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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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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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DNA Replication02:40

DNA Replication

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DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
DNA replication...
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The Replisome03:01

The Replisome

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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
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Lagging Strand Synthesis01:59

Lagging Strand Synthesis

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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.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
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Replication in Prokaryotes01:32

Replication in Prokaryotes

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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.
Many Proteins Work Together to Replicate the Chromosome
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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

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FORK-seq: Single-Molecule Profiling of DNA Replication.

Magali Hennion1, Bertrand Theulot2,3, Jean-Michel Arbona4

  • 1Epigenetics and Cell Fate, CNRS, Université de Paris, Paris, France.

Methods in Molecular Biology (Clifton, N.J.)
|May 6, 2022
PubMed
Summary
This summary is machine-generated.

FORK-seq is a new nanopore sequencing method that maps DNA replication in single cells. This technique reveals cell-to-cell differences in genome replication, overcoming limitations of population-based studies.

Keywords:
Convolutional neural networkDNA replicationNanopore sequencingReplication fork directionReplication originsSingle-molecule analysisTermination sitesWhole-genome

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Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay
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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Traditional genome replication mapping methods analyze cell populations, obscuring crucial cell-to-cell variability.
  • Understanding DNA replication heterogeneity is vital for comprehending genome stability and cellular processes.

Purpose of the Study:

  • To introduce FORK-seq, a novel single-molecule nanopore sequencing method for high-resolution DNA replication mapping.
  • To overcome the limitations of population-based approaches in studying DNA replication.

Main Methods:

  • FORK-seq utilizes nanopore sequencing to analyze DNA replication intermediates at 200-nucleotide resolution.
  • The method incorporates a nanopore current interpretation tool for quantifying BrdU incorporation.
  • Applied to Saccharomyces cerevisiae, FORK-seq analyzes pulse-chased replication intermediates.

Main Results:

  • FORK-seq successfully maps individual DNA replication tracks and allows for orientation.
  • The method accurately reproduces population-based replication directionality profiles.
  • FORK-seq enables the precise mapping of individual replication initiation and termination events.
  • The study demonstrates the capability of FORK-seq to uncover cell-to-cell heterogeneity in DNA replication.

Conclusions:

  • FORK-seq provides unprecedented insight into single-cell DNA replication dynamics.
  • This method significantly advances the study of genome replication by resolving cell-to-cell heterogeneity.
  • FORK-seq opens new avenues for investigating the complexities of DNA replication.