Abstract
A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.
MeSH Terms
CDC2 Protein Kinase/metabolism
Cell Cycle/drug effects
Cell Cycle Proteins/metabolism
Checkpoint Kinase 2
DNA Damage/drug effects
DNA Repair/genetics
DNA, Fungal/genetics,metabolism
Genes, Fungal/genetics
Mating Factor
Nocodazole/pharmacology
Peptides/pharmacology
Protein Serine-Threonine Kinases/metabolism
Recombination, Genetic/genetics
Saccharomyces cerevisiae/drug effects,enzymology,genetics
Saccharomyces cerevisiae Proteins/metabolism
Sequence Homology, Nucleic Acid
Chemicals
Cell Cycle Proteins
DNA, Fungal
Peptides
Saccharomyces cerevisiae Proteins
Mating Factor
Checkpoint Kinase 2
Protein Serine-Threonine Kinases
CDC2 Protein Kinase
RAD53 protein, S cerevisiae
Nocodazole
Authors & Affiliations
12 authors, click to expand affiliations / ORCID
Ira Grzegorz
Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
Pellicioli Achille
Balijja Alitukiriza
Wang Xuan
Fiorani Simona
Carotenuto Walter
Liberi Giordano
Bressan Debra
Wan Lihong
Hollingsworth Nancy M
Haber James E
Foiani Marco
References (29)
29 references, click to expand
-
Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination.
Mol Cell. 2002 Nov;10(5):1189-99
PMID: 12453425
-
Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase.
Mol Cell. 2002 Aug;10 (2):373-85
PMID: 12191482
-
In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination.
Mol Cell. 2003 Jul;12(1):209-19
PMID: 12887906
-
Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex.
Nat Cell Biol. 2001 Sep;3(9):844-7
PMID: 11533665
-
Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects.
Science. 2002 Jul 26;297(5581):599-602
PMID: 12142537
-
Cdc2-cyclin B kinase activity links Crb2 and Rqh1-topoisomerase III.
Genes Dev. 2002 May 15;16(10):1195-208
PMID: 12023299
-
Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase.
EMBO J. 1999 Nov 15;18(22):6561-72
PMID: 10562568
-
CDC5 and CKII control adaptation to the yeast DNA damage checkpoint.
Cell. 1997 Sep 19;90(6):1097-106
PMID: 9323137
-
Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest.
Mol Cell. 2001 Feb;7(2):293-300
PMID: 11239458
-
The Mre11 complex: at the crossroads of dna repair and checkpoint signalling.
Nat Rev Mol Cell Biol. 2002 May;3(5):317-27
PMID: 11988766
-
Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes.
Mol Cell. 2001 Nov;8(5):1105-15
PMID: 11741545
-
Role of Saccharomyces single-stranded DNA-binding protein RPA in the strand invasion step of double-strand break repair.
PLoS Biol. 2004 Jan;2(1):E21
PMID: 14737196
-
Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere.
Curr Biol. 2001 Sep 4;11(17):1336-40
PMID: 11553326
-
Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre.
Nat Cell Biol. 2003 Jun;5(6):572-7
PMID: 12766777
-
Cyclin A/CDK2 regulates V(D)J recombination by coordinating RAG-2 accumulation and DNA repair.
Immunity. 1999 Dec;11(6):771-81
PMID: 10626899
-
Enhancement of Saccharomyces cerevisiae end-joining efficiency by cell growth stage but not by impairment of recombination.
Genetics. 2002 Jul;161(3):1015-27
PMID: 12136007
-
A chemical switch for inhibitor-sensitive alleles of any protein kinase.
Nature. 2000 Sep 21;407(6802):395-401
PMID: 11014197
-
Pathways of DNA double-strand break repair during the mammalian cell cycle.
Mol Cell Biol. 2003 Aug;23(16):5706-15
PMID: 12897142
-
Targets of the cyclin-dependent kinase Cdk1.
Nature. 2003 Oct 23;425(6960):859-64
PMID: 14574415
-
Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism.
Mol Cell Biol. 2004 Apr;24(8):3277-85
PMID: 15060150
-
Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity.
EMBO J. 2000 Sep 15;19(18):5027-38
PMID: 10990466
-
Cell cycle-dependent phosphorylation and dephosphorylation of the yeast DNA polymerase alpha-primase B subunit.
Mol Cell Biol. 1995 Feb;15(2):883-91
PMID: 7823954
-
S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast.
Nature. 2002 Feb 7;415(6872):651-5
PMID: 11807498
-
At the heart of the budding yeast cell cycle.
Trends Genet. 1996 Oct;12(10):405-12
PMID: 8909137
-
Role of the Saccharomyces cerevisiae Rad9 protein in sensing and responding to DNA damage.
Biochem Soc Trans. 2003 Feb;31(Pt 1):242-6
PMID: 12546694
-
Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage.
Cell. 1998 Aug 7;94(3):399-409
PMID: 9708741
-
Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.
Mol Cell Biol. 1996 May;16(5):2164-73
PMID: 8628283
-
The yeast Xrs2 complex functions in S phase checkpoint regulation.
Genes Dev. 2001 Sep 1;15(17 ):2238-49
PMID: 11544181
-
Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.
Science. 2003 Jun 6;300(5625):1542-8
PMID: 12791985