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Updated: Jul 9, 2026
Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
Published on: June 8, 2018
Two checkpoint complexes are independently recruited to sites of DNA damage in vivo
J A Melo1, J Cohen, D P Toczyski
1Mt. Zion Cancer Research Institute, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94115, USA.
Abstract:
The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by "sensing" DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1-GFP or Ddc2-GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2-GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.
Insights
DNA damage checkpoint proteins Ddc1 and Ddc2 are recruited to DNA damage sites through independent mechanisms. Visualization of these proteins in living cells aids in DNA damage analysis.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The Ddc1/Rad17/Mec3 complex and Rad24 are known DNA damage checkpoint components.
- Mec1 kinase phosphorylates Ddc2 upon DNA damage, suggesting Mec1/Ddc2 as potential initial sensors.
- Previous models proposed direct sensing of DNA damage by checkpoint factors.
Purpose of the Study:
- To investigate the roles of Ddc1 and Ddc2 in sensing and responding to DNA damage.
- To determine the mechanisms of Ddc1 and Ddc2 recruitment to DNA damage sites.
- To explore the utility of visualizing checkpoint proteins at damage sites in living cells.
Main Methods:
- Utilized green fluorescent protein (GFP) fusions to Ddc1 and Ddc2 to visualize their localization in living cells.
- Induced DNA damage using endonucleolytic breaks and other damaging agents.
- Analyzed Ddc1 and Ddc2 localization in wild-type, mec1, and other checkpoint mutant cells.
Main Results:
- Ddc1-GFP and Ddc2-GFP localize to subnuclear foci at sites of DNA damage.
- Ddc2 localization is Mec1-dependent but independent of other known checkpoint genes.
- Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1, indicating independent recognition mechanisms.
- Ddc1 remains localized after checkpoint adaptation, while Ddc2 foci diminish, suggesting regulation during cell cycle resumption.
Conclusions:
- Ddc1 and Ddc2 recognize DNA damage via independent pathways, supporting a model of multi-complex assembly at damage sites.
- Visualization of Ddc1-GFP and Ddc2-GFP provides a valuable tool for analyzing DNA damage in real-time.
- Ddc2 localization appears to be downregulated during checkpoint adaptation and cell division resumption.
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