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Related Concept Videos

The DNA Replication Fork01:02

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...
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During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
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Homologous Recombination02:31

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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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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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Replication in Prokaryotes01:32

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DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell's circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
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Updated: Sep 30, 2025

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay
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Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

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Studying Single-Stranded DNA Gaps at Replication Intermediates by Electron Microscopy.

Jessica Jackson1, Alessandro Vindigni2

  • 1Division of Oncology, Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 15, 2022
PubMed
Summary
This summary is machine-generated.

This study details a method for visualizing single-stranded DNA gaps at replication forks using electron microscopy. This technique aids in understanding DNA replication stress and fork dynamics.

Keywords:
DNA replicationDNA replication stressElectron microscopyReplication structuresssDNA gaps

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Single-stranded DNA gaps frequently form on newly synthesized DNA during replication stress.
  • Identifying these gaps is crucial for understanding how DNA replication overcomes obstacles and lesions.
  • DNA fiber assays can detect replication fork perturbations but lack direct visualization of gaps.

Purpose of the Study:

  • To provide a detailed electron microscopy (EM) method for visualizing single-stranded DNA gaps at replication forks.
  • To enable direct visualization and localization of these critical DNA structures.
  • To compare EM with DNA fiber assays for studying replication fork dynamics.

Main Methods:

  • Psoralen cross-linking of cultured mammalian cells.
  • Genomic DNA extraction and enrichment of replication intermediates.
  • DNA spreading and platinum rotary shadowing for electron microscopy.

Main Results:

  • Electron microscopy allows direct visualization and precise localization of single-stranded DNA gaps at replication forks.
  • The described method enables detailed analysis of gap structures and their context within replication forks.
  • Comparison highlights EM's advantage for direct visualization over indirect methods like DNA fiber assays.

Conclusions:

  • Electron microscopy offers a powerful, direct approach for visualizing single-stranded DNA gaps at replication forks.
  • This method enhances the study of DNA replication stress and fork dynamics at the molecular level.
  • EM provides complementary insights to DNA fiber assays, improving our understanding of genome stability mechanisms.