Single-Molecule Visualization of DNase I-Mediated DNA Cleavage by High-Speed Atomic Force Microscopy

DNase I is a nonspecific endonuclease that preferentially cleaves double-stranded DNA (dsDNA) over single-stranded DNA (ssDNA) in the presence of Ca²⁺ and Mg²⁺. Although the structure and biochemical properties of DNase I are well-characterized, the catalytic process remains poorly understood, as earlier studies primarily inferred cleavage from end point fragments or time-averaged measurements rather than direct visualization. Here, we employ high-speed atomic force microscopy (HS-AFM) to directly visualize DNase I activity on linear dsDNA, circular dsDNA plasmids, and circular ssDNA plasmids. DNase I, observed as monomeric particles, dimers, and trimeric or higher-order aggregates, dynamically binds to and slides along DNA substrates while inducing both single-strand and double-strand cleavage events. These observations reveal that DNase I-mediated cleavage is not restricted to an isolated monomeric state and that sliding-like DNA-bound motion can accompany nonspecific nuclease activity. DNase I exhibits significantly higher cleavage efficiency toward dsDNA than ssDNA. Together, these results provide direct mechanistic insights into DNase I-mediated nucleic acid degradation, with implications for its biochemical functions and therapeutic applications.