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. 1997 Dec 23;94(26):14361-6.
doi: 10.1073/pnas.94.26.14361.

The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway (VSports在线直播)

O Cohen-Fix  1 , D Koshland
Affiliations

The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway

O Cohen-Fix et al. Proc Natl Acad Sci U S A. .

Abstract

Inhibition of DNA replication and physical DNA damage induce checkpoint responses that arrest cell cycle progression at two different stages. In Saccharomyces cerevisiae, the execution of both checkpoint responses requires the Mec1 and Rad53 proteins. This observation led to the suggestion that these checkpoint responses are mediated through a common signal transduction pathway. However, because the checkpoint-induced arrests occur at different cell cycle stages, the downstream effectors mediating these arrests are likely to be distinct VSports手机版. We have previously shown that the S. cerevisiae protein Pds1p is an anaphase inhibitor and is essential for cell cycle arrest in mitosis in the presence DNA damage. Herein we show that DNA damage, but not inhibition of DNA replication, induces the phosphorylation of Pds1p. Analyses of Pds1p phosphorylation in different checkpoint mutants reveal that in the presence of DNA damage, Pds1p is phosphorylated in a Mec1p- and Rad9p-dependent but Rad53p-independent manner. Our data place Pds1p and Rad53p on parallel branches of the DNA damage checkpoint pathway. We suggest that Pds1p is a downstream target of the DNA damage checkpoint pathway and that it is involved in implementing the DNA damage checkpoint arrest specifically in mitosis. .

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Figures

Figure 1
Figure 1
Pds1p-HA is phosphorylated in the presence of DNA damage but not when DNA replication is inhibited. (A) Wild-type OCF1522 cells grown at 30°C were arrested in mitosis with nocodazole (lanes 2–7) and exposed to either UV- or γ-radiation at the indicated doses. Protein samples were prepared 20 min after irradiation and processed for Western blot analysis. The sample from the cycling cells (lane 1) was prepared from cells of the same culture taken prior to the addition of nocodazole. The protein bands corresponding to Pds1p-HA are indicated. Occasionally, a fast-migrating HA-cross-reacting band appears (data not shown). (B) An enlargement of the Pds1-HA bands from lanes 2 and 3 of A. (C) Densitometry scan of lanes 2, 4 (Left), 5, and 7 (Right) of A. The dashed and solid lines are of Pds1p-HA from nonirradiated and irradiated cells, respectively. Scans were preformed from bottom to top. (D) Protein extracts were prepared from nocodazole-arrested wild-type OCF1522 cells that were UV-irradiated at 80 J/m2 as described above. Reactions were carried out in the absence (−) or presence of calf intestine alkaline phosphatase (AP; +, 40 units per reaction; ++, 80 units per reaction) and in the absence (−) or presence (+) of the alkaline phosphatase inhibitor β-glycerophosphate (β-glyc.). (E) Wild-type OCF1522 cells grown at 30°C were not treated (−) or treated with 0.1 M HU for 2 h, after which protein extracts were prepared and examined by Western blot analysis for Pds1p-HA (Left) and Rad53p (Right). Identical results were obtained when cells were arrested with 0.2 M HU (data not shown). The S phase arrest after the HU treatment was verified by flow cytometry analysis (data not shown).
Figure 2
Figure 2
DNA-damage-dependent phosphorylation of Pds1p-HA is Mec1p- and Rad9p-dependent. (A) Wild-type (OCF1522), mec1–1 (OCF1523), and mec1–1 cells carrying a centromere-based plasmid (pRS313, indicated as vector) or a pRS313 derivative carrying a copy of the wild-type MEC1 gene (pOC75, indicated as MEC1) were grown at 30°C and were arrested in G1 phase with the α mating pheromone. When more than 85% of the cells appeared arrested morphologically (shmooed), the cultures were either left untreated (−) or exposed to γ-radiation (+, 4 krad). After the irradiation, the cultures were released into medium containing Pronase and nocodazole. Samples for protein extracts were taken 2 h after the release from G1 phase and analyzed by Western blot analysis. At the time when the sample were taken, approximately 90% of the cells in all cultures had a mitotic arrest phenotype (large budded cell with a single nucleus) as determined by 4′,6-diamidino-2-phenylindole (DAPI) staining. (B) Wild-type cells (OCF1522), rad9 cells (OCF1544), and rad9 cells carrying a centromere-based plasmid (pRS314, indicated as vector) or a pRS314-derived plasmid containing a copy of the RAD9 wild-type gene (pOC81, indicated as RAD9) were treated as described in A.
Figure 3
Figure 3
DNA-damage-dependent phosphorylation of Pds1p is independent of Rad53p function. (A) Wild-type (OCF1522), mec1–1 (OCF1523), and rad53–21 (OCF1524) strains were arrested in G1 phase, irradiated, and released into medium containing nocodazole as described in Fig. 2A. (B) Wild-type (OCF1522), mec1–1 (OCF1523), rad53–21 (OCF1524), and pds1Δ (OCF1525) strains were arrested in G1 phase, irradiated, and released into medium containing nocodazole as described in Fig. 2A, except that samples were taken for both Western blot analysis (Top) and Northern blot analysis (Middle and Bottom), which were carried out as described (19).
Figure 4
Figure 4
Pds1p-HA is phosphorylated after the UV-irradiation of S phase-arrested cells. (A) Wild-type (OCF1522) cells were arrested in S phase with HU (0.1 M) or in mitosis with nocodazole (NZ, 15 μg/ml) and UV-irradiated at the indicated doses. Twenty minutes after irradiation, the cells were harvested and analyzed by Western blot analysis for Pds1p-HA. (B) Densitometry scans of the lanes indicated by asterisks in A: HU-treated cells, 0 and 160 J/m2; NZ-treated cells, 0 and 160 J/m2. Scans were done as described in Fig. 1B. The dashed and solid lines are of Pds1p-HA from nonirradiated and irradiated cells, respectively. (C) Wild-type (OCF1522) cells were arrested in S phase with HU (0.2 M) and UV-irradiated at 160 J/m2. The irradiated cells were harvested 20 min after irradiation and processed for phosphatase treatment. The phosphatase reaction were carried out in the presence (+) or absence (−) of 40 units of calf intestine alkaline phosphatase (AP) and 0.2 M β-glycerophosphate. (D) Wild-type (OCF1522) and rad9 (OCF1544) cells were arrested in S phase with HU (0.1 M) and UV-irradiated at the indicated UV doses as described in A.
Figure 5
Figure 5
Model for the signal transduction pathways of the DNA damage and replication checkpoint. DNA damage is represented by a double-strand break. In the presence of DNA damage, Mec1 and/or its downstream target associate with DNA-alteration-specific determinant X (Dasd X), leading to the phosphorylation of Pds1p and Rad53p. Inhibition of DNA replication activates a different Dasd (Dasd Y) that will not promote the phosphorylation of Pds1p but perhaps the phosphorylation of an S phase-specific mitotic inhibitor. The proximity of different proteins indicates their possible localization relative to the DNA and does not imply physical interactions between the proteins. Symbols are as follows: 9, Rad9p; 17, Rad17p; 24, Rad24p; 3, Mec3p; ɛ, polɛ; 5, Rfc5p; 11, Dpb11p.
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