DNA damage-induced phosphorylation of Rad55 protein as a sentinel for DNA damage checkpoint activation in S. cerevisiae

• Published in: Methods in enzymology Vol. 409; p. 166
• Main Authors: Bashkirov, Vladimir I, Herzberg, Kristina, Haghnazari, Edwin, Vlasenko, Alexey S, Heyer, Wolf-Dietrich
• Format: Journal Article
• Language: English
• Published: United States 2006
• Subjects: Cell Cycle Proteins - metabolism; Checkpoint Kinase 2; Dimerization; DNA Damage; DNA-Binding Proteins - metabolism; Immunoprecipitation; Phosphorylation; Protein-Serine-Threonine Kinases - metabolism; Saccharomyces cerevisiae Proteins - metabolism
• ISSN: 0076-6879
• DOI: 10.1016/S0076-6879(05)09010-5

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.