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First published online 14 November 2007
doi: 10.1242/jcs.018366

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The processing of double-strand breaks and binding of single-strand-binding proteins RPA and Rad51 modulate the formation of ATR-kinase foci in yeast

Karine Dubrana^*, Haico van Attikum, Florence Hediger and Susan M. Gasser^¶

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland

^¶ Author for correspondence (e-mail: susan.gasser{at}fmi.ch)

Accepted 20 September 2007

Double-strand breaks (DSB) in yeast lead to the formation of repair foci and induce a checkpoint response that requires both the ATR-related kinase Mec1 and its target, Rad53. By combining high-resolution confocal microscopy and chromatin-immunoprecipitation assays, we analysed the genetic requirements for and the kinetics of Mec1 recruitment to an irreparable HO-endonuclease-induced DSB. Coincident with the formation of a 3' overhang, the Mec1-Ddc2 (Lcd1) complex is recruited into a single focus that colocalises with the DSB site and precipitates with single-strand DNA (ssDNA). The absence of Rad24 impaired cut-site resection, Mec1 recruitment and focus formation, whereas, in the absence of yKu70, both ssDNA accumulation and Mec1 recruitment was accelerated. By contrast, mutation of the N-terminus of the large RPA subunit blocked Mec1 focus formation without affecting DSB processing, arguing for a direct involvement of RPA in Mec1-Ddc2 recruitment. Conversely, loss of Rad51 enhanced Mec1 focus formation independently of ssDNA formation, suggesting that Rad51 might compete for the interaction of RPA with Mec1-Ddc2. In all cases, Mec1 focus formation correlated with checkpoint activation. These observations led to a model that links end-processing and competition between different ssDNA-binding factors with Mec1-Ddc2 focus formation and checkpoint activation.

Key words: Double-strand break, ATR-related kinase, Checkpoint, Single-stranded DNA, Rad24, RPA, Rad51