<p>DNA bendability plays a critical role in stabilizing nucleosome assembly, yet its contribution to nucleosome dynamics in vivo remains poorly understood. Here, we applied chemical mapping to generate high-resolution nucleosome positioning maps at single-base-pair resolution from human interphase and metaphase chromosomes, revealing distinct patterns of nucleosome organization between the two states. Notably, we observed a unifying pattern of nucleosome positioning near euchromatic landmarks, including promoters, enhancers, and insulators, during mitosis. Interphase nucleosomes exhibited extensive repositioning, marked by increased nucleosome density, reduced spacing between nucleosomes, and the appearance of additional fragile nucleosomes compared to metaphase. Furthermore, our results show that metaphase nucleosomes display significantly higher DNA cyclizability around the dyad axis, whereas interphase nucleosomes, particularly those near regulatory regions, tend to position DNA with greater cyclizability at the edges of the nucleosome. Together, these findings highlight a dynamic interplay between DNA mechanics and nucleosome organization during the cell cycle.</p>