Related Experiment Video
Updated: Aug 4, 2025
Visualization of DNA Repair Proteins Interaction by Immunofluorescence
Published on: June 26, 2020
A DNA damage-induced phosphorylation circuit enhances Mec1ATR Ddc2ATRIP recruitment to Replication Protein A
Luke A Yates1, Elias A Tannous2, R Marc Morgan3
1Section of Structural Biology, Department of Infectious Disease, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.
Abstract:
The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are vital for the DNA damage and replication stress response. Mec1-Ddc2 "senses" single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA damage-induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2-RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1-Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modeling approaches, we propose that Mec1-Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1-Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signaling.
Insights
DNA damage response relies on Mec1-Ddc2 recruitment to RPA-ssDNA. Phosphorylation of Ddc2 enhances this interaction, promoting checkpoint signaling and DNA repair in yeast.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Mec1 (ATR) and Ddc2 (ATRIP) are key kinases in DNA damage and replication stress responses.
- Mec1-Ddc2 complex binds single-stranded DNA (ssDNA) via Replication Protein A (RPA) through Ddc2.
Purpose of the Study:
- To investigate the role of DNA damage-induced phosphorylation in Mec1-Ddc2 checkpoint recruitment.
- To elucidate the molecular mechanisms by which Ddc2 phosphorylation enhances Mec1-Ddc2 binding to RPA-ssDNA.
Main Methods:
- Biochemical assays to study Ddc2-RPA interactions and RPA-ssDNA association.
- Phosphorylation analysis of RPA and Ddc2.
- X-ray crystallography to determine the structure of phosphorylated Ddc2 peptide with RPA.
- Electron microscopy and structural modeling to visualize Mec1-Ddc2-RPA complexes.
Main Results:
- DNA damage induces a phosphorylation circuit that modulates Mec1-Ddc2 recruitment.
- Ddc2-RPA interactions influence RPA-ssDNA binding, and Rfa1 phosphorylation aids Mec1-Ddc2 recruitment.
- Ddc2 phosphorylation significantly enhances Mec1-Ddc2 recruitment to RPA-ssDNA in yeast.
- Structural data reveals Zn2+ involvement in enhanced checkpoint recruitment.
- Phosphorylated Mec1-Ddc2 can form higher-order assemblies with RPA, suggesting supramolecular complex formation.
Conclusions:
- Phosphorylation acts as a critical regulator of Mec1-Ddc2 recruitment to DNA damage sites.
- Enhanced recruitment via Ddc2 phosphorylation promotes efficient clustering of damage foci, facilitating robust checkpoint signaling.
- The findings provide molecular insights into Mec1 recruitment and DNA damage response mechanisms.
Related Concept Videos
02:37DNA Damage can Stall the Cell Cycle
02:37Restarting Stalled Replication Forks
02:31Homologous Recombination
02:38S-Cdk Initiates DNA Replication
Two states at the origin of replication
In eukaryotes, the initiation of replication occurs at many sites on the chromosomes, called the origins of...
01:38Nucleotide Excision Repair
Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...
02:10Translesion DNA Polymerases
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...