In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1 is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1⌬ mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1⌬ cells, as in mec1⌬ cells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of the pie1⌬ mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1⌬ mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.When DNA replication is blocked and DNA damage occurs, checkpoints arrest the cell cycle, allowing DNA replication and repair to take place (13,19). Loss of checkpoint control results in cell death or genetic instability that can lead to cancer. Checkpoint pathways are an evolutionarily conserved feature of eukaryotic cells. This conservation is exemplified by the family of genes encoding high-molecular-weight protein kinases, including ATM (mammals), ATR (mammals), MEC1 (Saccharomyces cerevisiae), TEL1 (S. cerevisiae), rad3 ϩ (Schizosaccharomyces pombe), mei-41 (Drosophila melanogaster), and uvsB (Aspergillus nidulans) (6,9,18,22,23,30,37,40,48). Each of these genes falls into two family groups based on homology; ATM is related most closely to TEL1, while ATR is more related to MEC1, rad3 ϩ , mei-41, and uvsB (6, 40). This homology is not restricted to the kinase domain at the carboxyl terminus but extends over the length of the protein. The carboxyl-terminal kinase domain is structurally related to the catalytic domain of the phosphatidylinositol (PI) 3-kinases. Despite this similarity, none of these proteins has been shown to phosphorylate lipids. ATM, ATR, and Rad3 are all capable of phosphorylating protein substrates (5,8,28,29). However, it remains to be determined how the kinase activity of these proteins is controlled in checkpoint responses. Moreover, little is known about whether these proteins form a complex with other proteins, although Rad3 has been recently shown to form a complex with Rad26 (12). The only Rad26 homolog identified so far is A. nidulans UVSD (40), but it has not be...

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae. We have previously shown that a temperature-sensitive (ts) rfc5-1 mutation is impaired in the S-phase checkpoint. In this report, we show that the rfc5-1 mutation is sensitive to DNA-damaging agents. RFC5 is necessary for slowing the S-phase progression in response to DNA damage. The phosphorylation of the essential central transducer, Rad53 protein kinase, is reduced in response to DNA damage in rfc5-1 mutants during the S phase. Furthermore, the inducibility of RNR3 transcription in response to DNA damage is dependent on RFC5. It has been shown that phosphorylation of Rad53 is controlled by Mec1 and Tel1, members of the subfamily of ataxiatelangiectasia mutated (ATM) kinases. We also demonstrate that overexpression of TEL1 suppresses the ts growth defect and DNA damage sensitivity of rfc5-1 mutants and restores phosphorylation of Rad53 and RNR3 induction in response to DNA damage in rfc5-1. Our results, together with the observation that overexpression of RAD53 suppresses the defects of the rfc5-1 mutation, suggest that Rfc5 is part of a mechanism transducing the DNA damage signal to the activation of the central transducer Rad53.In eukaryotic cells, successful mitotic division requires the events of the cell cycle to be ordered into dependent pathways in which the initiation of late cycle events is dependent on the completion of early events. The mechanisms which ensure that cell division does not occur before completion of such prerequisite steps have been termed checkpoint controls (8). Checkpoint controls ensure that cells remain in S phase before completion of DNA replication. DNA damage also activates checkpoint controls to provide enough time to complete DNA repair. Defects of these DNA-related checkpoints result in increased genomic instability and mutagenesis (5, 21).In the budding yeast Saccharomyces cerevisiae, checkpoint pathways induce cell cycle arrest in G 1 or G 2 /M and retard Sphase progression in response to DNA damage. Other checkpoints prevent cells with incompletely replicated DNA from exiting the S phase (5, 21). A number of genes that are involved in the DNA damage checkpoint and/or the replication checkpoint have been identified (5, 21). These include RAD9, RAD17, RAD24, POL2, MEC1/ESR1, RAD53/SPK1/MEC2/ SAD1, and MEC3 (1,10,17,28,[33][34][35]. Among these genes, RAD9, RAD17, RAD24, and MEC3 are involved not only in the G 2 /M-phase but also in the G 1 -and S-phase DNA damage checkpoints (12,20,(25)(26)(27)(33)(34)(35). POL2, which encodes a large subunit of DNA polymerase ε (pol ε), is proposed to sense DNA damage and replication block in S phase (16,17). MEC1 and RAD53 are necessary for checkpoints operating in response to both DNA damage and incomplete DNA replication (1, 35). RAD53 encodes a dual-specificity protein kinase (28), and Mec1 belongs to the phosphatidylinositol kinase family that includes S. cerevisiae Tel1 and human ATM proteins (10,15,24). MEC1 and TEL1 share some...

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage. Here we describe the isolation of RAD24, known to play a role in the DNA damage checkpoint, as a dosagedependent suppressor of rfc5-1. RAD24 overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation. Rad24, like Rfc5, is required for the regulation of Rad53 phosphorylation in response to DNA damage. The Rad24 protein, which is structurally related to the RFC subunits, interacts physically with RFC subunits Rfc2 and Rfc5 and cosediments with Rfc5. Although the rad24⌬ mutation alone does not cause a defect in the replication block checkpoint, it does enhance the defect in rfc5-1 mutants. Furthermore, overexpression of RAD24 suppresses the rfc5-1 defect in the replication block checkpoint. Taken together, our results demonstrate a physical and functional interaction between Rad24 and Rfc5 in the checkpoint pathways.The survival of eucaryotes depends on the accurate transmission of genetic information from one generation to the next. Successful mitotic division requires the events of the cell cycle to be ordered such that the initiation of late cycle events is dependent on the completion of early events. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls (7). When DNA replication is delayed and DNA damage occurs, checkpoint controls activate cell cycle arrest enough to complete DNA replication and repair DNA damage (4, 18).In the budding yeast Saccharomyces cerevisiae, checkpoint pathways induce cell cycle arrest in G 1 or G 2 /M and retard Sphase progression in response to DNA damage. Other checkpoints prevent cells with incompletely replicated DNA from exiting the S phase. A number of genes that are involved in the DNA damage checkpoint and/or the replication block checkpoint have been identified elsewhere (4, 18). These include RAD9, RAD17, RAD24, POL2, MEC1/ESR1, RAD53/SPK1/ MEC2/SAD1, RFC5, MEC3, and DDC1. Among these genes, RAD9, RAD17, RAD24, MEC3, and DDC1 are involved not only in the G 2 /M-phase but also in the G 1 -and S-phase DNA damage checkpoints (11,12,17,(22)(23)(24)(31)(32)(33). POL2, encoding a large subunit of DNA polymerase (Pol) ε, is required for the checkpoints responding to replication block and DNA damage in S phase (15, 16). MEC1 and RAD53 are necessary for the checkpoints operating in response to both DNA damage and incomplete DNA replication (1, 33). RAD53 encodes a dual-specificity protein kinase (25), and Mec1 belongs to the phosphatidylinositol kinase family that includes human ataxiatelangiectasia-mutated (ATM) proteins (9, 21). Rad53 is phosphorylated in response to DNA damage and DNA replication block in a MEC1-dependent manner (20,29).Replication factor C (RFC) is required for DNA replication and repair and consists of one large and four small...

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We studied the mechanism of post-overdrive suppression in superfused rabbit sinus node pacemaker cells. Small specimens of sinus node tissue isolated from rabbit hearts were driven at a fast rate (overdrive) for 10-120 seconds using single sucrose gap methods. During the control perfusion (35 degrees C Tyrode's solution), overdrive caused a progressive decrease in maximum diastolic potential (MDP), overshoot (OS), and maximum rate of depolarization at phase 0 [dV/dt)max]. After cessation of the overdrive, the rate of diastolic depolarization decreased, and the spontaneous activity was suppressed temporarily (post-overdrive suppression). MDP, OS, (dV/dt)max, and the spontaneous activity returned within a few seconds to the level observed before overdrive. Atropine (2 x 10(-6) g/ml) did not influence the effects of overdrive. After ouabain administration (3 x 10(-7) g/ml) or in low temperature perfusate (25 degrees C), the effects of overdrive were accentuated, and a marked suppression of spontaneous activity with a long pause of over several seconds was seen following the overdrive. These results suggest that the post-overdrive suppression of sinus node is attributable, at least in part, to ionic shifts following overdrive, and may be potentiated by metabolic dysfunction of pacemaker cells.

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