RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

Abstract

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

A p53-independent damage-sensing mechanism that functions as a checkpoint at the G 1 /S transition in Chinese hamster ovary cells

“…Thus, it seems unlikely that every origin in the cell could be directly down-regulated by a cis-acting structural hindrance to initiation. We consider it more likely that this regulatory mechanism(s) involves a trans-acting signal transduction pathway(s), and compelling evidence for this proposal has been obtained (35)(36)(37). In combination with data showing that the repair machinery is activated immediately after DNA damage (29,30), we propose that this G 1 ͞S checkpoint may represent a major control mechanism connecting the replication machinery with damage-sensing and repair pathways.…”

“…However, there are precedents for both types of checkpoints in Saccharomyces cerevisiae. One operates at START in mid-G 1 and is apparently mediated by the RAD9 gene product (37,38). A second checkpoint functions near the G 1 ͞S transition and acts between the cell cycle steps effected by the DBF4 and CDC7 gene products (36,37).…”

“…One operates at START in mid-G 1 and is apparently mediated by the RAD9 gene product (37,38). A second checkpoint functions near the G 1 ͞S transition and acts between the cell cycle steps effected by the DBF4 and CDC7 gene products (36,37). Interestingly, a defect in the G 1 ͞S checkpoint in yeast dramatically reduces cell survival rates after a radiation challenge (36,37).…”

“…A second checkpoint functions near the G 1 ͞S transition and acts between the cell cycle steps effected by the DBF4 and CDC7 gene products (36,37). Interestingly, a defect in the G 1 ͞S checkpoint in yeast dramatically reduces cell survival rates after a radiation challenge (36,37). It will be important to determine whether loss of the mammalian G 1 ͞S checkpoint is equally sensitizing to radiation-induced cell killing, since this pathway appears to be intact in most human tumors (21), and therefore may protect against radiation-based therapeutic strategies.…”

RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae.

“…These include RAD9,RAD17,RAD24,and MEC3, which are required for cell-cycle arrest in G~ (Siede et al 1993 or G2 Hartwell 1988, 1993;Weinert et al 1994) in response to DNA damage; MEC1/ SAD3/ESR1 and RAD53/SAD1/MEC2/SPK1, which are required for the S-phase checkpoint, the transcriptional response, and G1 and G2 arrest (Allen et al 1994;Kato and Ogawa 1994;Weinert et al 1994); POL2/DUN2, which is required for the S-phase checkpoint (Navas et al 1995); and DUN1, which encodes a protein kinase that is activated in response to DNA damage and is necessary for the transcriptional response . MEC1 and RAD53 are central transducers of the signal.…”