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Related Concept Videos

The DNA Replication Fork01:02

The DNA Replication Fork

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 Replisome03:01

The Replisome

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 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...
Lagging Strand Synthesis01:59

Lagging Strand Synthesis

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...
DNA Replication02:40

DNA Replication

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 uses a large number of...
Chromosome Replication02:31

Chromosome Replication

Before a cell can divide, it must accurately replicate all of its chromosomes, including the DNA and its associated histone and non-histone proteins.  This process begins at numerous origins of replication during the S phase of the cell cycle in each of a cell’s chromosomes simultaneously. Certain nucleotides can act as origins of replication, but these sequences are not well defined - especially in complex, multi-cellular, eukaryotic species. The length of DNA that spans an origin of...

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Related Experiment Video

Updated: Jun 22, 2026

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
15:57

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

Published on: October 9, 2009

Electron microscopy methods for studying in vivo DNA replication intermediates.

Massimo Lopes1

  • 1Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland.

Methods in Molecular Biology (Clifton, N.J.)
|July 1, 2009
PubMed
Summary
This summary is machine-generated.

Understanding DNA replication requires structural details. Psoralen crosslinking and electron microscopy visualize in vivo DNA replication intermediates, revealing duplication mechanisms and genotoxic effects.

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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Detailed understanding of DNA replication necessitates structural insights.
  • Psoralen crosslinking and electron microscopy are key techniques for studying DNA replication intermediates.
  • This methodology has been crucial for elucidating DNA duplication mechanisms and the impact of genotoxic agents.

Purpose of the Study:

  • To outline procedures for visualizing in vivo DNA replication intermediates.
  • To provide a method for stabilizing and extracting replication structures for electron microscopy.
  • To enable structural analysis of genomic DNA replication in various cell types.

Main Methods:

  • In vivo stabilization of DNA replication structures using psoralen crosslinking.
  • Extraction and enrichment of crosslinked DNA replication intermediates.
  • Visualization of intermediates using transmission electron microscopy.
  • Application to budding yeast and cultured mammalian cells.

Main Results:

  • Successful visualization of in vivo DNA replication intermediates.
  • Structural insights into basic DNA duplication mechanisms.
  • Understanding of how genotoxic treatments perturb DNA replication.

Conclusions:

  • Psoralen crosslinking combined with electron microscopy is a powerful approach for studying DNA replication.
  • This technique provides essential structural information about DNA replication intermediates.
  • The outlined procedures are applicable to different biological systems, including yeast and mammalian cells.