ABSTRACT
The topology of cellular DNA is carefully controlled by enzymes called topoisomerases. By using single-molecule techniques, we monitored the activity of two type IA topoisomerases in real time under conditions in which single relaxation events were detected. The strict one-at-a-time removal of supercoils we observed establishes that these enzymes use an enzyme-bridged strand-passage mechanism that is well suited to their physiological roles and demonstrates a mechanistic unity with type II topoisomerases.
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , Biophysical Phenomena , Biophysics , DNA, Superhelical/chemistry , DNA, Superhelical/metabolism , Escherichia coli/enzymology , Kinetics , Models, Biological , Nucleic Acid Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thermotoga maritima/enzymology , Thermotoga maritima/geneticsABSTRACT
Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.
Subject(s)
Computer Simulation , DNA Topoisomerases, Type II/metabolism , DNA, Circular/metabolism , Models, Molecular , DNA Topoisomerases, Type II/ultrastructure , DNA, Circular/chemistry , DNA, Circular/ultrastructure , Microscopy, ElectronABSTRACT
Xenopus 13S condensin converts interphase chromatin into mitotic-like chromosomes, and, in the presence of ATP and a type I topoisomerase, introduces (+) supercoils into DNA. The specific production of (+) trefoil knots in the presence of condensin and a type II topoisomerase shows that condensin reconfigures DNA by introducing an ordered, global, (+) writhe. Knotting required ATP hydrolysis and cell cycle-specific phosphorylation of condensin. Condensin bound preferentially to (+) supercoiled DNA in the presence of ATP but not in its absence. Our results suggest a mechanism for the compaction of chromatin by condensin during mitosis.
Subject(s)
Adenosine Triphosphatases/metabolism , Chromosomes/enzymology , DNA, Superhelical/chemistry , DNA-Binding Proteins/metabolism , Nucleic Acid Conformation , Adenosine Triphosphate/metabolism , Animals , Cell Cycle/physiology , Chromosomes/chemistry , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type II/metabolism , DNA, Superhelical/metabolism , DNA, Superhelical/ultrastructure , Hydrolysis , Kinetics , Microscopy, Electron , Mitosis/physiology , Multiprotein Complexes , Oocytes/physiology , XenopusABSTRACT
Type II DNA topoisomerases catalyze the interconversion of DNA topoisomers by transporting one DNA segment through another. The steady-state fraction of knotted or catenated DNA molecules produced by prokaryotic and eukaryotic type II topoisomerases was found to be as much as 80 times lower than at thermodynamic equilibrium. These enzymes also yielded a tighter distribution of linking number topoisomers than at equilibrium. Thus, topoisomerases do not merely catalyze passage of randomly juxtaposed DNA segments but control a global property of DNA, its topology. The results imply that type II topoisomerases use the energy of adenosine triphosphate hydrolysis to preferentially remove the topological links that provide barriers to DNA segregation.
Subject(s)
DNA Topoisomerases, Type II/metabolism , DNA, Circular/metabolism , DNA, Superhelical/metabolism , Nucleic Acid Conformation , Adenosine Triphosphate/metabolism , Animals , DNA Topoisomerase IV , DNA Topoisomerases, Type II/chemistry , DNA, Circular/chemistry , DNA, Superhelical/chemistry , DNA, Viral/chemistry , DNA, Viral/metabolism , Drosophila melanogaster/enzymology , Escherichia coli/enzymology , Humans , Hydrolysis , Models, Chemical , Myoviridae/enzymology , Saccharomyces cerevisiae/enzymology , ThermodynamicsABSTRACT
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.
Subject(s)
DNA, Superhelical/chemistry , Magnesium Chloride/pharmacology , Nucleic Acid Conformation/drug effects , Sodium Chloride/pharmacology , Blotting, Southern , Electrophoresis, Gel, Two-Dimensional , Static ElectricityABSTRACT
We studied the conformations of supercoiled DNA as a function of superhelicity and ionic conditions by determining its sedimentation coefficient both experimentally and by calculation. To cancel out unknown parameters from both calculations and experiments, we determined the ratio of the sedimentation coefficient, s, to that of open circular DNA, s(oc). Calculations of the sedimentation coefficient were based on direct solution of the Burgers-Oseen problem for an equilibrium set of DNA conformations generated for each condition by the Metropolis Monte Carlo procedure. There were no adjustable parameters in the Monte Carlo simulations because all three parameters of the DNA model used, bending and torsional elasticity of DNA and DNA effective diameter specifying electrostatic interactions, were known from independent data. The good agreement between measured and calculated values of s/s(oc) allowed us to interpret the sedimentation results in terms of DNA conformations, with particular emphasis on the marked effect of ionic conditions. As NaCl concentration decreases, s/s(oc) increases because the superhelix becomes less regular and more compact. In the presence of just 10 mM MgCl(2), supercoiled DNA adopts essentially the same set of conformations as in moderate to high concentrations of NaCl. Our simulations showed that s is a strong function of the superhelix branching frequency. At near physiological ionic conditions, there are about four branches in the 7 kb DNA molecule used in this work. We found no indication of superhelix collapse in any ionic conditions even remotely approaching physiological ones. For all ionic conditions studied, we conclude that the electrostatic interaction of DNA segments specified by the DNA effective diameter is the primary determinant of supercoiled DNA conformations.
Subject(s)
DNA, Superhelical/chemistry , Magnesium Chloride/pharmacology , Nucleic Acid Conformation , Sodium Chloride/pharmacology , Centrifugation, Density Gradient , Chemical Phenomena , Chemistry, Physical , Computer Simulation , Models, Molecular , Monte Carlo Method , Particle Size , Plasmids , Spermidine/pharmacologyABSTRACT
We studied the equilibrium formation of DNA catenanes to assess the conformational properties of supercoiled DNA as a function of ionic conditions and supercoiling density. Catenanes were formed by cyclizing linear DNA with long cohesive ends in the presence of supercoiled molecules. The efficiency of the catenation depends on the distance between opposing segments of DNA in the interwound superhelix. The fraction of cyclizing molecules that becomes topologically linked with the supercoiled DNA is the product of the concentration of the supercoiled DNA and a proportionality constant, B, that depends on conformations of supercoiled DNA. In parallel with these experimental studies, we calculated the values of B using Monte Carlo simulations of the equilibrium distribution of DNA conformations. There were no adjustable parameters in the calculations because all three parameters of the DNA model, bending and torsional elasticity of DNA and DNA effective diameter, specifying intersegment interactions, were known from independent studies. We found very good agreement between measured and simulated values of B for all the ionic conditions and DNA superhelix densities studied; the discrepancy was less than a factor of 2 over the 200-fold variation in B. The value of B decreases nearly exponentially with increasing superhelicity, this dependence being especially strong at low salt concentration. The dependence of B on the concentration of NaCl, MgCl(2), and spermidine can be described with good accuracy in terms of changes of the DNA effective diameter. We found no indication of superhelix collapse under any ionic conditions studied. We discuss, in light of these results, the biological importance of the effect of DNA supercoiling on the unlinking of the products of DNA replication.
Subject(s)
DNA, Superhelical/chemistry , Magnesium Chloride/pharmacology , Nucleic Acid Conformation , Sodium Chloride/pharmacology , Computer Simulation , DNA, Circular/chemistry , Electrophoresis, Agar Gel , Models, Molecular , Monte Carlo Method , Plasmids , Spermidine/pharmacologyABSTRACT
During the random cyclization of long polymer chains, knots of different types are formed. We investigated experimentally the distribution of knot types produced by random cyclization of phage P4 DNA via its long cohesive ends. The simplest knots (trefoils) predominated, but more complex knots were also detected. The fraction of knots greatly diminished with decreasing solution Na+ concentration. By comparing these experimental results with computer simulations of knotting probability, we calculated the effective diameter of the DNA double helix. This important excluded-volume parameter is a measure of the electrostatic repulsion between segments of DNA molecules. The calculated effective DNA diameter is a sensitive function of electrolyte concentration and is several times larger than the geometric diameter in solutions of low monovalent cation concentration.
Subject(s)
Coliphages/metabolism , DNA, Viral/chemistry , Nucleic Acid Conformation , Base Sequence , DNA, Viral/isolation & purification , Electrophoresis, Agar Gel , Escherichia coli/metabolism , Models, Structural , Molecular Sequence Data , Monte Carlo Method , Oligodeoxyribonucleotides , ProbabilityABSTRACT
Different methods of determination of effective diameter of DNA double helix under different ionic conditions in solution are reviewed. This value is a characteristic of excluded volume effects in DNA and allows for electrostatic repulsion between segments of the molecule. The values of the effective diameter found by completely different methods are in excellent agreement. This allows to conclude that in wide range of ionic and DNA concentrations a model of unpermeable cylinders provides adequate description of DNA conformational properties. The effective double helix diameter grows rapidly with decreasing salt concentration and can be several times higher than the geometric one. Even in solutions of physiological ionic strength corresponding to 200 mM NaCl the value of the effective diameter is 5 nm. Such a sharp dependence of DNA effective diameter on salt concentration should be taken into account when analyzing conformational properties of superhelical DNA.
