Intra-G ₁ arrest in response to UV irradiation in fission yeast

Abstract

G1 is a crucial phase of cell growth because the decision to begin another mitotic cycle is made during this period. Occurrence of DNA damage in G 1 poses a particular challenge, because replication of damaged DNA can be deleterious and because no sister chromatid is present to provide a template for recombinational repair. We therefore have studied the response of Schizosaccharomyces pombe cells to UV irradiation in early G 1 phase. We find that irradiation results in delayed progression through G 1, as manifested most critically in the delayed formation of the pre-replication complex. This delay does not have the molecular hallmarks of known checkpoint responses: it is independent of the checkpoint proteins Rad3, Cds1, and Chk1 and does not elicit inhibitory phosphorylation of Cdc2. Irradiated cells eventually progress into S phase and arrest in early S by a rad3-and cds1-dependent mechanism, most likely the intra-S checkpoint. Caffeine alleviates both the intra-G 1-and intra-S-phase delays. We suggest that intra-G 1 delay may be widely conserved and discuss significance and possible mechanisms.W hen supplied with appropriate nutrients, all growing cells, from simple prokaryote to complex eukaryote, go through an obligatory cell cycle consisting of alternating periods of DNA replication (S phase), segregation of the chromosomes and nuclear division (mitosis), and cell division. The order of events is crucial and is monitored by molecular mechanisms termed checkpoints. A checkpoint inhibits or delays an event of the cell cycle if an upstream event has not been completed properly or if the DNA is damaged (1).Several different DNA damage and DNA replication checkpoints have been identified and characterized in the fission yeast Schizosaccharomyces pombe (2, 3). They include mechanisms to inhibit mitosis when the DNA is damaged (the G 2 ͞M checkpoint) or when S phase has not been completed (the S͞M checkpoint), as well as a mechanism to inhibit ongoing DNA replication when the DNA is damaged (the intra-S checkpoint). These three checkpoints have several molecular determinants in common. First, all of them depend on a set of genes called the checkpoint rad genes. Second, they all are mediated by one or both of two protein kinases, Chk1 and Cds1. Third, a downstream target for all of them is the Cdc2 kinase, which is kept phosphorylated on tyrosine-15 and, thus, is inactivated when the checkpoints are induced.The Cds1 and Chk1 protein kinases are central players in all of the DNA damage and replication checkpoints characterized in S. pombe, but they have different functions in the different checkpoints. The S͞M and intra-S checkpoints both depend on Cds1, which is activated only in S phase (4, 5). The Cds1 protein is phosphorylated by a process dependent on Rad3 and Mrc1 (6, 7). Chk1 is phosphorylated after DNA damage in late S or in G 2 phase in a process dependent on , and this phosphorylation has been taken as a diagnostic sign of checkpoint activation. In human cells, the Rad3 homolog ATR binds and phosphorylate...

Induction of a G 1 -S checkpoint in fission yeast

“…This event occurred at about the same time as Cdc2 became phosphorylated (Fig. 1D), consistent with the preRC data and with the conclusion that this is the time when unirradiated cells enter S phase (9). In cells exposed to UVC, Rum1 disappeared later, 90 min after irradiation, and Cdc2 phosphorylation was detectable only after 120 min ( Fig.…”

“…UVC Irradiation. A dose of 1,100 J/m 2 of UVC irradiation (254 nm) was given at an incident dose rate of approximately 250 J/m 2 per min in a thin, stirred suspension in EMM medium, as described (9). G 1 -synchronized cells were irradiated immediately after the release from the cdc10 block.…”

“…These checkpoints are largely conserved through evolution. We have described a G 1 -S checkpoint in fission yeast that delays formation of the prereplicative complex (preRC) at chromosomal replication origins (9) and absolutely depends on the Gcn2 kinase (10), which is a regulator of translation. The only known mechanism of action of Gcn2 is phosphorylation of the initiation factor eIF2α, which inhibits initiation of translation when phosphorylated.…”

The G1-S checkpoint in fission yeast is not a general DNA damage checkpoint

“…These checkpoints are all dependent on the checkpoint Rad proteins and on one or both of the protein kinases Cds1 and Chk1 that inhibit Cdc2 activity by promoting its phosphorylation. We have recently described a novel checkpoint in S. pombe regulating the G1-S transition (Nilssen et al, 2003;Tvegård et al, 2007), where G1 cells are delayed in Sphase entry after exposure to ultraviolet light (254 nm; UVC). In contrast to the other checkpoints listed above the G1-S checkpoint acts independently of Cds1 and Chk1 and does not lead to Cdc2 phosphorylation.…”

New roles of the fission yeast eIF2α kinases Hri1 and Gcn2 in response to nutritional stress

“…Previous studies showed that ultraviolet (UV) irradiation of fission yeast cells in G1 phase induced a transient delay in S-phase entry (Nilssen et al, 2003). This UV-induced G1-S checkpoint was totally dependent on the Gcn2 kinase and, additionally, UV light led to activation of Gcn2, which in turn resulted in phosphorylation of eIF2a.…”