Two checkpoint complexes are independently recruited to sites of DNA damage in vivo
Open accessAbstract
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.
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Two complexes are independently recruited to lesion sites: the ATR/ATRIP or Mec1/Ddc2 complex, and the 9-1-1 checkpoint clamp complex.
“Two complexes are independently recruited at the lesion sites [17]: the human ATR/ATRIP or Saccharomyces cerevisiae Mec1/Ddc2 complex, and the 9-1-1 checkpoint clamp complex, composed of Rad9-Rad1-Hus1 in human or their orthologue subunits Rad17-Mec3-Ddc1 in yeast.”
From Mind the gap: keeping UV lesions in check (2011) · cites this paper as [17]
The ATR-ATRIP complex and the 9-1-1 complex are recruited to RPA-coated single-stranded DNA independently and are bridged by TopBP1.
“The ATR-ATRIP complex and 9-1-1 complexes are recruited to RPA-ssDNA independently and are bridged by the multiple-function protein TopBP1 (Topoisomerase IIβ binding protein 1) [106–110].”
From Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress (2014) · cites this paper as [107]
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“Two complexes are independently recruited t the lesion sites [17]: the human ATR/ATRIP or Saccharomyces”
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- Authors
- Melo, J. A., Cohen, J., & Toczyski, D. P.
- Affiliations
- University of California, San Francisco (US)
- Venue
- Genes & Development
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2001-11-01
- Volume
- 15
- Issue
- 21
- Pages
- 2809-2821
- ISSN
- 0890-9369 (ISSN-L)
- 1549-5477
- PMC
- PMC312815
- OpenAlex
- W2107130407
- Cited by
- 384
- References
- 36
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 9 · 2023: 5 · 2022: 3 · 2021: 2 · 2020: 3 · 2019: 6 · 2018: 5 · 2017: 7 · 2016: 7 · 2015: 11 · 2014: 11 · 2013: 11 · 2012: 6
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genes & Development (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- University of California, San Francisco
- National Institute of General Medical Sciences (GM59691-01, R01GM059691)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- DNA damage checkpoint, Rad24, Mec1 kinase, checkpoint adaptation, cell cycle re-entry, Saccharomyces cerevisiae
- MeSH terms
- Chromatin, DNA Damage, DNA-Binding Proteins, Fungal Proteins, Genes, Fungal, Luminescent Proteins, Microscopy, Fluorescence, Mitosis, Models, Biological, Nuclear Proteins, Phosphoproteins, Phosphorylation, Precipitin Tests, Protein Binding, Recombinant Fusion Proteins, Temperature, Time Factors, Down-Regulation, Protein Serine-Threonine Kinases, Cell Cycle Proteins, Saccharomyces cerevisiae Proteins, Intracellular Signaling Peptides and Proteins, Adaptor Proteins, Signal Transducing, Green Fluorescent Proteins
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