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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...

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...

The DNA Replication Fork

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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...

The DNA Replication Fork

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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...

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...

Homologous Recombination

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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 5, 2026

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

Published on: February 13, 2019

FBH1 and RAD54L directly interact and cooperate to drive replication fork reversal.

Mollie E Uhrig¹, Courtney N Johnson², Jordi Gomez¹

¹Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, 80523, USA.

Biorxiv : the Preprint Server for Biology

|June 4, 2026

Summary

FBH1 and RAD54L protein cooperation is essential for replication fork reversal, a process that maintains genome stability. This study reveals their direct interaction and sequential action, clarifying their molecular mechanism in alleviating DNA replication stress.

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Related Experiment Videos

Last Updated: Jun 5, 2026

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

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Published on: February 13, 2019

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Published on: April 29, 2010

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Published on: September 11, 2022

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Replication fork reversal is crucial for managing DNA replication stress and ensuring genome stability.
Previous research indicated a cooperative role for FBH1 and RAD54L in fork reversal, but the underlying molecular mechanisms were not fully understood.

Purpose of the Study:

To elucidate the molecular basis of the cooperation between FBH1 and RAD54L in promoting replication fork reversal.
To identify the specific domains and interactions involved in the FBH1-RAD54L complex formation.

Main Methods:

Co-immunoprecipitation and in vitro binding assays to demonstrate physical interaction between FBH1 and RAD54L.
Site-directed mutagenesis and mapping studies to identify critical interaction domains (RAD54L Lobe 1 and FBH1 2B subdomain).
Biochemical reconstitution assays using purified proteins to assess the functional synergy of FBH1 and RAD54L in fork reversal.

Main Results:

A direct physical and functional interaction between purified FBH1 and RAD54L was identified, forming a complex at stalled replication forks.
The RAD54L Lobe 1 domain is critical for binding to the FBH1 2B subdomain.
Complex formation is enhanced in RAD51-deficient cells, and purified RAD54L shows higher affinity for RAD51 than FBH1.
F H1 and RAD54L act sequentially, with FBH1 preceding RAD54L, to promote fork reversal more efficiently than either protein alone.

Conclusions:

RAD54L is an essential functional partner for FBH1 in the process of replication fork reversal.
The findings provide mechanistic insights into the sequential coordination of FBH1 and RAD54L activities, crucial for maintaining genome stability during replication stress.