truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae

• Published in: Nucleic acids research Vol. 38; no. 7; pp. 2302 - 2313
• Main Authors: Janke, Ryan, Herzberg, Kristina, Rolfsmeier, Michael, Mar, Jordan, Bashkirov, Vladimir I, Haghnazari, Edwin, Cantin, Greg, Yates, John R. III, Heyer, Wolf-Dietrich
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-04-2010
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Cell Cycle Proteins - metabolism; Checkpoint Kinase 2; DNA Breaks, Double-Stranded; DNA Repair; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Electrophoretic Mobility Shift Assay; G1 Phase - genetics; Genome Integrity, Repair and; Histones - metabolism; Intracellular Signaling Peptides and Proteins - metabolism; Phosphorylation; Protein-Serine-Threonine Kinases - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Serine - metabolism; Signal Transduction
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkp1222

In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55-S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases.