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 yeast1,2,3,4,5,6. 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 protein7,8 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.
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Acknowledgements
The order of the co-first authors (G.I. and A.P.) and the order of the second two authors (A.B. and X.W.) were decided alphabetically. A.B. and X.W. made substantial and equal contributions to this work. We thank C. Zhang and K. Shokat (supported by NIH grant AI44009) for the cdc28-as1 allele and the 1-NMPP1 inhibitor. We note the ownership of the ASKA technology by Cellular Genomics Inc. and thank CGI for granting us the rights to perform the experiments discussed in the publication. We thank J. Petrini, J. Diffley, K. Labib, M. P. Longhese, S. Brill and the IFOM antibody facility for reagents. We are grateful to C. Lucca, E. Pandiani, M. B. Vaze, S. Francia, S. Lovett, M. Lichten and the members of our laboratories for discussions and technical support. N.M.H. was supported by the NIH. M.F. and A.P. were supported by grants from Associazione Italiana per la Ricerca sul Cancro and M.F. from Telethon-Italy and the European Community. G.I. was supported by Charles A. King Trust Postdoctoral Fellowship. G.I. is on leave from N. Copernicus University, Torun, Poland. J.E.H. was supported by the NIH and the DOE.
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Ira, G., Pellicioli, A., Balijja, A. et al. DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431, 1011–1017 (2004). https://doi.org/10.1038/nature02964
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DOI: https://doi.org/10.1038/nature02964
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