Type II topoisomerases, such as yeast topoisomerase II (topo II), have been increasingly demonstrated to be vital for relaxing torsional stress on DNA resulting from both transcription and replication. The strand-passage mechanism of topo II requires two disparate segments of DNA to closely approach each other and form a crossing so that both segments can interact with an individual enzyme. Using novel single-molecule experiments, we examine how the distinct mechanical properties of different substrates impact topo II activity by determining the availability of DNA crossings. In the context of replication over chromatin, we demonstrate through direct torque measurements that the intrinsic mechanical properties of chromatin drive DNA supercoiling into the single chromatin fiber in front of a replication fork instead of the braided fiber behind. We further show that topo II displays a strong preference for a single chromatin fiber over a braided fiber, suggesting a synergistic coordination—the mechanical properties of chromatin inherently suppress precatenane formation during replication elongation by driving DNA supercoiling ahead of the fork, where supercoiling is more efficiently removed by topo II. Next, we examine how topo II interacts with naked DNA and single chromatin fibers under varying levels of torsional stress. In the context of high torsional stress, we show that topo II can be immensely more processive on plectonemic DNA than previously measured. We then demonstrate that topo II can relax non-plectonemic DNA under low torsional stress but with greatly reduced processivity, suggesting a mechanism by which topo II may target genomic regions where topological problems are most severe. Furthermore, we map the relaxation rate of topo II as a function of torsional stress in both the high and low torsional stress regimes. The relaxation rate of DNA was independent of supercoiling density under high torsional stress, suggesting that topo II is able to efficiently capture DNA crossings in plectonemic DNA regardless of plectoneme size. In contrast, relaxation of non-plectonemic DNA slowed down with decreasing supercoiling density, suggesting that topo II captures spontaneous loops to relax DNA under low torsional stress. Finally, we map the relaxation rate of topo II on single chromatin fibers as a function of supercoiling density, demonstrating that the rate is determined by topological stress in a chromatin compaction dependent manner. This work demonstrates the active role of the DNA substrate in type II topoisomerase activity and reinforces the broad importance of substrate dynamics in fundamental processes.