Related Experiment Video

Updated: Jun 17, 2026

Visualization of DNA Repair Proteins Interaction by Immunofluorescence
07:55

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Published on: June 26, 2020

Enhanced H2AX phosphorylation, DNA replication fork arrest, and cell death in the absence of Chk1

Mary E Gagou1, Pedro Zuazua-Villar, Mark Meuth

  • 1Institute for Cancer Studies, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom.

Molecular Biology of the Cell
|January 8, 2010
PubMed

Insights

Phosphorylation of H2AX (gammaH2AX) indicates DNA damage. In Chk1-depleted cells, gammaH2AX forms at stalled replication forks but does not commit cells to death.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • DNA Damage Response

Background:

  • H2AX phosphorylation at serine 139 (gammaH2AX) is a key marker for DNA damage and replication stress.
  • The role of gammaH2AX in cell fate decisions following replication stress, particularly in the context of checkpoint inhibition, requires further elucidation.

Purpose of the Study:

  • To investigate the formation and significance of gammaH2AX foci in Chk1-depleted cells under replication stress.
  • To determine whether gammaH2AX accumulation precedes apoptosis and if these foci are associated with cell death.
  • To explore the relationship between gammaH2AX, replication fork stability, and the resumption of DNA synthesis after stress release.

Main Methods:

  • Utilized HCT116 and SW480 cell lines with Chk1 depletion.
  • Applied replication inhibitors and ionizing radiation to induce stress.
  • Monitored gammaH2AX foci formation and colocalization with RPA foci.
  • Assessed BrdU incorporation and activation of ATM/Chk2 kinases.
  • Investigated the impact of releasing cells from thymidine arrest.

Main Results:

  • GammaH2AX formation was significantly enhanced by replication inhibitors but not ionizing radiation in Chk1-depleted cells.
  • GammaH2AX foci colocalized with RPA foci and depended on Cdc45, indicating association with stalled replication forks.
  • Cells with gammaH2AX were not committed to apoptosis, and gammaH2AX persisted after stress release.
  • Resumed DNA synthesis was limited and did not occur at gammaH2AX foci sites in released cells.
  • Activated ATM and Chk2 kinases remained detectable in Chk1-depleted cells.

Conclusions:

  • GammaH2AX foci in Chk1-depleted cells likely represent persistent replication fork damage or abandonment.
  • These gammaH2AX sites are unable to resume DNA synthesis but do not directly mediate Chk1-suppressed cell death.
  • The findings differentiate the role of gammaH2AX in replication stress from its role in response to DNA double-strand breaks.

More Related Videos

Quantification of γH2AX Foci in Response to Ionising Radiation06:53

Quantification of γH2AX Foci in Response to Ionising Radiation

Published on: April 6, 2010

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage10:44

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Published on: January 31, 2018

Related Concept Videos

DNA Damage can Stall the Cell Cycle02:36

DNA Damage can Stall the Cell Cycle

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...
DNA Damage Can Stall the Cell Cycle02:36

DNA Damage Can Stall the Cell Cycle

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...
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...
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...
Homologous Recombination02:31

Homologous Recombination

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...
Inhibition of Cdk Activity02:34

Inhibition of Cdk Activity

The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex...