Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.The SOS response in Escherichia coli provides the coordination between DNA damage sensing and the cellular responses to DNA damage (reviewed in reference 22). The primary SOS signal, single-stranded DNA (ssDNA), activates RecA in a ternary complex with ATP as a transcriptional regulator (44) and as a DNA repair protein (reviewed in reference 41). The transcriptional induction of the SOS regulon leads to increased expression of certain DNA repair genes (including RecA itself) and also elicits transient cell cycle arrest by the expression of sfiA, a cell division inhibitor (22). The activation of RecA as a repair protein leads to immediate repair of the primary damage that initiated the SOS signal. Although different in mechanism, the DNA damage checkpoints could provide a similar coordination between DNA damage sensing and repair in eukaryotes. First conceptualized as an active cell cycle control system in response to DNA damage in Saccharomyces cerevisiae (29,89), DNA damage checkpoints were later shown to control also DNA damage-induced gene expression in this organism (3). DNA damage checkpoints and DNA repair serve a common purpose to secure survival and genomic stability after DNA damage. Indirect effects of the DNA damage checkpoints on DNA repair have been discussed before (reviewed in references 18, 85, and 87), but a direct coupling of the DNA damage sensing capabilities of the checkpoint system with DNA damage repair pathways has not been identified yet.The DNA damage checkpoints in eukaryotes relay a signal in response to DNA damage to transiently delay the entry into the S or M phases, to slow down the ongoing DNA replication, or to arrest in meiotic prophase (reviewed in references 29, 62, and 87). They also elicit DNA damage-induced transcription of many genes, including some coding for DNA repair proteins (87, 93). Moreover, a related DNA replication block checkpoint ensures the dependency of M phase on a...

. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G 2 /M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.DNA damage checkpoints coordinate the cellular responses to genotoxic stress and ensure genomic integrity (31,40,55,60). Besides cell cycle transitions, DNA damage checkpoints in the yeast Saccharomyces cerevisiae control damage-induced transcription; DNA replication; DNA repair and genomic stability; deoxynucleoside triphosphate metabolism; the relocalization of the Sir3/4, Ku80, and Rap1 proteins; and possibly other physiological responses to genotoxic stress (5,20,35,55,58,60,61).Central to the DNA damage checkpoints in S. cerevisiae is a branched kinase cascade consisting of five protein kinases (Mec1, Tel1, Rad53, Chk1, and Dun1) (55, 60). Mec1 and Tel1 are both high-molecular-weight phosphoinositide 3-kinase-related protein kinases that are activated by unknown mechanisms. Their human counterparts, ATM and ATR, are also essential for the human DNA damage checkpoints. Rad53 and Dun1 are related forkhead-associated (FHA) domain kinases (see Fig. 1) and have counterparts in other organisms, including fission yeast Cds1 and human Chk2 (40, 60). Finally, Chk1 kinase, as well as its fission yeast and human homologs, is critical for the G 2 cell cycle arrest in response to DNA damage (41). Genetic analysis of S. cerevisiae established that Mec1 controls the activities of the three downstream kinases Rad53, Dun1, and Chk1 (4,38,41,42,61). Under certain conditions, Tel1 controls the activation of Rad53 kinase in a Mec1-independent fashion (52). The exact mechanisms of how DNA damage checkpoints are activated and how the checkpoint kinases transmit and possibly amplify the signal, as well as control the effector pathways, are only beginning to be understood.Dun1 kinase functions in DNA damage-induced transcription of a subset of damage-inducible genes, including the RNR genes, by controlling the inactivating phosphorylation of the Crt1 transcriptional repressor (26,27,61). Mutations in DUN1 cause sensitivity to DNA damaging-agents and the replication inhibitor hydroxyurea (HU). This sensitivity can be partly suppressed by elevating the deoxynucleoside triphosphate pools through deletion of the ribonucleotide reductase inhibitor Sml1 or by overexpression of RNR1, the gene encoding the large subunit of ribonucleotide reductase (58, 61). In addition, Dun1 functions in one pathway with Rad53 kinase to cause a G 2 /M arrest in response to DNA damage by negatively regulating ...

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

A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55+ (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an Mr of 38,000. The rhp55Δ mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Δ cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55+ acts in one DNA repair pathway with rhp51+ and rhp54+, homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55+ is in a different epistasis group for repair of UV-, MMS-, or γ-ray-induced DNA damage than is rad22+, a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55+ is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved.

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