Knowledge of the elastic properties of DNA is required to understand the structural dynamics of cellular processes such as replication and transcription. Measurements of force and extension on single molecules of DNA have allowed direct determination of the molecule's mechanical properties, provided rigorous tests of theories of polymer elasticity, revealed unforeseen structural transitions induced by mechanical stresses, and established an experimental and conceptual framework for mechanical assays of enzymes that act on DNA. However, a complete description of DNA mechanics must also consider the effects of torque, a quantity that has hitherto not been directly measured in micromanipulation experiments. We have measured torque as a function of twist for stretched DNA--torsional strain in over- or underwound molecules was used to power the rotation of submicrometre beads serving as calibrated loads. Here we report tests of the linearity of DNA's twist elasticity, direct measurements of the torsional modulus (finding a value approximately 40% higher than generally accepted), characterization of torque-induced structural transitions, and the establishment of a framework for future assays of torque and twist generation by DNA-dependent enzymes. We also show that cooperative structural transitions in DNA can be exploited to construct constant-torque wind-up motors and force-torque converters.

The ATP-utilizing chromatin assembly and remodelling factor (ACF) functions to generate regularly spaced nucleosomes, which are required for heritable gene silencing. The mechanism by which ACF mobilizes nucleosomes remains poorly understood. Here we report a single-molecule FRET study that monitors the remodelling of individual nucleosomes by ACF in real time, revealing previously unknown remodelling intermediates and dynamics. In the presence of ACF and ATP, the nucleosomes exhibit gradual translocation along DNA interrupted by well-defined kinetic pauses that occurred after approximately 7 or 3 – 4 base pairs of translocation. The binding of ACF, translocation of DNA, and exiting of translocation pauses are all ATP-dependent, revealing three distinct functional roles of ATP during remodelling. At equilibrium, a continuously bound ACF complex can move the nucleosome back-and-forth many times before dissociation, indicating that ACF is a highly processive and bidirectional nucleosome translocase.

Escherichia coli topoisomerase (Topo) IV is an essential type II Topo that removes DNA entanglements created during DNA replication. Topo IV relaxes (؉) supercoils much faster than (؊) supercoils, promoting replication while sparing the essential (؊) supercoils. Here, we investigate the mechanism underlying this chiral preference. Using DNA binding assays and a single-molecule DNA braiding system, we show that Topo IV recognizes the chiral crossings imposed by the left-handed superhelix of a (؉) supercoiled DNA, rather than global topology, twist deformation, or local writhe. Monte Carlo simulations of braid, supercoil, and catenane configurations demonstrate how a preference for a single-crossing geometry during strand passage can allow Topo IV to perform its physiological functions. Single-enzyme braid relaxation experiments also provide a direct measure of the processivity of the enzyme and offer insight into its mechanochemical cycle.T he interwound structure of duplex DNA demands that the two DNA strands be unlinked from each other during semiconservative DNA replication in vivo. Unlinking is carried out primarily by the cooperation of helicases and topoisomerases (Topos). DNA helicases unwind the parental double helix, which generates (ϩ) supercoils or precatenanes (Fig. 1A) (1). Most of these topological entanglements are removed by Topos during replication, but those that remain form catenanes of circular genomes and braids of linear chromosomes. Both of these structures also are unlinked by Topos.Topos are divided into two classes depending on whether one (type I) or both (type II) strands of DNA are broken during topoisomerization (2). With type II enzymes, one DNA segment, called the transfer or T-segment, is passed through the gap of another DNA segment, designated the gate or G-segment (3). Type II Topos initially bind to the G-segment and subsequently capture the T-segment in an ATP-dependent manner. Next, the G-segment is transiently cleaved, to allow the passage of the T-segment. The free energy of ATP hydrolysis allows type II Topos to work energetically uphill to suppress DNA entanglements favored by the high concentrations of DNA found in vivo (4).Bacteria possess two type II Topos, DNA gyrase and Topo IV. Gyrase has the unique ability among all Topos to introduce (Ϫ) supercoils into DNA. A steady state of (Ϫ) supercoiling in the chromosome is required for DNA compaction and for metabolic processes that melt DNA (5, 6). The (Ϫ) supercoiling also cancels the (ϩ) supercoiling introduced by DNA replication. Topo IV ensures proper segregation of daughter chromosomes at the end of replication by removing any remaining (ϩ) intertwinings (1, 7). Topo IV also can relax supercoils generated during transcription and is capable of replacing gyrase during replication elongation in vitro and partially in vivo (8, 9).Topo IV relaxes (ϩ) supercoiled DNA far more efficiently than (Ϫ) supercoiled DNA (10), contributing to elongation of the replication fork without simultaneously removing the (Ϫ) supercoils ess...