Multiplexed single-molecule flow-stretching bead assay for DNA enzymology

Single-molecule techniques have been used successfully to visualize real-time enzymatic activities, revealing transient complex properties and heterogeneity of various biological events. Especially, conventional force spectroscopy including optical tweezers and magnetic tweezers has been widely used to monitor change in DNA length by enzymes with high spatiotemporal resolutions of similar to nanometers and similar to milliseconds. However, DNA metabolism results from coordination of a number of components during the processes, requiring efficient monitoring of a complex of proteins catalyzing DNA substrates. In this min-review, we will introduce a simple and multiplexed single-molecule assay to detect DNA substrates catalyzed by enzymes with high-throughput data collection. We conclude with a perspective of possible directions that enhance capability of the assay to reveal complex biological events with higher resolution.11Ysciescopuskc

Dynamics of Proofreading by E.Coli Pol III Replicase.

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Single-molecule assay for UvrD helicase activity.

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A correlative Single-molecule Technique.

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Strand excision in E.Coli DNA mismatch repair.

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Single-molecule DNA flow-stretching force spectroscopy with higher resolution using dark-field microscopy.

Single-molecule force spectroscopy often refers to the measurement of the enzymatic activities coupled to the mechanical properties of biomolecules. We apply a simple dark-field microscope to a single-molecule flow-stretching method that quantitatively measures thermal fluctuations of the micron bead linked to DNA under a hydrodynamic force. Using various lengths of DNA attached to the bead, we measured the external force exerted on DNA immobilized on the surface and quantitatively showed the anticorrelation between the thermal fluctuation and the length of DNA. We propose a multiplexed single-molecule flow-stretching technique with better spatial resolution.11Nsciescopuskc

Single-molecule screening of mismatch recognition function of cells

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Dynamics of Proofreading by the E. coli Pol III Replicase

The αεθ core of Escherichia coli DNA polymerase III (Pol III) associates with the β2 sliding clamp to processively synthesize DNA and remove misincorporated nucleotides. The α subunit is the polymerase while ε is the 3\u27 to 5\u27 proofreading exonuclease. In contrast to the polymerase activity of Pol III, dynamic features of proofreading are poorly understood. We used single-molecule assays to determine the excision rate and processivity of the β2 -associated Pol III core, and observed that both properties are enhanced by mutational strengthening of the interaction between ε and β2. Thus, the ε-β2 contact is maintained in both the synthesis and proofreading modes. Remarkably, single-molecule real-time fluorescence imaging revealed the dynamics of transfer of primer-template DNA between the polymerase and proofreading sites, showing that it does not involve breaking of the physical interaction between ε and β2