a, b, Example snapshots (left) and kymographs (right) for the single-tethered DNA molecules showing single loop (a) and Z-loops into which DNA is pulled from two sides (b). As in the kymograph shown in a, single loops grow either towards the direction of the free end or towards the tethered end, whereas the length of DNA strand on the opposite side remains constant. When, on reeling in DNA, the single loop reaches the free end of the DNA, it collapses within a single frame (white arrow). In the case of Z-loops into which DNA is pulled from both sides as shown in b, both sides of the DNA are shortened. When the Z-loop reaches the free end of the DNA, it collapses into a single loop within a single frame, near one of the edge of the Z-loop. These data for a single loop and a Z-loop reeling in DNA from two sides were measured at 1 nM and 10 nM condensin, respectively. Data represent two (a) and three (b) independent experiments, respectively. c, Visual explanation of how the final length of a Z-loop relates to the initial single loop size l in our double-tether DNA assay, where the condensins stall owing to high DNA tension. d, Final Z-loop size versus single-loop size before the Z-loop formation. A linear relation is observed, with 30% of molecules exhibiting the maximum possible Z-loop size, 1.5 times the size of the initial single loop (n = 32 molecules, 10 independent experiments). This size estimate of 1.5 times is determined by the nature of our specific set-up, in which loops reach only a finite size because of stalling, and therefore cannot be generalized. In our double-tether DNA assay, strain on the DNA increases rapidly as soon as the initial loop expands, making the first condensin typically inactive before the start of the Z-loop, thus limiting the length of the Z-loop to at most 1.5 times that of the initial loop. However, Z-loops can, in principle, grow infinitely long if no tension is built up during the growth of the loop, as shown in b. e, Visual explanation of the definition of maximum fold compaction (FC). f, g, Maximum fold compaction (f) and the corresponding stall force (g; estimated from the known force–extension relation28) for single loops (n = 49 molecules) and Z-loops (n = 22 molecules). The similar fold compaction, as measured in the double-tether assay, which exhibits a high internal tension, shows that two condensins forming a Z-loop do not linearly compact DNA much more than two condensins that form separate single loops in this condition. Data were obtained from ten independent datasets. Boxes span 25 to 75th percentiles, centre line represents the median and whiskers show maximum and minimum data points. h, Snapshots (left) and kymograph (middle) of a DNA molecule showing a Z-loop that exhibits random diffusion along the DNA tether, presumably owing to a ‘tug of war’ between the two partly slipping condensin motors, which occurs when Z-loops reeling in DNA from both sides fully extend along the double-tethered DNA. Representative of three independent datasets. Right, time traces of centre-of-mass positions of Z-loops that diffuse along DNA (n = 6 molecules from 3 independent datasets).