Interaction of diverse voltage sensor homologs with lipid bilayers revealed by self-assembly simulations

Homologous pairing and braiding (supercoiling) have crucial effects on genome organization, maintenance, and evolution. Generally, the pairing and braiding processes are discussed in different contexts, independently of each other. However, analysis of electrostatic interactions between DNA double helices suggests that in some situations these processes may be related. Here we present a theory of DNA braiding that accounts for the elastic energy of DNA double helices as well as for the chiral nature of the discrete helical patterns of DNA charges. This theory shows that DNA braiding may be affected, stabilized, or even driven by chiral electrostatic interactions. For example, electrostatically driven braiding may explain the surprising recent observation of stable pairing of homologous double-stranded DNA in solutions containing only monovalent salt. Electrostatic stabilization of left-handed braids may stand behind the chiral selectivity of type II topoisomerases and positive plasmid supercoiling in hyperthermophilic bacteria and archea.     

Tryptic fragments of the Escherichia coli DNA gyrase A protein

Treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two large fragments which are stable to further digestion. The molecular masses of these fragments are 64 and 33 kDa, and they are shown to be derived from the N terminus and the C terminus of the A protein, respectively. These fragments could represent structural and/or functional domains within the A subunit of DNA gyrase. The trypsin-cleaved A protein (A'), in combination with the B subunit of gyrase, can support ATP-dependent supercoiling of relaxed DNA and other reactions of DNA gyrase. The isolated 64-kDa fragment will also catalyse DNA supercoiling in the presence of the B protein, but the 33-kDa fragment shows no enzymic activities. We conclude that the N-terminal 64-kDa fragment represents the DNA breakage/reunion domain of the A protein, while the 33-kDa fragment may contribute to the stability of the gyrase-DNA complex.     

A role of basic residues and the putative intercalating phenylalanine of the HMG-1 box B in DNA supercoiling and binding to four-way DNA junctions

HMG (high mobility group) 1 is a chromosomal protein with two homologous DNA-binding domains, the HMG boxes A and B. HMG-1, like its individual HMG boxes, can recognize structural distortion of DNA, such as four-way DNA junctions (4WJs), that are very likely to have features common to their natural, yet unknown, cellular binding targets. HMG-1 can also bend/loop DNA and introduce negative supercoils in the presence of topoisomerase I in topologically closed DNAs. Results of our gel shift assays demonstrate that mutation of Arg(97) within the extended N-terminal strand of the B domain significantly (>50-fold) decreases affinity of the HMG box for 4WJs and alters the mode of binding without changing the structural specificity for 4WJs. Several basic amino acids of the extended N-terminal strand (Lys(96)/Arg(97)) and helix I (Arg(110)/Lys(114)) of the B domain participate in DNA binding and supercoiling. The putative intercalating hydrophobic Phe(103) of helix I is important for DNA supercoiling but dispensable for binding to supercoiled DNA and 4WJs. We conclude that the B domain of HMG-1 can tolerate substitutions of a number of amino acid residues without abolishing the structure-specific recognition of 4WJs, whereas mutations of most of these residues severely impair the topoisomerase I-mediated DNA supercoiling and change the sign of supercoiling from negative to positive.     

Topological effects of the TATA box binding protein on minicircle DNA and a possible thermodynamic linkage to chromatin remodeling

DNA ring closure experiments on short restriction fragments ( approximately 160 bp) bound by the TATA box binding protein (TBP) have demonstrated the formation of negative topoisomers, consistent with crystallographically observed TBP-induced DNA untwisting but in contrast to most previous results on topological effects in plasmid DNA. The difference may be due to the high free energy cost of substantial writhe in minicircles. A speculative mechanism for the loss of TBP-induced writhe suggests that TBP is capable of inducing DeltaTw between 0 and -0.3 in minicircles, via loss of out-of-plane bending upon retraction of intercalating Phe stirrups, and that TBP can thus act as a "supercoil shock absorber". The proposed biological relevance of these observations is that they may model the behavior of DNA in constrained chromatin environments. Irrespective of the detailed mechanism of TBP-induced supercoiling, its existence suggests that chromatin remodeling and enhanced TBP binding are thermodynamically linked. Remodeling ATPases or histone acetylases release some of the negative supercoiling previously restrained by the nucleosome. When TBP takes up the supercoiling, its binding should be enhanced transiently until the unrestrained supercoiling is removed by diffusion or topoisomerases. The effect is predicted to be independent of local remodeling-induced changes in TATA box accessibility.     

Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling.

ATP alpha S (Rp) has been shown to support the supercoiling of plasmid pBR322 catalyzed by Escherichia coli DNA gyrase at comparable rates to the natural substrate ATP and is able to promote the introduction of one more superhelical turn than ATP. The difference in free energy change between consecutive rounds of supercoiling in gyrase-mediated reactions is calculated to be 2.6 kJ mol-1. The difference in free energy of hydrolysis of ATP and ATP alpha S (Rp) has been determined from the difference in the equilibrium constants for the phosphorylation of arginine established by arginine kinase. This equilibrium constant has been found to be displaced by a factor of about 1.5, corresponding to a greater free energy of hydrolysis of ATP alpha S (Rp) compared to ATP of approximately 1 kJ mol-1. This difference in free energy can be tentatively ascribed to a relative destabilization of the MgATP alpha S (Rp) complex with respect to MgATP. Assuming that the stoichiometry of the coupled reactions requires two ATPs hydrolyzed per round of supercoiling, ATP alpha S (Rp) should be capable of providing an additional ca. 2 kJ mol-1 of free energy for DNA supercoiling, which is in good agreement with estimates for the additional free energy required to achieve a further round of supercoiling. These results provide direct evidence to support the proposal that the extent of DNA supercoiling by DNA gyrase is limited by the free energy of hydrolysis of the nucleotide.     

DNA gyrase can supercoil DNA circles as small as 174 base pairs.

DNA gyrase introduces negative supercoils into closed-circular DNA using the free energy of ATP hydrolysis. Consideration of steric and thermodynamic aspects of the supercoiling reaction indicates that there should be a lower limit to the size of DNA circle which can be supercoiled by gyrase. We have investigated the supercoiling reaction of circles from 116-427 base pairs (bp) in size and have determined that gyrase can supercoil certain relaxed isomers of circles as small as 174 bp, dependent on the final superhelix density of the supercoiled product. Furthermore, this limiting superhelical density (-0.11) is the same as that determined for the supercoiling of plasmid pBR322. We also find that although circles in the range 116-152 bp cannot be supercoiled, they can nevertheless be relaxed by gyrase when positively supercoiled. These data suggest that the conformational changes associated with the supercoiling reaction can be carried out by gyrase in a circle as small as 116 bp. We discuss these results with respect to the thermodynamics of DNA supercoiling and steric aspects of the gyrase mechanism.     

The effect of ionic conditions on DNA helical repeat, effective diameter and free energy of supercoiling.

We determined the free energy of DNA supercoiling as a function of the concentration of magnesium and sodium chloride in solution by measuring the variance of the equilibrium distribution of DNA linking number,<(DeltaLk)2>. We found that the free energy of supercoiling changed >1.5-fold over the range of ionic conditions studied. Comparison of the experimental results with those of computer simulations showed that the ionic condition dependence of<(DeltaLk)2>is due mostly to the change in DNA effective diameter, d, a parameter characterizing the electrostatic interaction of DNA segments. To make this comparison we determined values of d under all ionic conditions studied by measuring the probability of knot formation during random cyclization of linear DNA molecules. From the topoisomer distributions we could also determine the changes in DNA helical repeat, gamma, in mixed NaCl/MgCl2 solutions. Both gamma and d exhibited a complex pattern of changes with changing ionic conditions, which can be described in terms of competition between magnesium and sodium ions for binding to DNA.     

The C-terminal domain of the Escherichia coli DNA gyrase A subunit is a DNA-binding protein.

We have constructed a clone which over-produces a 33 kDa protein representing the C-terminal portion of the Escherichia coli DNA gyrase A subunit. This protein has no enzymic activity of its own, but will form a complex with a 64 kDa protein (representing the N-terminal part of the A subunit) and the gyrase B subunit, that will efficiently catalyse DNA supercoiling. We show that the 33 kDa protein can bind to DNA on its own in a manner which induces positive supercoiling of the DNA. We propose that the 33 kDa protein represents a domain of the gyrase A subunit which is involved in the wrapping of DNA around DNA gyrase.     

Analysis of the eukaryotic topoisomerase II DNA gate: a single-molecule FRET and structural perspective

Type II DNA topoisomerases (topos) are essential and ubiquitous enzymes that perform important intracellular roles in chromosome condensation and segregation, and in regulating DNA supercoiling. Eukaryotic topo II, a type II topoisomerase, is a homodimeric enzyme that solves topological entanglement problems by using the energy from ATP hydrolysis to pass one segment of DNA through another by way of a reversible, enzyme-bridged double-stranded break. This DNA break is linked to the protein by a phosphodiester bond between the active site tyrosine of each subunit and backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this enzymatic cleavage/religation of the backbone. This reversible DNA cleavage reaction is the target of a number of anticancer drugs, which can elicit DNA damage by affecting the cleavage/religation equilibrium. Because of its clinical importance, many studies have sought to determine the manner in which topo II interacts with DNA. Here we highlight recent single-molecule fluorescence resonance energy transfer and crystallographic studies that have provided new insight into the dynamics and structure of the topo II DNA gate.     

Single-molecule studies on the mechanical interplay between DNA supercoiling and H-NS DNA architectural properties

The Escherichia coli H-NS protein is a major nucleoid-associated protein that is involved in chromosomal DNA packaging and gene regulatory functions. These biological processes are intimately related to the DNA supercoiling state and thus suggest a direct relationship between H-NS binding and DNA supercoiling. Here, we show that H-NS, which has two distinct DNA-binding modes, is able to differentially regulate DNA supercoiling. H-NS DNA-stiffening mode caused by nucleoprotein filament formation is able to suppress DNA plectoneme formation during DNA supercoiling. In contrast, when H-NS is in its DNA-bridging mode, it is able to promote DNA plectoneme formation during DNA supercoiling. In addition, the DNA-bridging mode is able to block twists diffusion thus trapping DNA in supercoiled domains. Overall, this work reveals the mechanical interplay between H-NS and DNA supercoiling which provides insights to H-NS organization of chromosomal DNA based on its two distinct DNA architectural properties.     

The influence of salt on the structure and energetics of supercoiled DNA.

We present a detailed computational study of the influence of salt on the configurations, energies, and dynamics of supercoiled DNA. A potential function that includes both elastic and electrostatic energy components is employed. Specifically, the electrostatic term, with salt-dependent coefficients, is modeled after Stigter's pioneering work on the effective diameter of DNA as a function of salt concentration. Because an effective charge per unit length is used, the electrostatic formulation does not require explicit modeling of phosphates and can be used to study long DNAs at any desired resolution of charge. With explicit consideration of the electrostatic energy, an elastic bending constant corresponding to the nonelectrostatic part of the bending contribution to the persistence length is used. We show, for a series of salt concentrations ranging from 0.005 to 1.0 M sodium, how configurations and energies of supercoiled DNA (1000 and 3000 base pairs) change dramatically with the simulated salt environment. At high salt, the DNA adopts highly compact and bent interwound states, with the bending energy dominating over the other components, and the electrostatic energy playing a minor role in comparison to the bending and twisting terms. At low salt, the DNA supercoils are much more open and loosely interwound, and the electrostatic components are dominant. Over the range of three decades of salt examined, the electrostatic energy changes by a factor of 10. The buckling transition between the circle and figure-8 is highly sensitive to salt concentration: this transition is delayed as salt concentration decreases, with a particularly sharp increase below 0.1 M. For example, for a bending-to-twisting force constant ratio of A/C = 1.5, the linking number difference (delta LK) corresponding to equal energies for the circle and figure-8 increases from 2.1 to 3.25 as salt decreases from 1.0 to 0.005 M. We also present in detail a family of three-lobed supercoiled DNA configurations that are predicted by elasticity theory to be stable at low delta Lk. To our knowledge, such three-dimensional structures have not been previously presented in connection with DNA supercoiling. These branched forms have a higher bending energy than the corresponding interwound configurations at the same delta Lk but, especially at low salt, this bending energy difference is relatively small in comparison with the total energy, which is dominated by the electrostatic contributions. Significantly, the electrostatic energies of the three-lobed and (straight) interwound forms are comparable at each salt environment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct observation of positive supercoils introduced by reverse gyrase through atomic force microscopy

Reverse gyrase is a hyperthermophilic enzyme that can introduce positive supercoiling in substrate DNA. It is showed in our studies that positive DNA supercoils were induced in both pBR322 vector and an artificially synthesized mini-plasmid DNA by reverse gyrase. The left-handed structures adopted by positively supercoiled DNA molecules could be identified from their right-handed topoisomers through atomic force microscopic examination. Additional structural comparisons revealed that positively supercoiled DNA molecule AFM images exhibited increased contour lengths. Moreover, enzymatic assays showed that the positively supercoiled DNA could not be cleaved by T7 endonuclease. Together, this suggests that the overwound structure of positive supercoils could prevent genomic duplex DNA from randomly forming single-stranded DNA regions and intra-stranded secondary structures.