INO80 rapidly shuttles nucleosomes between chromatin barriers

ATP-dependent chromatin remodelers establish nucleosome organization, but how they move nucleosomes over extended distances and respond to neighboring chromatin remains unclear. Here, we combine correlative optical tweezers and fluorescence microscopy, and single-molecule FRET to visualize human INO80 activity on naked and chromatinized DNA. hINO80 undergoes free one-dimensional diffusion along DNA with multiple, nucleotide-regulated, diffusive states. Upon engaging a nucleosome, hINO80 drives rapid, processive nucleosome sliding over thousands of base pairs in bidirectional bursts. Neighboring nucleosomes prevent passage and redirect translocation, confining mobile nucleosomes to repeated movement between chromatin boundaries. Single-molecule FRET reveals transitions consistent with INO80 switching between opposing nucleosome-binding orientations, while fluorescence stoichiometry and mass photometry show that two hINO80 complexes can simultaneously occupy one nucleosome. These findings identify non-exclusive mechanisms for directional reversal and reveal how long-range hINO80 translocation is converted into boundary-constrained nucleosome repositioning, providing a dynamic framework for understanding nucleosome organization within chromatin.