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 [1][2][3][4][5][6] . 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 7,8 results in a compensatory increase in nonhomologous 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.In budding yeast, a chromosomal DSB created by HO endonuclease has been used both to study the kinetics and efficiency of DSB repair and to analyse the induction of the DNA damage checkpoint dependent on Mec1 (an ATR homologue). In cells carrying HML or HMR mating-type switching donor sequences, a DSB at the MAT locus is efficiently repaired by gene conversion. In strains lacking donor sequences, induction of an unrepairable DSB causes arrest of cell cycle progression before anaphase 1,2 . In bothCorrespondence and requests for materials should be addressed to M.F. (marco.foiani@ifom-ieo-campus.it) or J.E.H. † Present address: Rockefeller University, 1230 York Avenue, New York, New York 10021-6399, USA. ★ These authors contributed equally to this work Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. instances, a key step is the 5′ to 3′ resection of DSB ends to produce single-stranded DNA (ssDNA), which is bound by the RPA complex. RPA binding is essential both for association of Mec1 checkpoint kinase 9 and for loading of Rad51 recombination protein 6 . HHS Public AccessActivation of the Mec1-dependent DNA damage checkpoint after a DSB is regulated by the cell cycle 3 , with no activation in G1-arrested cells. A DSB induced in cells that have been arrested in G1, and then released into S phase, results in hyperphosphorylation of the Mec1 target Rad53 after the completion of S phase, in G2 ( Supplementary Fig. S1a). To test whether the checkpoint depends on the activity of cyclin-dependent kinases, we inactivated CDK1 in nocodazole-blocked G2 cells. We overexpressed the CDK1/Clb inhibitor, Sic1 (ref. 10), in G2 cells at the same time that an unrepairable DSB was induced at MAT. CDK1 i...

In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase. Active Rad53 slows DNA synthesis when DNA is damaged and prevents firing of late origins of replication. Further, rad53 mutants are unable to recover from a replication block. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes. Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.

In Saccharomyces strains in which homologous recombination is delayed sufficiently to activate the DNA damage checkpoint, Rad53p checkpoint kinase activity appears 1 hr after DSB induction and disappears soon after completion of repair. Cells lacking Srs2p helicase fail to recover even though they apparently complete DNA repair; Rad53p kinase remains activated. srs2Delta cells also fail to adapt when DSB repair is prevented. The recovery defect of srs2Delta is suppressed in mec1Delta strains lacking the checkpoint or when DSB repair occurs before checkpoint activation. Permanent preanaphase arrest of srs2Delta cells is reversed by the addition of caffeine after cells have arrested. Thus, in addition to its roles in recombination, Srs2p appears to be needed to turn off the DNA damage checkpoint.