In response to DNA damage, eukaryotic cells activate checkpoint pathways that arrest cell cycle progression and induce the expression of genes required for DNA repair. In budding yeast, the homothallic switching (HO) endonuclease creates a site-specific double-strand break at the mating type (MAT) locus. Continuous HO expression results in the phosphorylation of Rad53, which is dependent on products of the ataxia telangiectasia mutated-related MEC1 gene and other checkpoint genes, including DDC1, RAD9, and RAD24. Chromatin immunoprecipitation experiments revealed that the Ddc1 protein associates with a region near the MAT locus after HO expression. Ddc1 association required Rad24 but not Mec1 or Rad9. Mec1 also associated with a region near the cleavage site after HO expression, but this association is independent of Ddc1, Rad9, and Rad24. Thus, Mec1 and Ddc1 are recruited independently to sites of DNA damage, suggesting the existence of two separate mechanisms involved in recognition of DNA damage.

RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24⌬ mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12⌬ nor rad24⌬ single mutants are defective, chl12⌬ rad24⌬ double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.Eukaryotic cells employ a set of surveillance mechanisms to coordinate the onset of one event and the completion of the preceding event during the cell cycle. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls in eukaryotes (11). When DNA is damaged or DNA replication is blocked, the activation of checkpoint pathways arrests the cell cycle and induces the transcription of genes that facilitate DNA repair and/or replication (5, 33).Checkpoint pathways are an evolutionarily conserved feature of eukaryotic cells. This feature is typified in the ATM and ATR family genes which encode phosphatidylinositol 3-kinaserelated proteins possessing protein kinase activity (33). In the budding yeast Saccharomyces cerevisiae, MEC1 encodes an ATR-related protein and plays a critical role in checkpoint controls (14,17,32). Mec1 physically interacts with Pie1 (also called Lcd1 or Ddc2), a protein that exhibits limited homology to the fission yeast Rad26 protein (17,19,32). Likewise, in fission yeast the ATR family protein Rad3 forms a complex with Rad26 (4). DNA damage responses have been well characterized in budding yeast and consist of the G 1 -, S-, and G 2 /M-phase damage checkpoints (14). Both Mec1 and Pie1 are essential for all three DNA damage checkpoints, as well as the DNA replication block checkpoint.In addition to MEC1 and PIE1, a number of genes that control the checkpoints in budding yeast have been identified. These include DDC1, MEC3, RAD9, RAD17, RAD24, and RAD53 (5,14,33). RAD53 encodes a protein kinase and functions downstream of MEC1 in the checkpoint pathway. Like Mec1, Rad53 plays an essential role in both the replication block and DNA damage checkpoints. Following DNA damage and replication block, the Rad53 protein is hyperphosphorylated and activated by a mechanism dependent on Mec1 (20, 26). Thus, Mec1 and Rad53 constitute a central checkpoint pathway in budding yeast. RAD9, RAD17, MEC3, DDC1, and RAD24 are also required for DNA damage checkpoints. Rad9 is hyperphosphorylated following DNA damage, and the phosphorylated Ra...

Adipocyte differentiation is regulated by a complex array of extracelluar signals, intracellular mediators and transcription factors. Here we describe suppression of adipocyte differentiation by TRBs, mammalian orthologs of Drosophila Tribbles. Whereas all the three TRBs were expressed in 3T3-L1 preadipocytes, TRB2 and TRB3, but not TRB1, were immediately downregulated by differentiation stimuli. Forced expression of TRB2 and TRB3 inhibited adipocyte differentiation at an early stage. Akt activation is a key event in adipogenesis and was severely inhibited by TRB3 in 3T3-L1 cells. However, the inhibition by TRB2 was mild compared with severe inhibition by TRB3, though TRB2 suppressed adipogenesis as strongly as TRB3. Interestingly, TRB2 but not TRB3 reduced the level of C/EBP␤, a transcription factor required for an early stage of adipogenesis, through a proteasome-dependent mechanism. Furthermore, knockdown of endogenous TRB2 by siRNA allowed 3T3-L1 cells to differentiate without full differentiation stimuli. These results suggest that inhibition of Akt activation in combination with degradation of C/EBP␤ is the basis for the strong inhibitory effect of TRB2 on adipogenesis.

Adipocytes are a major energy reservoir, storing excess energy as lipids and releasing it on demand. In addition, adipocytes constitute an endocrine system by secreting soluble mediators known as adipokines, which regulate not only peripheral tissues such as muscles and adipose tissues but also the central nervous system (1). Disorders in adipose tissues are a major cause of the development of the metabolic syndrome, a common basis of type 2 diabetes and atherosclerotic vascular diseases (2). It is therefore important to understand the nature of adipocytes and the mechanism of adipocyte differentiation.

RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24⌬ mutants are defective in all the G 1 -, S-, and G 2 /M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine 115 residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G 1 -and G 2 /M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24 K115R fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G 1 -, S-and G 2 /M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.Eukaryotic cells employ a set of surveillance mechanisms to coordinate cell cycle events by permitting the onset of one event only after the completion of the preceding event. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls (10). DNA damage triggers the activation of checkpoint pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. Other checkpoints are activated when DNA replication is blocked. Failure to respond properly to DNA alterations may result in genomic instability, a mutagenic condition that predisposes organisms to cancer (5, 24).The cell cycle is transiently arrested at different stages depending on the phase at which DNA damage occurs. Three responses have been characterized in the budding yeast Saccharomyces cerevisiae, known as the G 1 -, S-and G 2 /M-phase DNA damage checkpoints (16). Genetic studies have identified genes that are involved in all three checkpoints. These include RAD9, RAD17, RAD24, MEC3, DDC1, MEC1(ESR1), and RAD53 (SPK1 or MEC2) (1,17,18,22,23,(30)(31)(32)(33)(43)(44)(45). Several lines of genetic evidence have suggested that RAD17, RAD24, MEC3, and DDC1 operate in the same checkpoint pathway, while RAD9 functions separately (17,18,20). Indeed, Ddc1, Mec3, and Rad17 physically interact with each other, suggesting that they function as a complex (13). RAD53 encodes a dual-specificity protein kinase (35), and Mec1 belongs to the ATM protein family (12,28). Rad53 is phosphorylated in response to DNA damage in a MEC1-dependent manner (26, 39). DNA damage-induced Rad53 phosphorylation is also dependent on R...