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.
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.
Dubrana
Karine
K
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.
van Attikum
Haico
H
Hediger
Florence
F
Gasser
Susan M
SM
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004249:DNA Damage; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000378:metabolism*; D011995:Recombination, Genetic; D051716:Replication Protein A / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D047908:Intracellular Signaling Peptides and Proteins; C440014:LCD1 protein, S cerevisiae; D010750:Phosphoproteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D051135:Rad51 Recombinase