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...

Left-handed Z-DNA is an extraordinary conformation of DNA, which can form by special sequences under specific biological, chemical or physical conditions. Human ADAR1, prototypic Z-DNA binding protein (ZBP), binds to Z-DNA with high affinity. Utilizing single-molecule FRET assays for Z-DNA forming sequences embedded in a long inactive DNA, we measure thermodynamic populations of ADAR1-bound DNA conformations in both GC and TG repeat sequences. Based on a statistical physics model, we determined quantitatively the affinities of ADAR1 to both Z-form and B-form of these sequences. We also reported what pathways it takes to induce the B–Z transition in those sequences. Due to the high junction energy, an intermediate B* state has to accumulate prior to the B–Z transition. Our study showing the stable B* state supports the active picture for the protein-induced B–Z transition that occurs under a physiological setting.

Despite recent genome-wide investigations of functional DNA elements, the mechanistic details about their actions remain elusive. One intriguing possibility is that DNA sequences with special patterns play biological roles, adopting non-B-DNA conformations. Here we investigated dynamics of thymine-guanine (TG) repeats, microsatellite sequences and recurrently found in promoters, as well as cytosine–guanine (CG) repeats, best-known Z-DNA forming sequence, in the aspect of Z-DNA formation. We measured the energy barriers of the B–Z transition with those repeats and discovered the sequence-dependent penalty for Z-DNA generates distinctive thermodynamic and kinetic features in the torque-induced transition. Due to the higher torsional stress required for Z-form in TG repeats, a bubble could be induced more easily, suppressing Z-DNA induction, but facilitate the B–Z interconversion kinetically at the transition midpoint. Thus, the Z-form by TG repeats has advantages as a torsion buffer and bubble selector while the Z-form by CG repeats likely behaves as torsion absorber. Our statistical physics model supports quantitatively the populations of Z-DNA and reveals the pivotal roles of bubbles in state dynamics. All taken together, a quantitative picture for the transition was deduced within the close interplay among bubbles, plectonemes and Z-DNA.