Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage

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. [Display omitted] •Crystal structures show Ddc2 homodimers bind Rfa1 by its N-terminal helix•Ddc2 RBD binds Rfa1 in a manner that is independent of the mutation rfa1-t11•Loss of Ddc2 homodimerization and Rfa1 binding compromises T-T dimer repair•Efficient long-patch NER requires Ddc2-RPA-ssDNA-mediated Mec1 activation Deshpande et al. report the co-crystal structure of homodimeric Ddc2 N terminus bound to two Rfa1 N-OB domains and show that Ddc2 recruitment and coiled-coil domains are important for cell survival after damage by UV light. A composite structural model of Mec1-Ddc2 assembly on ssDNA-RPA at DNA damage sites is presented.