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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. Type I...

Restarting Stalled Replication Forks

Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart, a...

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...

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...

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Related Experiment Video

Updated: Jun 15, 2026

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

Published on: April 4, 2025

Duplex unwinding with DEAD-box proteins.

Eckhard Jankowsky¹, Andrea Putnam

¹Department of Biochemistry & Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.

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

|March 13, 2010

Summary

DEAD-box proteins are key to RNA metabolism, performing ATP-driven unwinding of RNA structures. This chapter details methods for studying their unique strand separation activity.

Area of Science:

Molecular Biology
Biochemistry

Background:

DEAD-box proteins represent the largest helicase family, crucial for RNA metabolism.
They perform ATP-dependent functions, including unwinding RNA secondary structures.
Unlike other helicases, they directly load onto RNA helices for local unwinding.

Purpose of the Study:

To outline protocols for substrate design and unwinding assays for DEAD-box proteins.
To focus on the quantitative evaluation of DEAD-box protein unwinding activity.

Main Methods:

Development of specific substrate designs for DEAD-box protein assays.
Implementation of detailed unwinding protocols.
Quantitative analysis of protein activity.

Main Results:

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Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Published on: November 1, 2012

Related Experiment Videos

Last Updated: Jun 15, 2026

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

Published on: April 4, 2025

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Published on: November 1, 2012

Established protocols for studying DEAD-box protein unwinding.
Provided a framework for quantitative evaluation of their enzymatic activity.

Conclusions:

The outlined methods enable robust characterization of DEAD-box protein function.
Quantitative evaluation is essential for understanding these vital RNA helicases.