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PreprintCited by 3Force Microscopy Techniques and ApplicationsDNA Repair MechanismsDiffusion and Search Dynamics

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

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Xu, Longfu, Jin, Shikai, Urem, Mia, Lee, Seung-Joo, Lamers, Meindert H., Chen, Xun, Wolynes, Peter, Wuite, Gijs J. L.·2025·Nature Communications
DOI 10.1101/2025.03.07.642097PMC PMC12339929

Preprint — published as 10.1038/s41467-025-62531-1 · Paper page

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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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Metadata

Authors
Xu, L., Jin, S., Urem, M., Lee, S.-J., Lamers, M. H., Chen, X., Wolynes, P., & Wuite, G. J. L.
Affiliations
  • Vrije Universiteit Amsterdam (NL)
  • Center for Theoretical Biological Physics (US)
  • Leiden University (NL)
  • Harvard University (US)
  • Leiden University Medical Center (NL)
  • Nanjing Medical University (CN)
Corresponding author
  • Wolynes, Peter
  • Wuite, Gijs J. L.
ORCID
Venue
Nature Communications
Publisher
openRxiv
Published
2025-03-11
Volume
16
Issue
1
Pages
7431-7431
ISSN
2041-1723 (ISSN-L)
DOI
10.1101/2025.03.07.642097
PMC
PMC12339929
OpenAlex
W4413106364
Cited by
3
References
1
Citations by year
2026: 2 · 2025: 1
FWCI
1.08
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • VU Research Portal (publishedVersion, cc-by-nc-nd)
  • Leiden Repository (Leiden University) (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Science Foundation (2019745)
  • Rice University (PHY-2019745, 2019745, C-0016)
  • European Commission (859853, HORIZON2020, 883240, H2020)
  • China Scholarship Council (859853, 201704910912)
  • Horizon 2020 Framework Programme (859853, 883240)
Topics
  • Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Force Microscopy Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Diffusion and Search Dynamics
Keywords
DNA polymerase, single-stranded DNA binding protein, DNA replication, bacteriophage T7, protein-DNA interactions, molecular collisions, DNA secondary structure, polymerase processivity
MeSH terms
Single Molecule Imaging, DNA-Directed DNA Polymerase, DNA Replication, DNA-Binding Proteins, DNA, Single-Stranded, Escherichia coli, Protein Binding, Viral Proteins, Bacteriophage T7, Fluorescence Resonance Energy Transfer, Molecular Dynamics Simulation

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