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Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA.
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DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
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Analyzing and Building Nucleic Acid Structures with 3DNA
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Supercoiling DNA optically.

Graeme A King1,2, Federica Burla1,2, Erwin J G Peterman1,2

  • 1Department of Physics and Astronomy, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.

Proceedings of the National Academy of Sciences of the United States of America
|December 7, 2019
PubMed
Summary
This summary is machine-generated.

Researchers developed Optical DNA Supercoiling (ODS) to study supercoiled DNA dynamics. This new method allows detailed observation of DNA structures and protein interactions, revealing how DNA supercoiling influences transcription factors like TFAM.

Keywords:
DNA supercoilingDNA–protein interactionsfluorescence microscopyoptical tweezers

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Genomics

Background:

  • Cellular DNA experiences torsional stress during vital genomic processes like transcription and replication.
  • Existing single-molecule techniques for studying supercoiled DNA lack integrated spatial manipulation and fluorescence microscopy.
  • This limitation hinders the comprehensive study of complex supercoiled DNA interactions.

Purpose of the Study:

  • To introduce a novel single-molecule assay for controlled generation and study of negatively supercoiled DNA.
  • To combine force spectroscopy, whole-DNA fluorescence imaging, and rapid buffer exchange for comprehensive analysis.
  • To investigate the effects of ionic strength and DNA sequence on underwound DNA structures and protein dynamics.

Main Methods:

  • Development of Optical DNA Supercoiling (ODS) using a standard dual-trap optical tweezers instrument.
  • Generation of negatively supercoiled DNA with varying degrees of underwinding (<5% to 70% lower helical twist).
  • Application of ODS to study ionic strength and sequence effects, and protein diffusion on supercoiled DNA.

Main Results:

  • ODS successfully generates a wide range of supercoiled DNA states.
  • Revealed previously unobserved effects of ionic strength and sequence on underwound DNA structures.
  • Demonstrated ODS's capability to visualize and quantify protein dynamics, showing TFAM diffusion is hindered by underwound DNA.

Conclusions:

  • Optical DNA Supercoiling (ODS) is a versatile method for studying supercoiled DNA biophysics and interactions.
  • Supercoiling may regulate mitochondrial transcription by affecting transcription factor diffusion, as exemplified by TFAM.
  • ODS provides a powerful platform for advancing research into DNA supercoiling and its biological roles.