In mechanical manipulation experiments, a single DNA molecule overwound at constant force undergoes a discontinuous drop in extension as it buckles and forms a superhelical loop (a plectoneme). Further overwinding the DNA, we observe an unanticipated cascade of highly regular discontinuous extension changes associated with stepwise plectoneme lengthening. This phenomenon is consistent with a model in which the force-extended DNA forms barriers to plectoneme lengthening caused by topological writhe. Furthermore, accounting for writhe in a fluctuating solenoid gives an improved description of the measured force-dependent effective torsional modulus of DNA, providing a reliable formula to estimate DNA torque. Our data and model thus provide context for further measurements and theories that capture the structures and mechanics of supercoiled biopolymers.Supercoiling of DNA permits its compaction in a cell and is crucial to the biological activities of DNA interacting proteins. Understanding the structures and mechanics of DNA supercoiling is therefore a topic of fundamental interest [1]. Experimentally, the force and linking number of a topologically closed DNA double helix can be specified in a single-molecule manipulation experiment with torsional control [2]. Following a theorem that states that the imposed torsion or excess linking number (Lk) partitions into interconverting components of twist (Tw) and writhe (Wr) [3][4][5], the DNA molecule can either twist about its backbone or writhe into more complex shapes that depend on force and solution conditions. These DNA shapes produce mechanical signatures observable by experiment.At a critical torque, twisted DNA buckles: It loops upon itself and wraps into a superhelical writhed structure called a plectoneme (Fig. 1). The plectoneme lengthens with additional imposed turns. Although this is widely assumed to be a smooth and continuous process, here we present experimental evidence that a DNA plectoneme lengthens in discrete steps that occur with a periodicity that exceeds the characteristic plectoneme repeat length (Fig. 1a). This behavior indicates supercoiling involves unexpected periodic free-energy barriers of unknown origin.The observed plectoneme lengthening steps can be interpreted as discontinuous exchanges of twist for writhe. At fixed force and torsion, the conjugate variables, extension and torque, are free to fluctuate. Since torque increases with increasing twist, the discrete plectoneme lengthening phenomenon corresponds to a progressive buildup, then release, of DNA torque (Fig. 1b).To explain this phenomenon, we propose a model in which barriers to plectoneme lengthening arise from the force-extended DNA adopting a solenoid structure (Fig. 2). If the force-extended region were straight without writhe, it would have continuous translational symmetry and adding length to the plectoneme would increase energy smoothly. In contrast, a solenoid shape is periodic and has discrete translational symmetry, resulting in a periodic energy associated with addin...