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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

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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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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

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Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

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ruvA Mutants that resolve Holliday junctions but do not reverse replication forks.

Zeynep Baharoglu¹, Alison Sylvia Bradley, Marie Le Masson

¹CNRS, Centre de Génétique Moléculaire, UPR 2167, Gif-sur-Yvette, France.

|March 29, 2008

Summary

Researchers isolated two ruvA mutants that can resolve Holliday junctions (HJs) but not reverse replication forks. This separation of functions demonstrates distinct roles for RuvA in DNA repair and replication fork stability.

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Area of Science:

Molecular Biology
Genetics
DNA Repair Mechanisms

Background:

The RuvAB and RuvABC complexes are crucial for resolving Holliday junctions (HJs) during homologous recombination.
These complexes also process HJs formed by replication fork reversal at inactivated replication forks.
RuvAB has been implicated in directly catalyzing replication fork conversion into HJs.

Purpose of the Study:

To isolate and characterize separation-of-function ruvA mutants.
To investigate the distinct roles of RuvA in Holliday junction resolution and replication fork reversal.
To understand the mechanism by which RuvA interacts with DNA and RuvB helicase activity.

Main Methods:

Isolation and characterization of two ruvA mutants with separation-of-function phenotypes.
Assays for conjugational recombination and recombinational repair of DNA damage (UV, mitomycin C).
In vivo and in vitro experiments to assess DNA binding and RuvB helicase stimulation.

Main Results:

Two ruvA mutants were identified that can resolve HJs but are deficient in replication fork reversal.
These mutants retain the capacity for homologous recombination and repair of DNA lesions.
Evidence suggests the mutations impair RuvA's DNA binding and its stimulation of RuvB helicase activity.

Conclusions:

RuvA's functions in replication fork processing and Holliday junction resolution can be genetically separated.
Mutations affecting RuvA's interaction with DNA and RuvB helicase activity disrupt fork reversal but not HJ resolution.
This study clarifies the distinct roles of RuvA in maintaining genome stability during recombination and replication.