DNA Damage-Induced Phosphorylation of Rad55 Protein as a Sentinel for DNA Damage Checkpoint Activation in S. cerevisiae

Vladimir I. Bashkirov, Kristina Herzberg, Edwin Haghnazari, Alexey S. Vlasenko, Wolf Dietrich Heyer

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)166-182
Number of pages17
JournalMethods in Enzymology
Volume409
DOIs
StatePublished - 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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