Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G 1 phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1⌬ dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.The cellular response to DNA damage is based on signal transduction mechanisms that are essential for the maintenance of genome integrity. The molecules involved and the organization of the pathway are generally conserved in all eukaryotes (2,29,30,42). A major output of this response is a controlled delay in cell cycle progression that regulates the G 1 -S transition (G 1 checkpoint) or the G 2 -M transition (G 2 /M checkpoint; in budding yeast, this response does not regulate the passage from G 2 to M but prevents the anaphase-to-metaphase transition). This is achieved by regulating Cdk kinase or anaphase-promoting complex activities. The current model predicts that genotoxin treatments activate the DNA damage checkpoint response through the recruitment of the ATM and ATR phosphoinositide 3-kinase-related kinases to damaged chromatin (42, 51). The molecular details of the DNA damage signaling pathway in fission and budding yeasts have been mostly worked out by following the phosphorylation of critical kinase substrates in appropriately mutated genetic backgrounds (5, 25). In budding yeast, the prevalent apical kinase is represented by Mec1, which is associated with a Ddc2 subunit. Processing of DNA lesions by repair mechanisms generates single-stranded DNA (ssDNA) filaments that are rapidly coated by replication protein A (RPA). This structure seems to be responsible for the recruitment of 38,51). The first biochemical event in the signal transduction cascade seems to be the direct phosphorylation of Ddc2 (33, 37). A heterotrimeric complex (9-1-1) composed of Rad17, Mec3, and Ddc1 is loaded onto damaged DNA by a replication factor C-like complex and is itself phosphorylated by Mec1 on the Ddc1 subunit (25,28,34). Another Mec1 target is checkpoint factor Rad9, the orth...

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