Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity.
In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination.
Liberi
G
G
Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, via Celoria 26, 20133 Milan, Italy. giordano.liberi@unimi.it
Chiolo
I
I
Pellicioli
A
A
Lopes
M
M
Plevani
P
P
Muzi-Falconi
M
M
Foiani
M
M
D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002469:Cell Separation; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D047908:Intracellular Signaling Peptides and Proteins; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011995:Recombination, Genetic; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D013997:Time Factors
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C501395:RAD17 protein, S cerevisiae; C088385:RAD24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C061376:SRS2 protein, S cerevisiae; D008741:Methyl Methanesulfonate; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C067022:RAD53 protein, S cerevisiae; D010744:Phosphoric Monoester Hydrolases; D004265:DNA Helicases