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...

Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases are known to target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisae. Correlation of these motifs with kinase primary sequence has uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P−3 Arg specificity among members of the CMGC group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo, and we identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of new kinase substrates across eukaryotes.

Meiotic recombination differs from mitotic recombination in that DSBs are repaired using homologous chromosomes, rather than sister chromatids. This change in partner choice is due in part to a barrier to sister chromatid repair (BSCR) created by the meiosis-specific kinase, Mek1, in a complex with two other meiosis-specific proteins, Hop1 and Red1. HOP1 contains two functional domains, called the N and C domains. Analysis of a point mutation that specifically inactivates the C domain (hop1-K593A) reveals that the N domain is sufficient for Hop1 localization to chromosomes and for Red1 and Hop1 interactions. The C domain is needed for spore viability, for chromosome synapsis, and for preventing DMC1-independent DSB repair, indicating it plays a role in the BSCR. All of the hop1-K593A phenotypes can be bypassed by fusion of ectopic dimerization domains to Mek1, suggesting that the function of the C domain is to promote Mek1 dimerization. Hop1 is a DSB-dependent phosphoprotein, whose phosphorylation requires the presence of the C domain, but is independent of MEK1. These results suggest a model in which Hop1 phosphorylation in response to DSBs triggers dimerization of Mek1 via the Hop1 C domain, thereby enabling Mek1 to phosphorylate target proteins that prevent repair of DSBs by sister chromatids.

Using a screen designed to identify yeast mutants specifically defective in recombination between homologous chromosomes during meiosis, we have obtained new alleles of the meiosis-specific genes, HOP1, RED1, and MEK1. In addition, the screen identified a novel gene designated MSH5 (MutS homolog 51. Although Msh5p exhibits strong homology to the MutS family of proteins, it is not involved in DNA mismatch repair. Diploids lacking the MSH5 gene display decreased levels of spore viability, increased levels of meiosis I chromosome nondisjunction, and decreased levels of reciprocal exchange between, but not within, homologs. Gene conversion is not reduced. Msh5 mutants are phenotypicaUy similar to mutants in the meiosis-specific gene MSH4 (Ross-Macdonald and Roeder 1994}. Double mutant analysis using rash4 rash5 diploids demonstrates that the two genes are in the same epistasis group and therefore are likely to function in a similar process--namely, the facilitation of interhomolog crossovers during meiosis.

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SUMMARY A preference for homologs over sister chromatids in homologous recombination is a fundamental difference in meiotic versus mitotic cells. In budding yeast, the bias for interhomolog recombination in meiosis requires the Dmc1 recombinase and the meiosis-specific kinase, Mek1, which suppresses engagement of sister chromatids by the mitotic recombinase, Rad51. Here, a combination of proteomic, biochemical and genetic approaches has identified an additional role for Mek1 in inhibiting the activity of the Rad51 recombinase through phosphorylation of its binding partner, Rad54. Rad54 phosphorylation of threonine 132 attenuates complex formation with Rad51 and a negative charge at this position reduces Rad51 function in vitro and in vivo. Thus, Mek1 phosphorylation provides a dynamic means of controlling recombination partner choice in meiosis in two ways: (1) it reduces Rad51 activity through inhibition of Rad51/Rad54 complex formation and (2) it suppresses Rad51-mediated strand invasion of sister chromatids via a Rad54-independent mechanism.

Loss of VPS13 produces multiple phenotypes. This study implicates VPS13 in the function of membrane contact sites and suggests that different phenotypes of the mutant result from defects in different contact sites. In yeast, mutations found in the VPS13A gene of ChAc patients have specific defects in the mitochondrial aspect of VPS13 function.