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doi: 10.1242/10.1242/jcs.00004

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A novel chk1-dependent G1/M checkpoint in fission yeast

Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway

View larger version (32K): [in a new window]   Fig. 1. Activity of the ars3001 origin at restrictive temperature in the orp1-4 mutant. The orp1-4 cells were grown exponentially in EMM medium at 25°C before shift to 36°C. Samples were removed at the times indicated (in hours) at restrictive temperature, and the DNA was purified and subjected to 2D gel electrophoresis, blotted to a membrane and probed with radioactively labelled ars3001 DNA. In the top panel, the strong arc (the Y-arc) reflects passive replication of the region and the weaker arc above reflects the usage of the region as an origin of DNA replication.

View larger version (19K): [in a new window]   Fig. 2. The G1 arrest of orp1-4 cells is checkpoint dependent. (A) Exponentially growing orp1-4 and the indicated orp1-4 checkpoint double mutant cells were shifted to the restrictive temperature of 36°C and cutting was monitored by DAPI staining. (B) orp1-4 rad9-HA and cdc17 rad9-HA cells were shifted to the restrictive temperature for the indicated times and the phosphorylation of Rad9-HA was monitored by SDS-PAGE and western blotting. (C) orp1-4, cdc18-K46 orp1-4 and orp1-4 rad26::ura4+ cells were synchronised by lactose gradient centrifugation in G2, and then shifted to the restrictive temperature. Septation index was determined by Calcofluor staining; cutting was measured by DAPI staining.

View larger version (23K): [in a new window]   Fig. 3. Chk1, but not Cds1, is required to prevent cutting in orp1-4. (A) Exponentially growing orp1-4, orp1-4 chk1 and orp1-4 cds1 cells were shifted to the restrictive temperature for orp1-4. Cutting was monitored by DAPI staining. (B) orp1-4 cells were shifted to the restrictive temperature for the indicated times and protein extracts were prepared. Cds1 was immunoprecipitated and Cds1 kinase activity was measured using MBP as substrate. Hydroxyurea-treated wild-type cells serve as positive controls. The graph shows quantification of the kinase activity corrected for loading. The immunoprecipitated IgG was used as a loading control. (C) orp1-4 chk1-HA and orp1-4 cdc18-K46 chk1-HA cells were shifted to the restrictive temperature for the indicated times. Total protein was prepared and Chk1 phosphorylation was investigated by SDS-PAGE and western blot analysis.

View larger version (37K): [in a new window]   Fig. 4. Cdc2 is phosphorylated by Wee1 upon Chk1 phosphorylation in orp1-4. (A) orp1-4 chk1-HA cells were synchronised in early G2 by lactose gradient centrifugation and shifted to the restrictive temperature. Total protein extract was prepared and the amount of phosphorylated Cdc2 and Chk1 was investigated by SDS-PAGE and immunoblot analyses against Chk1, total Cdc2 and phosphorylated Cdc2 (Cdc2-YP). (B) Quantification of the blots (A) and comparison with cell cycle parameters. The levels of Cdc2 and Chk1 phosphorylation were corrected for loading. Septation index was determined by aniline blue staining; mitotic index was determined by DAPI staining. (C) Exponentially growing orp1-4, orp1-4 cdc2-1w and orp1-4 cdc2-3w cells were shifted to the restrictive temperature for orp1-4. Cutting was monitored by DAPI staining. (D) Exponentially growing orp1-4, orp1-4 mik1 and orp1-4 wee1-50 cells were shifted to the restrictive temperature for orp1-4. Cutting was monitored by DAPI staining. (E) The orp1-4 mik1-myc strain was shifted to the restrictive temperature for the indicated times. Total protein extract was prepared and the amount of mik1-myc was investigated by SDS-PAGE and western blot analysis. -tubulin was measured as a loading control. (F) The orp1-4 wee1-HA strain was shifted to the restrictive temperature for the indicated times. Total protein was extracted and the amount and phosphorylation state of Wee1 was investigated by SDS-PAGE and western blot analysis.

View larger version (21K): [in a new window]   Fig. 5. Activating a putative damage checkpoint in G1 delays cutting in orp1 cells. An orp1::ura4+/orp1-4 ura4-D18/ura4-D18 h+/h- diploid was sporulated and the spores were germinated in the absence of uracil at the restrictive temperature for orp1-4. Half of the culture was irradiated 4 hours after inoculation and cutting was followed by DAPI staining in both cultures.

View larger version (17K): [in a new window]   Fig. 6. Cutting in orp1-4 is initiated early. cdc10-129 orp1-4 and orp1-4 cells were synchronised by lactose gradient centrifugation in early G2, and then shifted to the restrictive temperature. Septation index is given as a measure of synchrony and was determined by Calcofluor staining (A). Aberrant mitoses (cutting) were detected by DAPI staining (B).

View larger version (28K): [in a new window]   Fig. 7. Cutting and cell cycle arrest in orp1-4 cells. The columns show the events taking place at a replication origin in wild-type, cutting and arrested orp1-4 cells. See discussion for details. Damaged DNA is shown here as a strand break, but we have no evidence that this is indeed the case.