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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 Damage can Stall the Cell Cycle02:36

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In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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DNA Damage Can Stall the Cell Cycle02:36

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In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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Translesion DNA Polymerases02:10

Translesion DNA Polymerases

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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
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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.
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Related Experiment Video

Updated: Feb 27, 2026

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts
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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Published on: November 5, 2012

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Xenopus laevis as Model System to Study DNA Damage Response and Replication Fork Stability.

Vincenzo Sannino1, Federica Pezzimenti1, Stefania Bertora1

  • 1DNA Metabolism Laboratory, IFOM-The FIRC Institute of Molecular Oncology, Milan, Italy.

Methods in Enzymology
|June 25, 2017
PubMed
Summary

This study uses Xenopus egg extract to investigate DNA damage response and repair mechanisms. It details protocols for studying chromosome breakage and analyzing replication intermediates using electron microscopy.

Keywords:
DNA damage checkpointsDNA replicationElectron microscopyReplication forksXenopus laevis

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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts
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Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells
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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • The biochemical roles of DNA damage response and repair proteins are not fully understood.
  • The Xenopus laevis egg extract system offers a cell-free environment for dissecting complex biological processes like DNA replication and cell cycle regulation.
  • Protein depletion in egg extracts enables the study of essential proteins whose inactivation is lethal.

Purpose of the Study:

  • To present protocols for recapitulating DNA damage response triggered by chromosome breakage in Xenopus egg extract.
  • To describe methods for isolating replication intermediates for electron microscopy analysis.
  • To enhance understanding of genome stability maintenance under normal and stressful conditions.

Main Methods:

  • Utilizing the Xenopus laevis egg extract cell-free system.
  • Employing protein depletion techniques to manipulate protein content.
  • Applying advanced electron microscopy for characterizing replication intermediates.
  • Using sperm nuclei or defined genomic substrates for analysis.

Main Results:

  • Established protocols to study DNA damage response and replication-repair coordination in egg extract.
  • Enabled visualization of replication intermediates formed in the absence of key DNA repair proteins.
  • Provided insights into genome stability mechanisms.

Conclusions:

  • The Xenopus egg extract system is a powerful tool for biochemical dissection of DNA damage response and repair.
  • Advanced imaging techniques combined with egg extracts facilitate the characterization of replication dynamics under DNA damage.
  • These methods contribute to understanding how cells preserve genome integrity.