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DNA Helicases

DNA Helicases

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...

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. Type I...

Single-Strand DNA Binding Proteins

Single-Strand DNA Binding Proteins

For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...

The DNA Replication Fork

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...

The DNA Replication Fork

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...

The DNA Helix

The DNA Helix

Deoxyribonucleic acid, or DNA, is the genetic material responsible for passing traits from generation to generation in all organisms and most viruses. DNA is composed of two strands of nucleotides that wind around each other to form a spring-like structure called a double helix. However, the double helix is not perfectly symmetrical. Instead, there are regularly occurring grooves in the structure. The major groove occurs where the sugar-phosphate backbones are relatively far apart. This space...

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Related Experiment Video

Updated: Jun 14, 2026

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Published on: September 27, 2024

Visualizing helicases unwinding DNA at the single molecule level.

Natali Fili¹, Gregory I Mashanov, Christopher P Toseland

¹MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

Nucleic Acids Research

|March 31, 2010

Summary

Researchers developed a new optical method to track DNA helicase activity in real time. This technique visualizes DNA unwinding and enzyme processivity at the single-molecule level.

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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

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Published on: April 4, 2025

Area of Science:

Biochemistry
Molecular Biology
Biophysics

Background:

DNA helicases are essential motor proteins that unwind double-stranded DNA using ATP hydrolysis.
Understanding their processive movement along DNA is crucial for molecular biology.
Single-molecule techniques offer high resolution for studying enzyme mechanisms.

Purpose of the Study:

To develop a novel optical method for real-time, simultaneous monitoring of multiple DNA unwinding events.
To investigate the mechanistic details of DNA helicase activity, including processivity and dynamics.
To provide a versatile tool for studying various DNA helicases and DNA substrates.

Main Methods:

Utilized total internal reflection fluorescence microscopy (TIRFm).
Measured the accumulation of fluorescent single-stranded DNA-binding protein on unwound DNA.
Immobilized either DNA or helicase to observe localized fluorescence changes.

Main Results:

Successfully tracked simultaneous unwinding of multiple DNA molecules in real time.
Quantified unwinding rates and enzyme processivity at the single-molecule level.
Observed dynamic unwinding behaviors, including pauses and bursts of activity.

Conclusions:

The developed optical assay is a versatile and powerful addition to the single-molecule toolkit for studying DNA processing enzymes.
This method provides unprecedented detail into the mechanics of DNA helicases.
Applicable to a wide range of DNA helicases and DNA templates, advancing molecular studies.