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

Ryan Janke, Kristina Herzberg, Michael Rolfsmeier, Jordan Mar, Vladimir I. Bashkirov, Edwin Haghnazari, Greg Cantin, John R. Yates, Wolf Dietrich Heyer

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article numbergkp1222
Pages (from-to)2302-2313
Number of pages12
JournalNucleic Acids Research
Volume38
Issue number7
DOIs
StatePublished - Jan 8 2010

    Fingerprint

ASJC Scopus subject areas

  • Genetics

Cite this

Janke, R., Herzberg, K., Rolfsmeier, M., Mar, J., Bashkirov, V. I., Haghnazari, E., Cantin, G., Yates, J. R., & Heyer, W. D. (2010). A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae. Nucleic Acids Research, 38(7), 2302-2313. [gkp1222]. https://doi.org/10.1093/nar/gkp1222