A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae.
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.
Janke
Ryan
R
Department of Microbiology, University of California, Davis, CA 95616-8665, USA.
Herzberg
Kristina
K
Rolfsmeier
Michael
M
Mar
Jordan
J
Bashkirov
Vladimir I
VI
Haghnazari
Edwin
E
Cantin
Greg
G
Yates
John R
JR
Heyer
Wolf-Dietrich
WD
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D064447:Checkpoint Kinase 2; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D016193:G1 Phase / Q000235:genetics; D006657:Histones / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C440014:LCD1 protein, S cerevisiae; C087191:RAD55 protein, S cerevisiae; C541026:RPA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae