DNA damage-induced phosphorylation of Rad55 protein as a sentinel for DNA damage checkpoint activation in S. cerevisiae.
Rad55 protein is one of two Rad51 paralogs in the budding yeast Saccharomyces cerevisiae and forms a stable heterodimer with Rad57, the other Rad51 paralog. The Rad55-Rad57 heterodimer functions in homologous recombination during the assembly of the Rad51-ssDNA filament, which is central for homology search and DNA strand exchange. Previously, we identified Rad55 protein as a terminal target of the DNA damage checkpoints, which coordinate the cellular response to genotoxic stress. Rad55 protein phosphorylation is signaled by a significant electrophoretic shift and occurs in response to a wide range of genotoxic stress. Here, we map the phosphorylation site leading to the electrophoretic shift and show that Rad55 protein is a bona fide direct in vivo substrate of the central DNA damage checkpoint kinase Mec1, the budding yeast equivalent of human ATM/ATR. We provide protocols to monitor the Rad55 phosphorylation status in vivo and assay Rad55-Rad57 phosphorylation in vitro using purified substrate with the Mec1 and Rad53 checkpoint kinases.
Bashkirov
Vladimir I
VI
Section of Microbiology, University of California, Davis, USA.
Herzberg
Kristina
K
Haghnazari
Edwin
E
Vlasenko
Alexey S
AS
Heyer
Wolf-Dietrich
WD
D018797:Cell Cycle Proteins / Q000378:metabolism; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D047468:Immunoprecipitation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C087191:RAD55 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae