same molecule. Overall, our results seem to support several important assumptions of the monomer redistribution model.     

Conformational studies of nucleic acids: IV. The conformational energetics of oligonucleotides: d(ApApApA) and ApApApA

Utilizing a new method for modeling furanose pseudorotation (D. A. Pearlman and S.-H. Kim, J. Biomol. Struct. Dyn. 3, 85 (1985)) and the empirical multiple correlations between nucleic acid torsion angles we derived in the previous report (D. A. Pearlman and S.-H. Kim, previous paper in this issue), we have made an energetic examination of the entire conformational spaces available to two nucleic acid oligonucleotides: d(ApApApA) and ApApApA. The energies are calculated using a semi-empirical potential function. From the resulting body of data, energy contour map pairs (one for the DNA molecule, one for the RNA structure) have been created for each of the 21 possible torsion angle pairs in a nucleotide repeating unit. Of the 21 pairs, 15 have not been reported previously. The contour plots are different from those made earlier in that for each point in a particular angle-angle plot, the remaining five variable torsion angles are rotated to the values which give a minimum energy at this point. The contour maps are overall quite consistent with the experimental distribution of oligonucleotide data. A number of these maps are of particular interest: delta (C5'-C4'-C3'-O3')-chi (O4'-C1'-N9-C4), where the energetic basis for an approximately linear delta-chi correlation can be seen: zeta (C3'-O3'-P-O5')-delta, in which the experimentally observed linear correlation between zeta and delta in DNA(220 degrees less than zeta less than 280 degrees) is clearly predicted; zeta-epsilon (C4'-C3'-O3'-P), which shows that epsilon increases with decreasing zeta less than 260 degrees; alpha (O3'-P-O5'-C5')-gamma (O5'-C5'-C4'-C3') where a clear linear correlation between these angles is also apparent, consistent with experiment; and several others. For the DNA molecule studied here, the sugar torsion delta is predicted to be the most flexible, while for the RNA molecule, the greatest amount of flexibility is expected to reside in alpha and gamma. Both the DNA and RNA molecules are predicted to be highly polymorphic. Complete energy minimization has been performed on each of the minima found in the energy searches and the results further support this prediction. Possible pathways for B-form to A-form DNA interconversion suggested by the results of this study are discussed. The results of these calculations support use of the new sugar modeling technique and torsion angle correlations in future conformational studies of nucleic acids.     

Role of DNA topology in Mu transposition: mechanism of sensing the relative orientation of two DNA segments

DNA strand transfer at the initiation of Mu transposition normally requires a negatively supercoiled transposon donor molecule, containing both ends of Mu in inverted repeat orientation. We propose that the specific relative orientation of the Mu ends is needed only to energetically favor a particular configuration that the ends must adopt in a synaptic complex. The model was tested by constructing special donor DNA substrates that, because of their catenation or knotting, energetically favor this same configuration of the Mu ends, even though they are on separate molecules or in direct repeat orientation. These structures are efficient substrates for the strand transfer reaction, whereas appropriate control structures are not. The result eliminates tracking or protein scaffold models for orientation preference. Several other systems in which the relative orientation of two DNA segments is sensed may utilize the same mechanism.     

Torque-limited RecA polymerization on dsDNA

The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA–DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 ± 0.7 k_bT. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.     

Conformational Studies of Nucleic Acids: IV. The Conformational Energetics of Oligonucleotides: d(ApApApA) and ApApApA

Abstract

Utilizing a new method for modeling furanose pseudorotation (D. A Pearlman and S.-H. Kim, J. Biomol. Struct. Dyn. 3, 85 (1985)) and the empirical multiple correlations between nucleic acid torsion angles we derived in the previous report (D. A Pearlman and S.-H. Kim, previous paper in this issue), we have made an energetic examination of the entire conformational spaces available to two nucleic acid oligonucleotides: d(ApApApA) and ApApApA The energies are calculated using a semi-empirical potential function. From the resulting body of data, energy contour map pairs (one for the DNA molecule, one for the RNA structure) have been created for each of the 21 possible torsion angle pairs in a nucleotide repeating unit. Of the 21 pairs, 15 have not been reported previously. The contour plots are different from those made earlier in that for each point in a particular angle-angle plot, the remaining five variable torsion angles are rotated to the values which give a minimum energy at this point. The contour maps are overall quite consistent with the experimental distribution of oligonucleotide data. A number of these maps are of particular interest: δ (C5′-C4′-C3′-03′)χ (04′-C1′-N9- C4), where the energetic basis for an approximately linear δ-χ correlation can be seen; ζ (C3′- 03′-P-05′)-δ, in which the experimentally observed linear correlation between ζ and δ in DNA (220° < ζ <280°) is clearly predicted; ζ-ε (C4′-C3′-03′-P), which shows that e increases with decreasing ζ <260°; α (03′-P-05′-C5′)-γ (05′-C5′-C4′-C3′) where a clear linear correlation between these angles is also apparent, consistent with experiment; and several others. For the DNA molecule studied here, the sugar torsion Ô is predicted to be the most flexible, while for the RNA molecule, the greatest amount of flexibility is expected to reside in a and y. Both the DNA and RNA molecules are predicted to be highly polymorphic. Complete energy minimization has been performed on each of the minima found in the energy searches and the results further support this prediction. Possible pathways for B-form to A-form DNA interconversion suggested by the results of this study are discussed. The results of these calculations support use of the new sugar modeling technique and torsion angle correlations in future conformational studies of nucleic acids.     

Conformational analysis of the 3'-5'-cyclic dinucleotide d less than pApA greater than by means of molecular mechanics

The conformational properties of the cyclic dinucleotide d less than pApA greater than were studied by means of molecular mechanics calculations in which a multiconformation analysis was combined with minimum energy calculations. In this approach models of possible conformers are built by varying the torsion angles of the molecule systematically. These models are then subjected to energy minimization; in the present investigation use was made of the AMBER Force field. It followed that the lowest energy conformer has a pseudo-two-fold axis of symmetry. In this conformer the deoxyribose sugars adopt a N-type conformation. The conformation of the sugar-phosphate backbone is determined by the following torsion angles: alpha +, beta t, gamma +, epsilon t and zeta +. The conformation of this ringsystem corresponds to the structure derived earlier by means of NMR spectroscopy and X-ray diffraction. The observation of a preference for N-type sugar conformations in this molecule can be explained by the steric hindrance induced between opposite H3' atoms when one sugar is switched from N- to S-type puckers. The sugars can in principle switch from N- to S-type conformations, but this requires at least the transition of gamma + to gamma -. In this process the molecule obtains an extended shape in which the bases switch from a pseudo-axial to a pseudo-equatorial position. The calculations demonstrate that, apart from the results obtained for the lowest energy conformation, the 180 degrees change in the propagation direction of the phosphate backbone can be achieved by several different combinations of the backbone torsion angles. It appeared that in the low energy conformers five higher order correlations are found. The combination of torsion angles which are involved in changes in the propagation direction of the sugar-phosphate backbone in DNA-hairpin loops and in tRNA, are found in the dataset obtained for cyclic d less than pApA greater than. It turns out, that in the available examples, 180 degrees changes in the backbone direction are localized between two adjacent nucleotides.     

Monte Carlo implementation of supercoiled double-stranded DNA

Metropolis Monte Carlo simulation is used to investigate the elasticity of torsionally stressed double-stranded DNA, in which twist and supercoiling are incorporated as a natural result of base-stacking interaction and backbone bending constrained by hydrogen bonds formed between DNA complementary nucleotide bases. Three evident regimes are found in extension versus torsion and force versus extension plots: a low-force regime in which over- and underwound molecules behave similarly under stretching; an intermediate-force regime in which chirality appears for negatively and positively supercoiled DNA and extension of underwound molecule is insensitive to the supercoiling degree of the polymer; and a large-force regime in which plectonemic DNA is fully converted to extended DNA and supercoiled DNA behaves quite like a torsionless molecule. The striking coincidence between theoretic calculations and recent experimental measurement of torsionally stretched DNA (Strick et al., Science. 271:1835, 1996; Biophys. J. 74:2016, 1998) strongly suggests that the interplay between base-stacking interaction and permanent hydrogen-bond constraint takes an important role in understanding the novel properties of elasticity of supercoiled DNA polymer.     

New coarse-grained DNA model

A new coarse-grained model of the DNA molecule has been proposed, which was elaborated on the basis of its all-atomic model analysis. The model has been shown to rather well reproduce the DNA structure under low and room temperatures. The Young's and torsion moduli calculated using the coarse-grained model are in close agreement with experimental data and the theoretical results of other authors. The model can be used for DNA fragments of several hundreds base pairs for rather long time scales (of the order of micros) and for simulating their interactions with other structures.     

Construction of linear functional expression elements with DNA and RNA hybrid primers: a flexible and fast method for proteomics

Two methods of constructing linear functional expression elements (LFEE) using hybrid DNA and RNA primers in DNA amplification for rapid gene expression are described. In both methods, it is not necessary to have additional transformation or bacterial propagation. The promoter, open reading frame (ORF) and terminator are amplified using Pfu or Taq DNA polymerase. Three elements containing DNA or RNA overhang are covalently ligated by T4 DNA ligase. The recombinant molecule is amplified with element-specific primers. The LFEE can be generated by both methods in a few hours and can be expressed in mammalian cells.     

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.     

New coarse-grained DNA model

A new coarse-grained model of the DNA molecule has been proposed, which was elaborated on the basis of its all-atomic model analysis. The model has been shown to rather well reproduce the DNA structure under low and room temperatures. The Young’s and torsion moduli calculated using the coarse-grained model are in close agreement with experimental data and the theoretical results of other authors. The model can be used for DNA fragments of several hundreds base pairs for rather long time scales (of the order of μs) and for simulating their interactions with other structures.     

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.     

Structure of a DNA intercalation complex as determined by NMR using a paramagnetic probe

Longitudinal relaxation rates of the protons of the 3,8-dimethyl-N-methyl-phenanthrolinium (DMP) cation in solutions containing DNA are strongly affected by the addition of the paramagnetic manganese (II) ions due to the electron-nuclear dipolar interaction in the ternary Mn-DNA-DMP complex. Two possible models for the DMP-DNA intercalation complex are examined and one of them is unequivocally discriminated on the basis of the proton relaxation data. It is concluded that in the intercalation complex the long axis of the DMP molecule is almost perpendicular to the hydrogen bonds of the DNA base-pairs.     

Structure of a DNA intercalation complex as determined by NMR using a paramagnetic probe.

Longitudinal relaxation rates of the protons of the 3,8-dimethyl-N-methyl-phenanthrolinium (DMP) cation in solutions containing DNA are strongly affected by the addition of the paramagnetic manganese (II) ions due to the electron-nuclear dipolar interaction in the ternary Mn-DNA-DMP Complex. Two possible models for the DMP-DNA intercalation complex are examined and one of them is unequivocally discriminated on the basis of the proton relaxation data. It is concluded that in the intercalation complex the long axis of the DMP molecule is almost perpendicular to the hydrogen bonds of the DNA base-pairs.     

2'-deoxyribonolactone lesion in DNA: refined solution structure determined by nuclear magnetic resonance and molecular modeling

The solution conformation of the DNA duplex d(C1G2C3A4C5L6C7A8C9G10C11).d(G12C13G14T15G16T17G18T19G20C21G22 ) containing the 2'-deoxyribonolactone lesion (L6) in the middle of the sequence has been investigated by NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances have been obtained by complete relaxation matrix analysis of the NOESY cross-peak intensities. These distances, along with torsion angles for sugar rings and additional data derived from canonical A- and B-DNA, have been used for structure refinement by restrained molecular dynamics (rMD). Six rMD simulations have been carried out starting from both regular A- and B-DNA forms. The pairwise rms deviations calculated for each refined structure are <1 A, indicating convergence to essentially the same geometry. The accuracy of the rMD structures has been assessed by complete relaxation matrix back-calculation. The average sixth-root residual index (Rx = 0.052 +/- 0.003) indicated that a good fit between experimental and calculated NOESY spectra has been achieved. Detailed analysis revealed a right-handed DNA conformation for the duplex in which both the T17 nucleotide opposite the abasic site and the lactone ring are located inside the helix. No kinking is observed for this molecule, even at the abasic site step. This structure is compared to that of the oligonucleotide with the identical sequence containing the stable tetrahydrofuran abasic site analogue that we reported previously [Coppel, Y., Berthet, N., Coulombeau, C., Coulombeau, Ce., Garcia, J., and Lhomme, J. (1997) Biochemistry 36, 4817-4830].     

DNA binding and recognition by the IIs restriction endonuclease MboII

The type IIs restriction endonuclease MboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca(2+) is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca(2+), possibly a (MboII)(2)-(DNA)(2) complex. Plasmids containing a single GAAGA site were hydrolyzed slowly by MboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme)(2)-(DNA)(2) complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.     

Molecular mechanics modeling of azobenzene-based photoswitches

We present an extension of the generalized amber force field to allow the modeling of azobenzenes by means of classical molecular mechanics. TD-DFT calculations were employed to derive different interaction models for 4-hydroxy-4'-methyl-azobenzene, including the ground (S(0)) and S(1) excited state. For both states, partial charges and the -N = N- torsion potentials were characterized. On this basis, we pave the way to large-scale model simulations involving azobenzene molecular switches. Using the example of an isolated molecule, the mechanics of cyclic switching processes are demonstrated by classical molecular dynamics simulations.