Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus. Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans. However, the cellular function of UBA domains is not completely understood. Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic control in yeast. The UBA domains of RAD23 and DDI1 are required for these interactions. Timely degradation of Pds1 via the ubiquitin/proteasome pathway allows anaphase onset and is crucial for chromosome maintenance. Here, we show that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control. Moreover, we show that a hydrophobic surface on the UBA domain, which from structural work had been predicted to be a protein-protein interaction interface, is indeed required for ubiquitin binding. By demonstrating that UBA domains interact with ubiquitin, we have provided the first indication of a cellular function for the UBA domain.

Abstract. Two peroxisomal targeting signals, PTS1 and

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In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1 mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact with PDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37°C. Genetic and functional interactions of two suppressors are described. RAD23 and DDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.When DNA is damaged or chromosomes are incompletely replicated, cells become checkpoint arrested. These checkpoints avoid replication of damaged template DNA and prevent aberrant segregation of damaged or partly replicated chromosomes. In budding yeast, proteolysis of anaphase inhibitors is regulated by these checkpoint systems. Progression from metaphase to anaphase is inhibited by Pds1p in Saccharomyces cerevisiae (6,7,29,30). Before anaphase, Pds1p binds to Esp1p, inhibiting its anaphase-promoting activity (3). During an unperturbed cell cycle, Pds1p becomes polyubiquitinated at the metaphase-to-anaphase transition by multienzyme anaphase-promoting complex (APC)-cyclosome complexes. The modified forms are then recognized and degraded by 26S proteasomes (7). Once released from Pds1p, Esp1p activity induces the onset of anaphase.pds1 mutants fail to execute checkpoint control in response to DNA damage, spindle poisons, or replication inhibition (4, 29, 30). Pds1p is required for replication checkpoint control only late in S phase, not in the context of an early S-phase replication block enforced by hydroxyurea (HU) (4,29,30). In the presence of 0.1 M HU, replication proceeds more slowly. Under these conditions, cells perform other aspects of cell cycle progression, budding, and spindle assembly as rapidly as in the absenc...

Cell cycle-specific proteolysis is critical for proper execution of mitosis in all eukaryotes. Ubiquitination and subsequent proteolysis of the mitotic regulators Clb2 and Pds1 depend on the cyclosome/APC and the 26S proteasome. We report here that components of the cell cycle machinery in yeast, specifically the cell cycle regulatory cyclin-dependent kinase Cdc28 and a conserved associated protein Cks1/Suc1, interact genetically, physically, and functionally with components of the 26S proteasome. A mutation in Cdc28 (cdc28-1N) that interferes with Cks1 binding, or inactivation of Cks1 itself, confers stabilization of Clb2, the principal mitotic B-type cyclin in budding yeast. Surprisingly, Clb2-ubiquitination in vivo and in vitro is not affected by mutations in cks1, indicating that Cks1 is not essential for cyclosome/APC activity. However, mutant Cks1 proteins no longer physically interact with the proteasome, suggesting that Cks1 is required for some aspect of proteasome function during M-phase-specific proteolysis. We further provide evidence that Cks1 function is required for degradation of the anaphase inbibitor Pds1. Stabilization of Pds1 is partially responsible for the metaphase arrest phenotype of cks1 mutants because deletion of PDS1 partially releaves the metaphase block in these mutants.

have occurred in random primordial genetic sequences (9). The prevailing interpretation of existing fossil evidence contradicts this conclusion that eukaryotic genes preceded those of prokaryotes, as prokaryotes occur 3.6 billion years ago, while the first unicellular eukaryotes do not appear until the Cambrian explosion, 3 billion years later. But a different interpretation of the same fossil record, compatible with the lost-introns hypothesis, seems more plausible than the astronomical improbability of a single contiguous gene coding for a specific protein (typical of prokaryotes) occurring purely by chance on Earth-or even in a random DNA molecule with the mass of the whole universe. Moreover, the high probability that fully formed split genes did occur by chance, in only a small amount of random primordial DNA (9), suggests a more likely scenario: While split genes did occur first, their expression into the first eukaryotic organisms could not have occurred, or the organisms themselves could not have survived in the oxygen-free atmosphere of primordial Earth. But the loss of introns from these genes by exon recombination and gene processing produced a new variety of genes that lacked introns (4, 9). These new, contiguously coded genes found immediate expression in prokaryotic cells, which were able to thrive without oxygen. Only when sufficient oxygen became available, by the beginning of the Cambrian period, did split genes finally find expression in the first viable eukaryotic organisms. And as the fossil record shows, the eukaryotes-unicellular and multicellular alike-veritably bloomed over a short time once the requisite oxygen became available (9).

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