Multiscale Conformational Dynamics of DNA Holliday Junctions Modulated by Ion Concentration: Insights from...

The DNA Holliday junction (HJ) is a central recombination intermediate formed by the association of two double-stranded DNA molecules during DNA repair and genetic recombination, with divalent cations such as Mg²⁺ playing a critical role in stabilizing its structure. However, the role of Mg²⁺ in governing single-molecule conformational dynamics of DNA Holliday junctions remains unresolved. Here, we use fluorescence correlation spectroscopy (FCS) to quantify microsecond-timescale conformational fluctuations and diffusion of DNA HJ. From the FCS measurements, it has been observed that increasing Mg²⁺ concentration gives rise to three resolvable microsecond-scale dynamic modes in DNA Holliday junctions, attributable to fast Cy3 dye motion (~1 µs), intermediate branch flexibility (~50 µs), and slower large-scale conformational rearrangements (~120 µs). These dynamic changes are accompanied by a 2.2-fold reduction in the diffusion coefficient and a decreased hydrodynamic radius, indicating enhanced structural compactness. Single-molecule time-resolved fluorescence anisotropy shows no significant change in rotational correlation times from 100 mM to 1.5 M Mg²⁺, ruling out oligomerization, while MSD analysis confirms reduced displacement consistent with monomeric Brownian diffusion. By directly linking Mg²⁺-dependent structural compaction to microsecond-scale dynamics, this work reveals fundamental physical principles governing the behavior of recombination intermediates at the single-molecule level.