Molecular crowding suppresses mechanical stress-driven DNA strand separation.

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.