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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...

Homologous Recombination

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...

Labeling DNA Probes

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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
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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...

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Related Experiment Video

Updated: Jun 18, 2026

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

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DNA helicases displace streptavidin from biotin-labeled oligonucleotides.

P D Morris¹, K D Raney

¹Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.

|April 23, 1999

Summary

Bacteriophage T4 helicases gp41 and Dda encircle single-stranded DNA and use ATP to rapidly displace blocking molecules from the DNA strand. These enzymes exhibit a directional bias, suggesting a 5’ to 3’ translocation mechanism on single-stranded DNA.

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Last Updated: Jun 18, 2026

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Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Published on: January 17, 2025

Area of Science:

Molecular Biology
Enzymology
Structural Biology

Background:

Helicases are essential enzymes that unwind double-stranded DNA using nucleoside triphosphate hydrolysis.
Bacteriophage T4 helicases, gp41 and Dda, are crucial for DNA metabolism and serve as model systems for enzymatic studies.
gp41 forms a hexamer in the presence of ATP, indicating potential structural changes during DNA binding.

Purpose of the Study:

To determine the binding topology of gp41 on single-stranded DNA (ssDNA).
To develop a novel assay for assessing the enzymatic activity of gp41 and Dda on ssDNA.
To investigate the force-generating capabilities and directional bias of these helicases on ssDNA.

Main Methods:

Protein cross-linking in the presence of linear or circular ssDNA substrates to analyze gp41 binding.
Development of an electrophoretic mobility shift assay to measure streptavidin dissociation from biotinylated oligonucleotides.
ATP-dependent enzymatic assays to quantify helicase activity in displacing streptavidin from DNA.

Main Results:

gp41 hexamer encircles ssDNA, similar to other hexameric helicases.
gp41 and Dda significantly enhance streptavidin dissociation from 3'-biotinylated DNA in an ATP-dependent manner.
Helicase-catalyzed dissociation rates are orders of magnitude faster than spontaneous dissociation, with Dda showing exceptionally high activity.
No significant enhancement of dissociation was observed for 5'-biotinylated DNA, indicating a 5’ to 3’ translocation bias.

Conclusions:

Bacteriophage T4 helicases gp41 and Dda bind ssDNA by encircling it.
These helicases can exert force to displace blocking molecules from DNA.
The observed 5’ to 3’ displacement bias suggests a directional translocation mechanism on ssDNA.