Mapping fast DNA polymerase exchange during replication

Abstract

Despite extensive studies on DNA replication, the exchange mechanisms of DNA polymerase during replication remain unclear. Existing models propose that this exchange is facilitated by protein partners like helicase. Here we present data, employing a combination of mechanical DNA manipulation and single fluorescent protein observation, that reveal DNA polymerase undergoing rapid and autonomous exchange during replication not coordinated by other proteins. The DNA polymerase shows fast unbinding and rebinding dynamics, displaying a preference for either exonuclease or polymerase activity, or pausing events, during each brief binding event. We also observed a 'memory effect' in DNA polymerase rebinding, i.e., the enzyme tends to preserve its prior activity upon reassociation. This effect, potentially linked to the ssDNA/dsDNA junction's conformation, might play a role in regulating binding preference enabling high processivity amidst rapid protein exchange. Taken together, our findings support an autonomous replication model that includes rapid protein exchange, burst of activity, and a 'memory effect' while moving processively forward. 

Data Set Description

The study presents findings on the autonomous exchange mechanisms of DNA polymerase at the replication fork. Employing a combination of mechanical DNA manipulation and single fluorescent protein observation, the research reveals rapid and autonomous exchange of DNA polymerase, independent of protein partners like helicase. It highlights the enzyme's preference for exonuclease or polymerase activity and a 'memory effect' during binding events. In this data repo, we provided all the raw data used to reproduce the findings.

Methodology

The single-molecule experiments were performed at room temperature in a 5-channel microfluidic flow cell using the LUMICKS C-Trap instrument that combines dual optical trapping, confocal microscopy, and microfluidics for single-molecule assays. Data analysis was conducted using Python, Origin, and MATLAB. Detailed methodology and instrument specifications are provided in the associated publication.

File Formats

All data is obtained from C-trap in .tdms format.

Usage Notes

The custom-written python scripts used in this study is available at https://github.com/longfuxu/DNAPolymeraseProject, under the MPL-2.0 license. The repository includes example dataset, example Jupiter notebook, along with a detailed README file for instructions on installation and usage.

Acknowledgments

We thank Seyda Aca and Sandrine D'Haene for assistance with protein purification and DNA construction, Noémie Danné for help with implementing the step-fitting algorithm. We thank Erwin Peterman for critical reading and constructive feedbacks of this manuscript. This work was financially supported by a PhD fellowship from China Scholarship Council (To L.X., funding No. 201704910912), the European Union H2020 Marie-Sklowdowska Curie International Training Network AntiHelix (To G.J.L.W., funding No. 859853), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program MONOCHROME (to G.J.L.W., funding No.883240).

Competing Interest

The combined optical tweezers and fluorescence technologies used in this article are patented and licensed to LUMICKS B.V., in which M.T.J.H., and G.J.L.W. declare a financial interest. All other authors declare that they have no competing interests.

Author Contributions

L.X. and G.J.L.W. conceptualized the research. L.X. prepared protein samples and collected single-molecule data and analyzed data; M.T.J.H. analyzed data; L.X., M.T.J.H. and G.J.L.W. wrote and edited the manuscript; G.J.L.W. supervised the project; the manuscript is read, revised, and confirmed by all the listed authors.