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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...

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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...

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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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Related Experiment Video

Updated: Jun 15, 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

Methods to study how replication fork helicases unwind DNA.

Daniel L Kaplan¹, Irina Bruck

¹Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA.

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

|March 13, 2010

Summary

This summary is machine-generated.

This study details methods for investigating replication fork helicase mechanisms, focusing on the eukaryotic Mcm4,6,7 complex. Understanding these DNA unwinding processes is crucial for DNA replication research.

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Published on: March 23, 2010

Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Replication fork helicases are essential enzymes that unwind DNA, enabling DNA replication.
In eukaryotes, the Mcm2-7 complex, along with Cdc45 and GINS, catalyzes fork unwinding, while archaea utilize a single Mcm protein.
The Mcm4,6,7 subcomplex offers a model for studying eukaryotic Mcm helicase mechanisms.

Purpose of the Study:

To describe detailed methods for investigating the mechanism of replication fork helicases.
To provide protocols for studying DNA unwinding and branch migration activities of Mcm helicases.

Main Methods:

Design of specific DNA substrates for unwinding and branch migration assays.
Protocols for annealing DNA substrates.
Methods for purification of replication fork helicase proteins.
Techniques for analyzing DNA unwinding activity in vitro.

Main Results:

The study provides a comprehensive set of methods for dissecting replication fork helicase function.
These methods enable the characterization of DNA unwinding by Mcm helicase complexes, including the Mcm4,6,7 subcomplex.

Conclusions:

The described methodologies are vital for advancing our understanding of eukaryotic Mcm helicase mechanisms.
This work facilitates future research into the intricate process of DNA replication fork progression.