Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-l-oxide (4NQO), or yradiation after release from G, arrest induced by a factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage (Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-3221, the normal execution of DNA damageinduced G, arrest depends on a functional yeast RAD9 gene.We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G, arrest point but prior to replicative DNA synthesis, S phaseassociated maximal induction of histone H2A mRNA, and bud emergence. Additional support for the notion of regulated checkpoints derives from several recent observations. A decrease in the fraction of S phase cells and an increase in the fraction of G, cells have been correlated with an increase in the level of p53 protein (11) in several mammalian cell lines following exposure to y irradiation. Furthermore, p53 mutant cells failed to arrest in G1 after y irradiation (12,13). An increase in p53 levels was not observed in irradiated AT cells (13). Hence, p53 and the AT gene(s) may participate in a signal transduction pathway that regulates cell cycle arrest after DNA damage.In S. cerevisiae nutrient deprivation or exposure to mating pheromone (a factor) results in the arrest of haploid cells in G1. This arrest is associated with a failure to activate the CDC28-encoded protein kinase, a homologue ofthe Cdc2 and p34 proteins in Schizosaccharomyces pombe and mammalian cells, respectively. In S. cerevisiae reentry to the cell cycle depends on a transition from this restriction point, termed START (14,15).In the present study we have investigated the effect of DNA damage on the progression of yeast cells through the cell cycle. We show that exposure of synchronized cells to UV radiation, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or 'y radiation results in G1 arrest. We additionally show that this arrest requires a functional RAD9 gene. The dependence of G1 arrest on a gene previously implicated in arrest in the G2 phase (5) suggests that arrest during G1 is a regulated phenomenon that operates as a cell cycle checkpoint in yeast cells exposed to various types of DNA damage. MATERIALS AND METHODS
Rab5GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.Rab proteins are critical regulators of the vesicle targeting and fusion events (reviewed in Refs. 1 and 2). Discrete classes of these small GTPases mediate very specific transport events, showing little if any functional overlap. For example, the Rab5 family members appear to be involved exclusively in targeting events within the early stages of the endocytic pathway (3, 4). The activation of Rab5 GTPases like all Rab proteins is dependent on the state of bound nucleotide, GDP or GTP. Two classes of proteins that modulate the Rab nucleotide occupancy are the GTPase activating proteins (GAPs) 1 and the guanine nucleotide exchange factors (GEFs). GAPs stimulate GTP hydrolysis, leaving the Rab in the GDP-bound, inactive state; conversely, GEFs initiate GDP release to permit GTP binding and thereby Rab activation. Multiple GAPs and GEFs for the Rab proteins have been identified, and an interesting distinction has been observed (reviewed in Ref. 5). The Rab GAPs share a conserved sequence motif and exhibit substrate promiscuity among the Rab families (6, 7) (reviewed in Ref. 8). In contrast, the GEF proteins for different Rab families are dissimilar at the sequence level and show great specificity for their cognate Rab proteins. Consequently, the GEFs appear to be the primary mechanism to control specific Rab activity.A number of exchange factors for Vps21p/Rab5 family members have been identified in mammalian and yeast systems. In yeast, Vps9p is the exchange factor for the Rab5 ortholog Vps21p (9). VPS9 was initially identified in genetic screens for mutants defective in vacuolar protein sorting (10). In vitro reconstitution of Vps9p-stimulated GDP release and GTP loading onto Vps21p demonstrated that Vps9p is the GEF for Vps21p (9). Concurrently, Rabex5 was identified as a Rab5-binding protein and demonstrated to exhibit in vitro GEF activity (11). In addition to conserved functions in activating Rab5 proteins, yeast Vps9p (451 amino acids) and human Rabex5 (491 amino acids) share 27% overall sequence identity (11). A peptide comprised of residues 158 -347 of Vps9p was identified as the domain necessary and sufficient for GEF catalytic activity. 2 Although Vps9p is the only known GEF for Vps21p in yeast, six human proteins have been identifi...
The delay of S-phase following treatment of yeast cells with DNA-damaging agents is an actively regulated response that requires functional RAD9 and RAD24 genes. An analysis of cell cycle arrest indicates the existence of (at least) two checkpoints for damaged DNA prior to S-phase; one at START (a G1 checkpoint characterized by pheromone sensitivity of arrested cells) and one between the CDC4- and CDC7-mediated steps (termed the G1/S checkpoint). When a dna1-1 mutant (that affects early events of replicon initiation) also carries a rad9 deletion mutation, it manifests a failure to arrest in G1/S following incubation at the restrictive temperature. This failure to execute regulated G1/S arrest is correlated with enhanced thermosensitivity of colony-forming ability. In an attempt to characterize the signal for RAD9 gene-dependent G1 and G1/S cell cycle arrest, we examined the influence of the continued presence of unexcised photoproducts. In mutants defective in nucleotide excision repair, cessation of S-phase was observed at much lower doses of UV radiation compared to excision-proficient cells. However, this response was not RAD9-dependent. We suggest that an intermediate of nucleotide excision repair, such as DNA strand breaks or single-stranded DNA tracts, is required to activate RAD9-dependent G1 and G1/S checkpoint controls.
A newly characterized rad1 missense mutation (rad1-20) in the yeast Saccharomyces cerevisiae maps to a region of the Rad1 polypeptide known to be required for Rad1-Rad10 complex formation. The UV sensitivity of the rad1-20 mutant can be partially and specifically corrected by overexpression of wild-type Rad10 protein. These results suggest that complex formation between the Rad1 and Rad10 proteins is required for nucleotide excision repair.
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