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 language | English (US) |
|---|---|
| Pages (from-to) | 166-182 |
| Number of pages | 17 |
| Journal | Methods in Enzymology |
| Volume | 409 |
| DOIs | |
| State | Published - 2006 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - DNA Damage-Induced Phosphorylation of Rad55 Protein as a Sentinel for DNA Damage Checkpoint Activation in S. cerevisiae
AU - Bashkirov, Vladimir I.
AU - Herzberg, Kristina
AU - Haghnazari, Edwin
AU - Vlasenko, Alexey S.
AU - Heyer, Wolf Dietrich
PY - 2006
Y1 - 2006
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33745198662&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33745198662&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(05)09010-5
DO - 10.1016/S0076-6879(05)09010-5
M3 - Article
C2 - 16793401
AN - SCOPUS:33745198662
VL - 409
SP - 166
EP - 182
JO - Methods
JF - Methods
SN - 0076-6879
ER -