DNA damage checkpoints coordinate the cellular response to genotoxic stress (10,45,52,82). In the budding yeast Saccharomyces cerevisiae, the DNA damage checkpoints are largely controlled by the phosphatidyl-inositol 3-kinase-like kinase Mec1, an ortholog of the human ATM and ATR kinases. Via the Rad9 and Mrc1 adaptor proteins, Mec1 controls the downstream kinases Chk1 and Rad53. This process amplifies the checkpoint response and transforms localized Mec1 activation into a pan-nuclear response regulating downstream effector pathways, including cell cycle control, transcription, DNA replication, and possibly DNA damage repair and DNA damage tolerance pathways.Checkpoint mutants fail to arrest their cell cycles in response to DNA damage and replication fork stalling, leading to damage sensitivity and genomic instability (49). However, extrinsically imposed cell cycle arrest does not rescue the damage sensitivity of S. cerevisiae rad53 or mec1 mutants (4, 65) or human ATM-deficient cells (73; reviewed in reference 24) and only partially rescues the sensitivity of S. cerevisiae rad9 cells (74), suggesting that DNA damage checkpoints also regulate mechanisms other than cell cycle arrest that are critical for survival and genome stability.Stalled replication forks are considered a major source of genomic instability (29), and multiple pathways operate at stalled forks, presumably in a hierarchy that is under active regulation. An analysis of a mec1 hypomorphic mutant demonstrated a central role of DNA damage checkpoints in preventing irreversible breakdown of stalled replication forks in budding yeast (66). The postreplication repair (PRR) controlled by the Rad6-Rad18 proteins is critical to budding yeast for the toleration of replication-blocking lesions (8). PRR comprises a number of pathways, which are incompletely understood at this moment, involving translesion synthesis (TLS) by DNA polymerases and template switching. TLS polymerases, including REV3, which encodes a subunit of DNA polymerase zeta (Pol), and RAD30, which encodes Pol in S. cerevisiae, accommodate damaged DNA templates, leading to bypass and damage tolerance. Template switching can occur by fork regression, a process that appears to be controlled by the Rad5 protein. However, the subpathways in PRR are complex and roles of Rad5 in conjunction with the TLS polymerase Rev3 have been identified (13,47). Template switching can also be catalyzed during gap repair by homologous recombination (HR) mediated by the RAD52 epistasis group (31).HR is a major pathway for the repair of DNA doublestranded breaks (DSBs) and other types of DNA damage. In bacteria, recombination is central in the recovery of stalled

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In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55–S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases.