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...

SummaryTranscription hinders replication fork progression and stability. The ATR checkpoint and specialized DNA helicases assist DNA synthesis across transcription units to protect genome integrity. Combining genomic and genetic approaches together with the analysis of replication intermediates, we searched for factors coordinating replication with transcription. We show that the Sen1/Senataxin DNA/RNA helicase associates with forks, promoting their progression across RNA polymerase II (RNAPII)-transcribed genes. sen1 mutants accumulate aberrant DNA structures and DNA-RNA hybrids while forks clash head-on with RNAPII transcription units. These replication defects correlate with hyperrecombination and checkpoint activation in sen1 mutants. The Sen1 function at the forks is separable from its role in RNA processing. Our data, besides unmasking a key role for Senataxin in coordinating replication with transcription, provide a framework for understanding the pathological mechanisms caused by Senataxin deficiencies and leading to the severe neurodegenerative diseases ataxia with oculomotor apraxia type 2 and amyotrophic lateral sclerosis 4.

SummaryTranscription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.

Mutations in the genes encoding the BLM and WRN RecQ DNA helicases and the MRE11-RAD50-NBS1 complex lead to genome instability and cancer predisposition syndromes. The Saccharomyces cerevisiae Sgs1 RecQ helicase and the Mre11 protein, together with the Srs2 DNA helicase, prevent chromosome rearrangements and are implicated in the DNA damage checkpoint response and in DNA recombination. By searching for Srs2 physical interactors, we have identified Sgs1 and Mre11. We show that Srs2, Sgs1, and Mre11 form a large complex, likely together with yet unidentified proteins. This complex reorganizes into Srs2-Mre11 and Sgs1-Mre11 subcomplexes following DNA damage-induced activation of the Mec1 and Tel1 checkpoint kinases. The defects in subcomplex formation observed in mec1 and tel1 cells can be recapitulated in srs2-7AV mutants that are hypersensitive to intra-S DNA damage and are altered in the DNA damage-induced and Cdk1-dependent phosphorylation of Srs2. Altogether our observations indicate that Mec1-and Tel1-dependent checkpoint pathways modulate the functional interactions between Srs2, Sgs1, and Mre11 and that the Srs2 DNA helicase represents an important target of the Cdk1-mediated cellular response induced by DNA damage.A well-characterized aspect of the DNA damage checkpoint response is the prompt activation of a set of highly conserved kinases (64,72). The activation of these checkpoint kinases, which include ATM and ATR and their orthologs in Saccharomyces cerevisiae, Tel1 and Mec1, respectively, induces changes in the phosphorylation state of several downstream targets (82), although the biological implications of such modifications are largely unknown. Several factors involved in DNA repair have been recently identified among the targets of checkpoint kinases (64, 72). Hence, one relevant role of the checkpointinduced phosphorylation cascade could be to directly modulate the activity of certain repair proteins implicated in the removal of the DNA lesions (8,59).Recent studies have shown that the checkpoint kinases, as well as their downstream repair targets, can be purified from large protein complexes (15,63,98). Notably, some of these factors colocalize at characteristic DNA spots, known as nuclear foci (98). The aggregation of repair proteins in nuclear foci is stimulated by DNA damage or replication stress and, at least under certain circumstances, depends on checkpoint activation and cell cycle progression, suggesting that these foci might indeed represent sites of repair and/or DNA damage signaling (6,25,30,56,61,65). Particularly in budding yeast, double-strand break repair centers have been recently described (41, 54, 55, 57, 69).The WRN and BLM RecQ helicases, altered in Werner's and Bloom's genome instability syndromes, also localize at DNA damage-induced nuclear foci (6,12,81,(97)(98)(99). RecQ proteins represent a highly conserved family of 3Ј to 5Ј DNA helicases that also includes Sgs1 in S. cerevisiae (34, 42). RecQ helicases have been implicated in several aspects of DNA metabolism, but the b...