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Related Concept Videos

Homologous Recombination02:31

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...
Restarting Stalled Replication Forks02:37

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

Published on: February 13, 2019

Assay for human Rad51-mediated DNA displacement loop formation.

Steven Raynard1, Patrick Sung

  • 1Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA.

Cold Spring Harbor Protocols
|February 12, 2010
PubMed
Summary

This study describes a biochemical assay to investigate how Rad51, a key protein in DNA repair, forms DNA displacement loops (D-loops) during homologous recombination. The protocol uses radiolabeled single-stranded DNA and supercoiled DNA as substrates. The reaction is analyzed using agarose gel electrophoresis and PhosphorImaging. Ancillary factors like the Hop2-Mnd1 complex or Rad54 are included to improve the efficiency of D-loop formation. The system allows researchers to study the molecular steps of homologous recombination in a controlled environment. The findings confirm that Rad51 forms a nucleoprotein filament in the presence of ATP and that ancillary factors enhance the reaction. This assay provides a valuable tool for investigating DNA repair mechanisms.

Keywords:
Rad51 recombinaseDNA displacement loophomologous recombinationD-loop assay

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Published on: June 19, 2018

Area of Science:

  • Molecular genetics within DNA repair mechanisms
  • Biochemistry of recombinase function
  • Cell biology of homologous recombination

Background:

Homologous recombination plays a central role in DNA repair and chromosome maintenance. It is essential for resolving stalled replication forks and repairing double-strand breaks. The Rad51 recombinase is a key player in this process. Rad51 forms a nucleoprotein filament on single-stranded DNA in the presence of ATP. This filament can search for homologous DNA sequences and drive strand invasion. The resulting DNA displacement loop, or D-loop, is a critical intermediate in homologous recombination. Prior research has shown that D-loop formation is a conserved process across eukaryotes. However, the precise biochemical mechanisms remain incompletely understood. This gap motivated the development of a reconstituted in vitro system to study D-loop formation in detail.

Purpose Of The Study:

The aim of this work is to establish a biochemical assay for Rad51-mediated D-loop formation. This protocol allows for the study of homologous recombination in a controlled environment. The study addresses the need for a reproducible method to analyze Rad51 activity. The protocol uses radiolabeled ssDNA and supercoiled duplex DNA as substrates. Agarose gel electrophoresis and PhosphorImaging are employed for product detection. The inclusion of ancillary factors like the Hop2-Mnd1 complex or Rad54 enhances reaction efficiency. This system enables researchers to dissect the molecular steps of homologous recombination. The study provides a tool to investigate the role of Rad51 and its cofactors in DNA repair.

Main Methods:

The assay employs a radiolabeled ssDNA oligonucleotide and a nonlabeled supercoiled duplex DNA as substrates. The reaction is carried out in vitro under controlled conditions. Rad51 is incubated with ATP to form a nucleoprotein filament on the ssDNA. The filament is then allowed to locate and invade a homologous duplex DNA. Agarose gel electrophoresis separates the reaction products. PhosphorImaging detects the radiolabeled D-loop structures. Ancillary factors such as the Hop2-Mnd1 complex or Rad54 are added to improve reaction efficiency. This approach allows for the biochemical dissection of D-loop formation.

Main Results:

The protocol successfully detects D-loop formation in vitro using radiolabeled ssDNA and supercoiled DNA. Rad51 forms a nucleoprotein filament that mediates DNA invasion. The presence of ATP is necessary for Rad51 filament formation. Agarose gel electrophoresis and PhosphorImaging confirm D-loop production. The Hop2-Mnd1 complex or Rad54 significantly enhances the efficiency of the reaction. The system allows for the detection of D-loops with high sensitivity. The assay provides a reliable method to study homologous recombination. The use of ancillary factors improves the yield and reproducibility of the D-loop formation.

Conclusions:

The described protocol provides a biochemical means to study Rad51-mediated D-loop formation. The system allows for the analysis of homologous recombination in vitro. The use of radiolabeled substrates and PhosphorImaging ensures accurate detection. The inclusion of ancillary factors like Hop2-Mnd1 or Rad54 improves reaction efficiency. The assay can be adapted to investigate the role of Rad51 and its cofactors. The study confirms that ATP is essential for Rad51 filament formation. The system enables the dissection of molecular steps in homologous recombination. The protocol offers a valuable tool for researchers in DNA repair and recombination.

The main outcome is the detection of DNA displacement loops (D-loops) formed by Rad51-mediated strand invasion using radiolabeled ssDNA and supercoiled DNA substrates.

The Hop2-Mnd1 complex or Rad54 enhances the efficiency of D-loop formation by acting as ancillary factors in the Rad51-mediated reaction.

Agarose gel electrophoresis separates reaction products, allowing visualization of D-loop formation through PhosphorImaging of radiolabeled DNA.

The radiolabeled ssDNA oligonucleotide enables the detection of D-loop formation via PhosphorImaging, ensuring high sensitivity in the assay.

ATP is necessary for Rad51 to polymerize on ssDNA and form the nucleoprotein filament that mediates DNA invasion.

The assay allows researchers to dissect the molecular mechanisms of homologous recombination by providing a reconstituted system to study D-loop formation in vitro.