DNA supercoiling by DNA gyrase

Using purified DNA gyrase to supercoil circular plasmid pBR322 DNA, we examined how the linking number attained at the steady state (‘static head’) varies with the concentrations of ATP and ADP, both in the absence and presence of spermidine. In the absence of spermidine at total adenine nucleotide concentrations between 0.35 and 1.4 mM, the static-head linking number was independent of the sum concentration of ATP and ADP, but depended strongly on the ratio of their concentrations. We established that the same linking number was attained independent of the direction from which the steady state was approached. The decrease in linking number at static head is more extensive when spermidine is present in the incubation, but remains a function of the [ATP]-to-[ADP] ratio. These results are discussed in terms of various kinetic schemes for DNA gyrase. We present one kinetic scheme that accounts for the experimental observations. According to this scheme our experimental results imply that there is significant slip in DNA gyrase when spermidine is absent. It is possible that spermidine acts through adjustment of the degree of coupling of DNA gyrase.     

DNA supercoiling inhibits DNA knotting

Despite the fact that in living cells DNA molecules are long and highly crowded, they are rarely knotted. DNA knotting interferes with the normal functioning of the DNA and, therefore, molecular mechanisms evolved that maintain the knotting and catenation level below that which would be achieved if the DNA segments could pass randomly through each other. Biochemical experiments with torsionally relaxed DNA demonstrated earlier that type II DNA topoisomerases that permit inter- and intramolecular passages between segments of DNA molecules use the energy of ATP hydrolysis to select passages that lead to unknotting rather than to the formation of knots. Using numerical simulations, we identify here another mechanism by which topoisomerases can keep the knotting level low. We observe that DNA supercoiling, such as found in bacterial cells, creates a situation where intramolecular passages leading to knotting are opposed by the free-energy change connected to transitions from unknotted to knotted circular DNA molecules.     

DNA supercoiling in vivo

DNA topoisomerase mutants of Escherichia coli and Saccharomyces cerevisiae were used to study the topological state of intracellular DNA. In E. coli, it is shown that switching off the gene topA encoding DNA topoisomerase I leads to an increase in the degree of negative supercoiling of intracellular DNA and inhibition of the growth of the cells: a d(pCpG)16.d(pCpG)16 sequence on a plasmid is also shown to flip from a right-handed B-helical structure to a left-handed Z-helical structure in vivo when topA is switched off. In S. cerevisiae, the topological state of intracellular DNA is little affected by the cellular levels of the topoisomerases.     

DNA supercoiling and relaxation by ATP-dependent DNA topoisomerases.

Bacterial DNA gyrase and the eukaryotic type II DNA topoisomerases are ATPases that catalyse the introduction or removal of DNA supercoils and the formation and resolution of DNA knots and catenanes. Gyrase is unique in using ATP to drive the energetically unfavourable negative supercoiling of DNA, an example of mechanochemical coupling: in contrast, eukaryotic topoisomerase II relaxes DNA in an ATP-requiring reaction. In each case, the enzyme-DNA complex acts as a 'gate' mediating the passage of a DNA segment through a transient enzyme-bridged double-strand DNA break. We are using a variety of genetic and enzymic approaches to probe the nature of these complexes and their mechanism of action. Recent studies will be described focusing on the role of DNA wrapping on the A2B2 gyrase complex, subunit activities uncovered by using ATP analogues and the coumarin and quinolone inhibitors, and the identification and functions of discrete subunit domains. Homology between gyrase subunits and the A2 homodimer of eukaryotic topo II suggests functional conservation between these proteins. The role of ATP hydrolysis by these topoisomerases will be discussed in regard to other energy coupling systems.     

Toroidal elastic supercoiling of DNA

Covalently closed circular DNA can exist in different configurations known as circular, toroidal, and interwound. Changes among these forms can be made in several ways, including the insertion of dye molecules between adjacent base pairs, which tends to untwist the double-helical structure. The aim of this paper is to discuss these configurations, and the changes among them, in the context of classical elastomechanics. The concepts of twisting, linkage and writhing are explained. Simple experiments on a twisted linear-elastic rod are described, and it is shown that although the circular and interwound forms may be modeled in this way, the toroidal form does not occur, being mechanically unstable. Theoretical energy calculations by Levitt on bent and twisted DNA show that DNA exhibits a particular kind of nonlinear elasticity in which there is an unusual coupling between bending and twisting. The aim of the paper is to show qualitatively that this special kind of elasticity can stabilize the toroidal form of closed circular DNA.     

Control of bacterial DNA supercoiling

Two DNA topoisomerases control the level of negative supercoiling in bacterial cells. DNA gyrase introduces supercoils, and DNA topoisomerase I prevents supercoiling from reaching unacceptably high levels. Perturbations of supercoiling are corrected by the substrate preferences of these topoisomerases with respect to DNA topology and by changes in expression of the genes encoding the enzymes. However, supercoiling changes when the growth environment is altered in ways that also affect cellular energetics. The ratio of [ATP] to [ADP], to which gyrase is sensitive, may be involved in the response of supercoiling to growth conditions. Inside cells, supercoiling is partitioned into two components, superhelical tension and restrained supercoils. Shifts in superhelical tension elicited by nicking or by salt shock do not rapidly change the level of restrained supercoiling. However, a steady-state change in supercoiling caused by mutation of topA does alter both tension and restrained supercoils. This communication between the two compartments may play a role in the control of supercoiling.     

Computer simulation of DNA supercoiling

We treat supercoiled DNA within a wormlike model with excluded volume. A modified Monte Carlo approach has been used, which allowed computer statistical-mechanical simulations of moderately and highly supercoiled DNA molecules. Even highly supercoiled molecules do not have a regular shape, though with an increase in writhing the chains look more and more like branched interwound helixes. The averaged writhing (Wr) approximately 0.7 delta Lk. The superhelical free energy F is calculated as a function of the linking number. Lk. The calculations have shown that the generally accepted quadratic dependence of F on Lk is valid for a variety of conditions, though it is by no means universal. Significant deviations from the quadratic dependence are expected at high superhelical density under ionic conditions where the effective diameter of DNA is small. The results are compared with the available experimental data.     

DNA supercoiling in gyrase mutants.

Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.     

DNA supercoiling by core histone fractions and protamine

The nuclear extract isolated from late Drosophila melanogaster embryos has Mg2+-dependent DNA topoisomerase 1 and nuclease activities. The extract facilitates the closed circular duplex DNA supercoiling in the presence of calf histone fractions H2A, H2B, H3 and H4, and fish protamine but not HI histone.     

Nucleotide- and stoichiometry-dependent DNA supercoiling by reverse gyrase

Reverse gyrase is a unique type IA topoisomerase that can introduce positive supercoils into DNA. We have investigated some of the biochemical properties of Archaeoglobus fulgidus reverse gyrase. It can mediate three distinct supercoiling reactions depending on the adenine nucleotide cofactor that is present in the reaction. Besides the ATP-driven positive supercoiling reaction, the enzyme can introduce negative supercoils with a nonhydrolyzable analog, adenylyl imidodiphosphate. In the presence of ADP the plasmid DNA is relaxed almost completely, leaving a very low level of positive supercoiling. Surprisingly, the final supercoiling extent for all three distinct reactions depends on the stoichiometry of enzyme to DNA. This dependence is not due to the difference of reaction rate, suggesting that the amount of enzyme bound to DNA is an important determinant for the final supercoiling state of the reaction product. Reverse gyrase also displays exquisite sensitivity toward temperature. Raising the reaction temperatures from 80 to 85 degrees C, both of which are within the optimal growth temperature of A. fulgidus, greatly increases enzyme activity for all the supercoiling reactions. For the reaction with AMPPNP, the product is a hypernegatively supercoiled DNA. This dramatic enhancement of the reverse gyrase activity is also correlated with the appearance of DNA in a pre-melting state at 85 degrees C, likely due to the presence of extensively unwound regions in the plasmid. The possible mechanistic insights from these findings will be presented here.