BPS2026 – Correlated dynamics and activity mapping (C-DAM) reveals rapid DNA polymerase exchange and SSB displacement at the replication fork

Single-molecule techniques have advanced our understanding of DNA-protein interactions, yet simultaneously resolving enzyme activity, position, and binding dynamics remains challenging. Here, we present correlated dynamics and activity mapping (C-DAM), integrating optical tweezers and confocal fluorescence microscopy to achieve concurrent, high-resolution correlation of enzyme activity with its precise DNA location. C-DAM employs dual-trap optical tweezers to track DNA extension changes during enzyme-mediated conversion between single- and double-stranded forms, achieving nanometer and millisecond resolution. This mechanical readout is synergistically correlated with fluorescence through “force-assisted imaging”—surpassing the diffraction limit by pinpointing fluorescently labeled proteins relative to mechanically defined activity sites. Unlike existing approaches, C-DAM leverages both techniques to directly map enzyme location, function, and binding dynamics. Using C-DAM, we challenge the stable replisome paradigm, revealing that DNA polymerase undergoes rapid, autonomous exchange at replication forks on second timescales, independent of helicase or other components. Despite this turnover, the fork maintains processivity through a “memory effect”—a departing polymerase leaves the primer-template junction catalytically competent, enabling an incoming polymerase to immediately resume synthesis. Dual-color C-DAM further revealed how polymerase navigates protein traffic: when encountering single-stranded DNA-binding proteins (SSBs), it employs sequential displacement, actively lowering the energy barrier for SSB dissociation through direct molecular collisions. C-DAM provides a powerful platform for quantitatively distinguishing protein-DNA binding modes, such as static binding versus diffusion. The method is broadly adaptable for dissecting complex molecular machineries, offering mechanistic insight into the protein traffic coordination essential for maintaining genomic integrity.