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Intercalation-Based Single-Molecule Fluorescence Assay To Study DNA Supercoil Dynamics.

Mahipal Ganji1, Sung Hyun Kim1, Jaco van der Torre1

  • 1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology , Van der Maasweg 9, Delft, 2629HZ, The Netherlands.

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|June 30, 2016
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Summary

A new assay visualizes DNA supercoiling in real-time. This method reveals DNA plectonemes are anchored to specific DNA sequence mispairs, advancing our understanding of DNA dynamics.

Keywords:
DNA supercoilingfluorescenceintercalationplectonemesingle-molecule

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Area of Science:

  • Molecular Biology
  • Biophysics

Background:

  • DNA supercoiling is vital for DNA replication, gene expression, and chromatin organization.
  • Limited experimental tools hinder mechanistic understanding of DNA supercoiling and related enzymes.

Purpose of the Study:

  • To develop a novel high-throughput assay for real-time visualization of supercoiled DNA molecules.
  • To characterize the dynamics and behavior of DNA plectonemes.

Main Methods:

  • Developed the Intercalation-induced Supercoiling of DNA (ISD) assay.
  • Utilized an intercalating dye to induce and visualize supercoiling and plectonemes on surface-attached DNA via epifluorescence microscopy.
  • Tracked individual plectonemes' positions, sizes, and dynamics without mechanical manipulation.

Main Results:

  • Demonstrated the ISD technique's capability for real-time visualization of supercoiled DNA.
  • Showed that DNA plectonemes nucleate, terminate, and diffuse.
  • Established that plectonemes are pinned to a specific 10-nucleotide long mispaired sequence in double-stranded DNA.

Conclusions:

  • The ISD assay provides a powerful new tool for studying DNA supercoiling and processing.
  • Plectoneme localization is sequence-dependent, influenced by DNA mispairs.
  • This finding offers insights into DNA organization and regulation at a molecular level.