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Molecular crowding suppresses mechanical stress-driven DNA strand separation.

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Molecular crowding affects DNA supercoiling. Polyethylene glycol suppresses strand separation, promoting plectoneme formation, while glycerol destabilizes DNA base pairs under supercoiling stress.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Genomics

Background:

  • Molecular crowding is prevalent in cells, influencing DNA mechanics and protein interactions.
  • Understanding crowding effects on DNA supercoiling is crucial for bridging in vitro and in vivo studies.
  • DNA supercoiling is a fundamental process affecting DNA accessibility and function.

Purpose of the Study:

  • To quantify the impact of molecular crowding on DNA supercoiling dynamics.
  • To investigate how different cosolutes (glycerol, polyethylene glycol) alter DNA supercoiling.
  • To provide insights into DNA behavior under conditions mimicking the cellular environment.

Main Methods:

  • Utilized single-molecule magnetic tweezers to apply stretching force (0.8 pN) and induce DNA supercoiling.
  • Studied DNA behavior in a 200 mM NaCl buffer with and without cosolutes.
  • Analyzed changes in DNA structure, including local melting and plectoneme formation.

Main Results:

  • In standard buffer, negatively supercoiled DNA forms locally melted regions to relieve torsional stress.
  • Glycerol, a dehydrating cosolute, further destabilizes base pairs in negatively supercoiled DNA.
  • Polyethylene glycol, a crowding agent, inhibits local strand separation, favoring plectoneme formation even under negative supercoiling.

Conclusions:

  • Molecular crowding significantly alters DNA supercoiling mechanisms.
  • Cosolutes differentially affect DNA stability and supercoiling pathways.
  • Findings advance the understanding of DNA supercoiling and DNA-protein interactions in cellular contexts.