Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase.
The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.
Pellicioli
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A
Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, via Celoria 26, 20133 Milan, Italy.
Lucca
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C
Liberi
G
G
Marini
F
F
Lopes
M
M
Plevani
P
P
Romano
A
A
Di Fiore
P P
PP
Foiani
M
M
D002453:Cell Cycle; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism; D016193:G1 Phase; D005838:Genotype; D008957:Models, Genetic; D016296:Mutagenesis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D004259:DNA-Directed DNA Polymerase