Dosage Suppressors of pds1 Implicate Ubiquitin-Associated Domains in Checkpoint Control

Abstract

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...

Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome

“…Rad23p is shown to negatively regulate the polyubiquitin pathway by inhibition of ubiquitylated chain assembly factor E4 (5, 43), whereas Dsk2p seems to function positively in polyubiquitylation. Overexpression of RAD23 stabilizes mutant Pds1p (44), and thus we tested whether, in contrast, overexpressed Dsk2p could stimulate Leu-␤-gal degradation. However, we cannot observe distinct effects of Leu-␤-gal degradation (data not shown), probably because an expressed Dsk2p to a single Leu-␤-gal substrate was titrated out by many other substrates present in cells.…”

DNA-Damage-Independent Checkpoints: Yeast and Higher Eukaryotes

“…Recent evidence has linked two yeast genes to regulation of Pds1 in this context. 76 Rad23 or Ddi1 overproduction was found to rescue the sensitivity of pds1 mutant cells to HU. Rad23 is a nucleotide excision repair protein, but recent studies suggest a novel role of Rad23 in regulating ubiquitin-dependent proteolysis.…”

“…Although this mechanism has not been tested directly in the context of S-phase checkpoint control, overexpression of Rad23 increases mutant pds1 protein levels in vivo. 76 These studies also spawned interest in a novel protein domain, the UBA (Ubiquitin Associated Domain), 81 recently shown to be non-covalent binding sites for ubiquitin. 77 The Rad23 UBA domains are needed for rescue of pds1 mutant strain HU sensitivity, 76 and are required for blocking the ubiquitin chain elongation on a test substrate in vitro.…”

“…76 These studies also spawned interest in a novel protein domain, the UBA (Ubiquitin Associated Domain), 81 recently shown to be non-covalent binding sites for ubiquitin. 77 The Rad23 UBA domains are needed for rescue of pds1 mutant strain HU sensitivity, 76 and are required for blocking the ubiquitin chain elongation on a test substrate in vitro. 79 Regulation of Rad23 function in this context could be mediated by dimerization.…”

“…Rad23 is a nucleotide excision repair protein, but recent studies suggest a novel role of Rad23 in regulating ubiquitin-dependent proteolysis. [76][77][78][79][80] Rad23 binds to mono-or di-ubiquitinated proteins, but cannot bind when the ubiquitin chains have been elongated. 78 Crucially, Rad23 blocks extension of the ubiquitin chains.…”