Grab, Manipulate and Watch Single DNA Molecule Replication

Abstract

Current single-molecule strategies provide us great details in kinetic motion of replicative proteins and dynamic DNA-protein interaction. However, many of these studies lack the possibility for direct observing individual molecules interacting with DNA. Here we present a Correlative Tweezers Fluorescence Microscopy (CTFM) strategy to observe, with high-temporal-resolution, multiple replicative protein interacting on single DNA molecule. First, the real-time movement of ssDNA/dsDNA junction due to the catalytic activity of DNA polymerase (DNAp) is measured with a mechanical study. This motion is then correlated with the fluorescent trajectories of labelled DNAp. The combination allows us to distinguish different binding dynamics of DNAp on ssDNA and dsDNA: DNAp diffuses along dsDNA while showing surprisingly static behaviour on ssDNA. Next, the observed trajectories are correlated back to the mechanical measurement to access high-resolution data (force acquisition rate, can be up to 2.5 MHZ) of the replicative polymerase. These events show high processivity and force-dependent proofreading or replication at the ssDNA/dsDNA junction. Activity pauses of DNAp are also correlated with the unbinding events and thus we demonstrate that unbinding events might lead to the apparent long pauses reported before. Furthermore, correlative mechanical fluorescence system is extended to multi-colour imaging. By adding another fluorescent SSB molecules, we observe DNAp interacting with SSB on single DNA molecule during replication. This dual-colour imaging of the dynamics of two molecules reveals that SSB remains stationary while DNAp moves toward SSB for replication. These observations support a hypothesis of SSB being removed one by one during replication. Taken together, the above-reported strategy allows us to track multiple replicative proteins on a single DNA molecule in great detail and in real-time, thus providing us unique kinetic information as well as a dynamic picture of DNA replication.

Longfu Xu

PhD in Biophysics

My research topics focus on exploring DNA-proteins interactions using single-molecule visualization and manipulation techniques, with a big passion for understanding life with physical principles.