Minute negative superhelicity is sufficient to induce the B-Z transition in the presence of low tension

Abstract

Left-handed Z-DNA has fascinated biological scientists for decades by its extraordinary structure and potential involvement in biological phenomena. Despite its instability relative to B-DNA, Z-DNA is stabilized in vivo by negative supercoiling. A detailed understanding of Z-DNA formation is, however, still lacking. In this study, we have examined the B-Z transition in a short guanine/cytosine (GC) repeat in the presence of controlled tension and superhelicity via a hybrid technique of single-molecule FRET and magnetic tweezers. The hybrid scheme enabled us to identify the states of the specific GC region under mechanical control and trace conformational changes synchronously at local and global scales. Intriguingly, minute negative superhelicity can facilitate the B-Z transition at low tension, indicating that tension, as well as torsion, plays a pivotal role in the transition. Dynamic interconversions between the states at elevated temperatures yielded thermodynamic and kinetic constants of the transition. Our single-molecule studies shed light on the understanding of Z-DNA formation by highlighting the highly cooperative and dynamic nature of the B-Z transition.NA is capable of adopting various conformations in addition to the conventional right-handed B-DNA double helix (1, 2). One of the most dramatic examples is Z-DNA, which is a lefthanded helical form with a zigzag backbone (3, 4). Since its discovery, Z-DNA has drawn considerable attention from a broad range of research areas, including physical biochemistry, structural biology, and molecular biology, because of its intriguing and unusual structural features (3). The bases in Z-DNA alternate in syn-and anti-conformations while all the bases in B-DNA adopt the anti-conformation. Because the syn conformation is more stable for purines (Pu) than for pyrimidines (Py), alternating Pu and Py sequences readily adopt the Z-DNA conformation. Another striking feature of Z-DNA is the overturning of base pairs, posing a steric dilemma known as the chain sense paradox (3, 5).Although Z-DNA is less stable than B-DNA at physiological ionic conditions, Z-DNA exists stably under certain conditions such as high ionic strength or negative supercoiling (4, 6-8).In the presence of a high-salt solution, the electrostatic repulsion between the phosphate backbones, which are closer in Z-DNA than in B-DNA, is reduced, stabilizing the Z-DNA conformation. Negative supercoiling induces the Z-DNA conformation even in physiological salt conditions because formation of Z-DNA significantly relieves torsional stress. Z-DNA has also been shown to exist stably in vivo in the presence of physiological negative supercoiling (9, 10).Although there were first doubts as to whether Z-DNA plays any biological role, considerable experimental evidence for biological functions of Z-DNA has accumulated over the past two decades (11). In addition to the discovery of antibodies and proteins that bind selectively to Z-DNA (12, 13), Z-DNA formation has been strongly correlated with transcriptional acti...

Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA

“…High salt conditions and negative torque induce Z-DNA formation in GC tracts (9,29,30). To investigate Z-DNA formation, we used SOIs containing either a 50 bp or a 22 bp GC repeat.…”

Programmed folding of DNA origami structures through single-molecule force control

“…1c). Magnetic tweezers can manipulate single DNA molecules with pico-newton force while probing their end-to-end distances with a nanometre resolution [17][18][19][20]26,[31][32][33][34][35][36] . In addition, different from other methods for single-molecule force spectroscopy, magnetic tweezers inherently permits multiplexed manipulation, which allows us to fold DNA nanostructures in a highly parallel manner in one experiment (see below).…”

Helicase Stepping Investigated with One-Nucleotide Resolution Fluorescence Resonance Energy Transfer

“…If a tension is exerted on the overhangs to suppress the fluctuations, the distance between the two fluorophores can be both stabilized and increased and the resolution of smFRET would thus be improved. The idea has led to the combination of optical or magnetic tweezers with smFRET in a few singemolecule studies [15][16][17][18][19]. These experiments, however, are even more difficult to perform because the fluorophores are prone to bleach during operations with the tweezers.…”