Long-lived pauses reveal tunable kinetic barriers during Holliday junction migration.

Holliday junctions (HJs) are key intermediates in homologous recombination, and their branch migration influences how recombination intermediates are positioned and processed. Although structural studies have shown that HJs interconvert between open and stacked-X conformations, and the stacked-X state has long been proposed to inhibit migration, it remains unclear whether such pausing creates a substantial kinetic barrier to long-range spontaneous branch migration. Here, we develop a magnetic-tweezer single-molecule assay that tracks spontaneous HJ migration over extended distances ($\sim 290$ bp) immediately after junction formation. We find that migration is discontinuous, alternating between active movement and long-lived pauses lasting seconds to tens of seconds. Focusing on pauses of at least 1 s, we show that Mg$^{2+}$ strongly increases both pause duration and pause frequency, whereas higher temperature and applied tension suppress long-lived pausing. Under high Mg$^{2+}$, low temperature, and low force, these pauses dominate total migration time and reduce effective migration several fold, establishing them as a major kinetic barrier. In addition, nucleoid-associated protein HU increases long-pause frequency and pause occupancy under conditions where intrinsic pausing is weak. Together, these results show that spontaneous HJ migration is governed by a tunable long-lived paused state consistent with a stacked-X-like migration-inhibitory conformation.