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bioRxiv · 10.1101/2025.03.07.642097

DNA polymerase actively and sequentially displaces single-stranded DNA-binding proteins

Xu, L.·Jin, S.·Urem, M.·Lee, S.-J.·Lamers, M.·Chen, X.·Wolynes, P.·Wuite, G. J. L.
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Abstract

Single-stranded DNA-binding proteins (SSBs) play a crucial role in stabilizing and protecting transiently exposed single-stranded DNA (ssDNA), yet the mechanisms governing their displacement by DNA polymerase (DNAp) during replication remain largely unexplored. Using bacteriophage T7 DNAp and its SSB, T7 gp2.5, we investigated the molecular mechanisms and visualized the dynamic process underlying SSB displacement. Our single-molecule force spectroscopy demonstrates that T7 SSB modulates DNA replication in an ssDNA conformation-dependent manner, regulated by tension applied to the DNA template. By integrating dual-color single-molecule imaging, we observe that T7 SSB remains stationary as DNAp approaches, indicating that SSB molecules are sequentially displaced rather than pushed forwards. Molecular dynamics (MD) simulations revealed reduced energy barriers for SSB dissociation in the presence of DNAp. This finding, combined with the detected FRET signals when DNAp approaches an SSB-bound ssDNA region and observations of faster replication rates compared to relative slow intrinsic SSB dissociation, collectively support an active displacement mechanism. Using both ensemble and single-molecule analyses, we demonstrated that SSB saturation of ssDNA is critical for optimal replication efficiency, with each SSB molecule contributing positively to the process. Taken together, the uncovered spatial-temporal coordination between SSB and DNAp is necessary for resolving molecular collisions during DNA replication, and may represent a universal strategy employed by other DNA translocating motors to ensure genomic integrity.

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Xu, L., Jin, S., Urem, M., Lee, S.-J., Lamers, M., Chen, X., Wolynes, P., Wuite, G. J. L.. 2025-03-11. DNA polymerase actively and sequentially displaces single-stranded DNA-binding proteins. https://doi.org/10.1101/2025.03.07.642097

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