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 Citations (Scopus)

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

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Phosphorylation
DNA Damage
Saccharomyces cerevisiae
Chemical activation
Yeast
Saccharomycetales
DNA
Proteins
Phosphotransferases
Homologous Recombination
Automatic teller machines
Substrates
Assays
cyhalothrin

ASJC Scopus subject areas

  • Biochemistry

Cite this

DNA Damage-Induced Phosphorylation of Rad55 Protein as a Sentinel for DNA Damage Checkpoint Activation in S. cerevisiae. / Bashkirov, Vladimir I.; Herzberg, Kristina; Haghnazari, Edwin; Vlasenko, Alexey S.; Heyer, Wolf Dietrich.

In: Methods in Enzymology, Vol. 409, 2006, p. 166-182.

Research output: Contribution to journalArticle

Bashkirov, Vladimir I. ; Herzberg, Kristina ; Haghnazari, Edwin ; Vlasenko, Alexey S. ; Heyer, Wolf Dietrich. / DNA Damage-Induced Phosphorylation of Rad55 Protein as a Sentinel for DNA Damage Checkpoint Activation in S. cerevisiae. In: Methods in Enzymology. 2006 ; Vol. 409. pp. 166-182.
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