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Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage
Ishan Deshpande1, Andrew Seeber2, Kenji Shimada2
1Friedrich Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, Faculty of Natural Sciences, Klingelbergstrasse 50, 4056 Basel, Switzerland.
Abstract:
Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage.
Insights
The Mec1-Ddc2 (ATR-ATRIP) complex, essential for DNA damage response, forms a homodimer at single-stranded DNA sites. This structural study reveals how Replication Protein A (RPA) facilitates this crucial homodimerization for DNA repair.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Mec1-Ddc2 (ATR-ATRIP) is a critical kinase for DNA damage response.
- Damage-specific recruitment of Mec1-Ddc2 is essential to prevent lethal activation without DNA damage.
- Mec1-Ddc2 homodimerization is a key mechanism for regulating kinase activation.
Purpose of the Study:
- To structurally, biochemically, and functionally characterize the yeast Mec1-Ddc2-RPA assembly.
- To elucidate the mechanisms of Mec1-Ddc2 homodimerization and DNA damage site targeting.
- To develop a composite structural model of the Mec1-Ddc2-RPA-ssDNA complex.
Main Methods:
- X-ray crystallography to obtain high-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 N-termini, and the Ddc2 coiled-coil domain (CCD).
- Biochemical assays to assess Mec1-Ddc2 homodimerization and RPA-dependent recruitment.
- Functional studies to evaluate the importance of Ddc2 domains in DNA damage survival.
Main Results:
- High-resolution structures reveal insights into Mec1-Ddc2 homodimerization and targeting.
- RPA-dependent recruitment of Mec1-Ddc2 is critical for maintaining its homodimeric state at single-stranded DNA.
- The Ddc2 recruitment domain and CCD are vital for Mec1-dependent survival following UV-induced DNA damage.
Conclusions:
- The study provides a structural and functional basis for Mec1-Ddc2-RPA interaction at DNA damage sites.
- RPA plays a crucial role in stabilizing Mec1-Ddc2 homodimers at ssDNA, ensuring damage-specific activation.
- Understanding these interactions is key to comprehending the DNA damage checkpoint response and developing therapeutic strategies.