Fluorescence imaging of protein dynamics on long single‐stranded DNA (549.7)

Abstract

Recent advances in single-molecule fluorescence imaging techniques have allowed the direct observation of protein dynamics on DNA, but the progress has been largely limited to double-stranded DNA (dsDNA) or short single-stranded DNA (ssDNA). Here, we present a single molecule imaging approach for observing dynamics of proteins on long ssDNA of thousands of nucleotides in length. Using a hybrid instrument combining single-molecule fluorescence and force spectroscopy, we could visualize the dynamics of proteins interacting with a long ssDNA similar in length to what’s generated during DNA metabolic processes. Our ultimate goal is to study many different proteins binding to the same long ssDNA and performing their function in coordination in each other. As a step toward the goal, here, we demonstrate the applicability of our methods to the dynamics of two different proteins: (1) the unidirectional motion of Escherichia coli (E. coli) UvrD helicase and (2) the diffusion of E. coli single-stranded DNA binding protein (SSB). With the multidimensional data obtained with our platform, we could capture the entire sequence of binding, translocation, unwinding initiation of UvrD helicase with single molecule resolution. With SSB, we found that the diffusion coefficient is at least 600 times higher than what was determined from SSB diffusion on short ssDNA suggesting that on long ssDNA that mimics physiological setting, SSB can migrate via a long range inter-segmental transfer. Force dependence of diffusion further supports the interpretation. Grant Funding Source: Supported by the US National Institutes of Health and by the National Science Foundation