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

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.

Yeast ATM and ATR use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

“…Ddc2 assembles at the DSB in a Mec1-dependent manner (26,27). Structural work on the Mec1-Ddc2 heterodimer suggests that it forms a stable focus at RPA-coated ssDNA with the coiled-coil domain of Ddc2 acting as a flexible linker so that Mec1 can freely phosphorylate nearby targets (28). If Mec1-Ddc2 remains at the break, then the looping mechanism would be the only possible option among the models that we considered, since the other mechanisms would require the kinase to detach from the DSB after recruitment.…”

“…In the tables displaying the Bayes factors(Table 1and S1), Model B is always the model with the highest marginal likelihood compared to the other models in the table.We use previous studies to estimate the range of possible parameter values -the integral in equation(28) is over this range. The previous studies measured the chromatin Kuhn length (95%…”

The Sir4 H‐ BRCT domain interacts with phospho‐proteins to sequester and repress yeast heterochromatin

“…Immunoprecipitation was performed as in Deshpande et al (2017) with the following modifications: Myc-Trap (chromotek-ytma-20) magnetic beads were used to pull-down Sir4-Myc. Roche complete protease inhibitor tablets (Sigma-04693116001) were used at twice the recommended concentration to prevent Sir4/Tom1 degradation.…”

Phosphoproteomics Reveals a Distinct Mode of Mec1/ATR Signaling in Response to DNA End Hyper-Resection

“…In eukaryotes, PI3K-like kinases (PIKKs) play essential roles in the sensing of DNA damage and the coordination of cell cycle checkpoint activation and DNA repair mechanisms to maintain genome stability (Savitsky et al, 1995; Shiloh, 2003). In Saccharomyces cerevisiae , the Mec1 PIKK (human ATR) plays a central role in coordinating the response to DNA lesions that result in single stranded DNA (ssDNA) exposure, including stalled replication forks and recessed double strand breaks (DSBs) (Cha & Kleckner, 2002; Deshpande et al, 2017; Segurado & Diffley, 2008; Tercero, Longhese, & Diffley, 2003). Mec1 senses ssDNA exposure through the recognition of replication protein A (RPA)-bound ssDNA via its obligate cofactor Ddc2 (human ATRIP) (Deshpande et al, 2017; Paciotti, Clerici, Lucchini, & Longhese, 2000; Zou & Elledge, 2003).…”

“…In Saccharomyces cerevisiae , the Mec1 PIKK (human ATR) plays a central role in coordinating the response to DNA lesions that result in single stranded DNA (ssDNA) exposure, including stalled replication forks and recessed double strand breaks (DSBs) (Cha & Kleckner, 2002; Deshpande et al, 2017; Segurado & Diffley, 2008; Tercero, Longhese, & Diffley, 2003). Mec1 senses ssDNA exposure through the recognition of replication protein A (RPA)-bound ssDNA via its obligate cofactor Ddc2 (human ATRIP) (Deshpande et al, 2017; Paciotti, Clerici, Lucchini, & Longhese, 2000; Zou & Elledge, 2003). Once recruited to ssDNA, activation of Mec1 requires the action of Mec1 activating proteins that contain Mec1 activation domains (MADs).…”