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The DNA Replication Fork

An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication forks, one in...
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Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase
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Winding single-molecule double-stranded DNA on a nanometer-sized reel.

Huijuan You1, Ryota Iino, Rikiya Watanabe

  • 1Department of Applied Chemistry, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan.

Nucleic Acids Research
|July 10, 2012
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Summary

Researchers developed a molecular reel from F(1)-ATPase to study DNA mechanics. This system precisely winds double-stranded DNA (dsDNA), revealing its bending stiffness and persistence length for micromechanics studies.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Nanotechnology

Background:

  • F(1)-ATPase is a rotary motor protein capable of mechanical work.
  • Understanding DNA mechanics at the nanoscale is crucial for molecular biology.
  • Previous methods for studying DNA micromechanics have limitations.

Purpose of the Study:

  • To develop a novel molecular system for studying the micromechanics of double-stranded DNA (dsDNA).
  • To measure the bending stiffness and persistence length of dsDNA using a nanometer-sized reel.
  • To create a platform for investigating DNA-associating proteins on sharply bent DNA.

Main Methods:

  • Construction of a molecular reel system using F(1)-ATPase.
  • Integration with magnetic and optical tweezers for single-molecule manipulation.
  • Winding single dsDNA molecules around the molecular reel and measuring winding tension and loop diameter.

Main Results:

  • Successfully wound dsDNA around the molecular reel with controlled tension (0.9-6.0 pN).
  • Determined dsDNA bending stiffness from winding parameters, yielding a persistence length of 54 ± 9 nm.
  • Results align with the conventional worm-like chain model for DNA elasticity.

Conclusions:

  • The developed molecular reel system provides a new platform for single-molecule studies of DNA micromechanics.
  • This system enables investigation of DNA behavior under sharp bending conditions.
  • It is expected to aid in elucidating the mechanisms of DNA-associating proteins on bent DNA strands.