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DNA Topoisomerases

DNA Topoisomerases

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.
Types and Mechanism of action
Topoisomerases are divided into two main types. ...

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Generating Negatively Supercoiled DNA Using Dual-Trap Optical Tweezers.

Graeme A King¹, Dian Spakman², Erwin J G Peterman³

¹Institute of Structural and Molecular Biology, University College London, London, UK. g.king@ucl.ac.uk.

Methods in Molecular Biology (Clifton, N.J.)

|September 5, 2022

Summary

Researchers developed Optical DNA Supercoiling (ODS) to study DNA topology. This single-molecule method uses optical tweezers to generate and analyze negatively supercoiled DNA structures and their interactions.

Keywords:

DNA structure DNA supercoiling DNA topology Microfluidics Optical tweezers

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Area of Science:

Genomics
Molecular Biology
Biophysics

Background:

Genomic processes create underwound (negatively supercoiled) and overwound (positively supercoiled) DNA.
DNA topological changes influence interactions with DNA-binding proteins, affecting gene regulation.
Understanding supercoiled DNA structure and protein interactions is crucial.

Purpose of the Study:

To introduce and detail the Optical DNA Supercoiling (ODS) method.
To enable advanced study of supercoiled DNA structure and protein interactions.
To provide a practical guide for generating and controlling supercoiled DNA.

Main Methods:

Developed a single-molecule approach called Optical DNA Supercoiling (ODS).
Utilized a standard dual-trap optical tweezers instrument.
Combined force spectroscopy, fluorescence imaging, and spatial control.

Main Results:

Enabled rapid generation of negatively supercoiled DNA (5-70% lower helical twist).
Facilitated combined force spectroscopy, fluorescence imaging, and spatial control of supercoiled DNA.
Provided detailed instructions for DNA substrate preparation and supercoiling calibration.

Conclusions:

ODS is a powerful method for studying DNA supercoiling.
The technique offers advantages in controlling and analyzing supercoiled DNA substrates.
This work advances the understanding of DNA topology and its biological implications.