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Related Concept Videos

Homologous Recombination02:31

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 DNA Replication Fork01:02

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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...
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Restarting Stalled Replication Forks02:37

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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,...
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Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

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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...
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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
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Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase
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Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

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Visualizing recombination intermediates with single-stranded DNA curtains.

Zhi Qi1, Eric C Greene1

  • 1Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, United States.

Methods (San Diego, Calif.)
|April 4, 2016
PubMed
Summary
This summary is machine-generated.

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) using Rad51 recombinase filaments. Single-molecule DNA curtain methods visualize these transient complexes, revealing HR mechanisms.

Keywords:
Homologous recombinationHomology searchPresynaptic complexesRad51RecASingle-molecule approachesTotal internal reflection fluorescence microscopy

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Homologous recombination (HR) is essential for repairing DNA double-strand breaks (DSBs), preventing genomic instability and cancer.
  • Rad51 recombinase proteins form nucleoprotein filaments (presynaptic complexes) on single-stranded DNA at DSBs, crucial for homologous pairing.
  • Traditional methods struggle to analyze the transient and heterogeneous intermediates in HR.

Purpose of the Study:

  • To explore the molecular mechanisms of homologous recombination.
  • To investigate the properties of individual Rad51 presynaptic complexes.
  • To highlight the utility of single-molecule techniques in studying DNA repair.

Main Methods:

  • Utilizing single-stranded DNA curtain technology.
  • Directly visualizing individual Rad51 presynaptic complexes.
  • Observing reaction intermediates in real time and in solution.

Main Results:

  • Demonstrated the capability of single-molecule DNA curtains to study Rad51 presynaptic complexes.
  • Provided insights into the transient and heterogeneous nature of HR intermediates.
  • Showcased a novel approach for dissecting complex DNA repair pathways.

Conclusions:

  • Single-molecule DNA curtain methods offer powerful new ways to study homologous recombination.
  • Direct visualization of intermediates is key to understanding complex DNA repair mechanisms.
  • This approach advances our knowledge of DNA double-strand break repair and genomic stability.