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

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

Description of the data and file structure

This dataset was generated to investigate the molecular mechanism by which DNA polymerase (DNAp) displaces single-stranded DNA-binding proteins (SSBs) during DNA replication. The central question was to determine whether the polymerase passively waits for SSBs to dissociate on their own or actively participates in their removal.

To address this, we employed a multi-faceted approach combining several biophysical techniques:

Single-molecule force spectroscopy: To quantify the kinetics of this process, we used a correlated optical tweezers fluorescence microscopy. These experiments allowed us to monitor the progression of a single bacteriophage T7 DNA polymerase along a DNA template under precisely controlled tension. The data was collected to measure how the presence of T7 SSB modulates replication velocity and pausing behavior across a range of opposing forces.

Dual-color fluorescence imaging: To directly visualize the spatiotemporal dynamics of the protein encounter, we utilized dual-color imaging microscopy. By fluorescently labeling both the T7 DNA polymerase and T7 SSB, we could track their relative positions in real-time. This effort was designed to distinguish between competing models of displacement, such as a sequential displacement model versus a co- translocation model.

Computational and proximity-sensing experiments: To understand the underlying energetics, we performed molecular dynamics (MD) simulations. These simulations were designed to probe the energy landscape of SSB dissociation from DNA and to test the hypothesis that the polymerase actively lowers this energy barrier. These computational efforts were complemented by single-molecule Förster Resonance Energy Transfer (smFRET) experiments, which were conducted to confirm the close physical proximity between the polymerase and SSB during the displacement event.

Bulk primer extension assays: To complement the single-molecule findings with ensemble-level kinetics, we performed real-time fluorescence-based primer extension assays in a plate reader. These experiments measured the overall DNA synthesis activity of T7 DNA polymerase on a fluorescently labeled DNA substrate. Crucially, activity was compared in the absence of SSB, and in the presence of either the wild-type T7 SSB or a mutant lacking the C-terminal tail (Δ21C). This was critical for assessing the functional role of the SSB C-terminal tail in modulating polymerase activity.

Collectively, the data in this repository provides a multi-faceted view of the DNAp-SSB interaction, from macroscopic kinetics and spatiotemporal dynamics down to the underlying molecular energetics, supporting a model of active and sequential SSB displacement by the polymerase.

Files and variables

File: Data_Repo_for_DNAp_SSB.zip

Description: This compressed zip folder contains all raw data supporting the manuscript. The data is organized into folders corresponding to the figures in the main text.

Folder: Figure 1 Data Repo

Description: This folder contains data demonstrating that the effect of wild-type T7 SSB on the DNA replication rate is dependent on the ssDNA template conformation, which is modulated by applied force. It includes single-molecule force spectroscopy data comparing replication with and without SSB.

Folder: Figure 2 Data Repo

Description: This folder contains data from dual-color microscopy. These experiments visualize the dynamic interplay between T7 DNA polymerase and T7 SSB, revealing that SSBs are displaced sequentially from the DNA template.

Folder: Figure 3 Data Repo

Description: This folder contains data probing the molecular mechanism of SSB displacement. It includes single-molecule FRET (smFRET) data showing the close proximity of the proteins during displacement .

Folder: Figure 4 Data Repo

Description: This folder contains data validating the critical role of the SSB's C-terminal tail in the displacement process. It includes force spectroscopy and bulk assay data comparing the activity of wild-type T7 SSB with a C-terminal truncated variant (mut T7 SSB).

Code/software

The custom-written python scripts for analyzing basepair-time traces, for analyzing the displacement of SSB and for analyzing the real-time DNA primer extension data are available from Github: https://github.com/longfuxu/Interplay_Between_DNAPol_and_SSB, under the MPL-2.0 license. The script for analzying the MD simulation result and the Weighted Histogram Analysis Method (WHAM) are available from GitHub: https://github.com/CryoSky/MD_simulation_DNAp_SSB.  The repository includes example dataset, example Jupiter notebook, along with a detailed README file for instructions on installation and usage.

Access information

Other publicly accessible locations of the data:

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Data was derived from the following sources:

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