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First published online 15 July 2003
doi: 10.1242/jcs.00677

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Delineating the position of rad4⁺/cut5⁺ within the DNA-structure checkpoint pathways in Schizosaccharomyces pombe

Sheila Harris^1,*,, Caroline Kemplen^1,*, Thomas Caspari^2,*,, Christopher Chan¹, Howard D. Lindsay², Marius Poitelea², Antony M. Carr² and Clive Price^1,3,¶

¹ Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, PO Box 594, Western Bank, Sheffield S10 2TN, UK
² Genome Damage and Stability Centre, School of Biological Sciences, University of Sussex, Brighton BN1 9RQ, UK
³ Department of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK

View larger version (44K): [in a new window]   Fig. 1. Rad4 is required for activation of Cds1 and Chk1 kinases. (A) Wild-type and rad4-116 cells were synchronized in G2, released into pre-warmed media and grown in the absence (-) or presence (+) of 10 mM HU at the temperatures indicated. Whole cell extracts from S-phase cells were prepared and assayed for Cds1p kinase activity. The data show that Cds1p kinase activity is severely reduced or absent in rad4-116 cells at 32°C and 36°C, respectively, indicating that Rad4p is required for the activation of Cds1p kinase. (B) Wild-type and rad4-116 cells carrying an integrated HA-chk1+ allele were grown overnight to mid-log and then released into pre-warmed media and grown in the absence (-) or presence (+) of 20 µg/ml bleomycin sulphate or irradiated with 150 J/m2 UV. Cells were also exposed to either a fixed dose of -irradiation 125 Gy (C) or doses in the range 0-125 Gy (D) at the temperatures indicated. Total protein was prepared from all cells and Chk1 phosphorylation analysed by immunoblot using anti-HA antibody (B-D).

View larger version (35K): [in a new window]   Fig. 2. Phosphorylation of Rad9, Hus1 and Rad26 in response to DNA damage and/or inhibition of DNA replication is independent of Rad4. Wild-type and rad4-116 cells carrying integrated, epitope-tagged alleles of hus1, rad9 or rad26 were used in these experiments. Cells were grown overnight to mid-log, harvested and inoculated into fresh pre-warmed media in the absence (-) or presence (+) of 20 µg/ml bleomycin sulphate; (A) Hus1-myc, (B) Rad9-HA, (C) Rad26-myc. (D) Exponentially growing Hus1-myc cells were treated with 10 mM HU for 5 hours. Total protein was prepared from all cells and phosphorylation analysed by immunoblot using either anti-HA or anti-myc antibodies.

View larger version (32K): [in a new window]   Fig. 3. A dominant allele of rad4+ suppresses the DNA damage and HU-sensitive phenotypes of checkpoint mutants. (A). Survival rates are shown for wild-type and rad3-56 cells as well as rad3-56 cells carrying either pREP41-HA (p182) or pREP41-HA-rad4+ (p247) following exposure to 10 mM HU, 20 µg/ml bleomycin sulphate and UV irradiation (0-150 J/m2). The presence of multi-copy HA-rad4+ suppresses the sensitivity of rad3-56 cells to all three agents, restoring viability to wild-type levels. (B-D) Equivalent experiments for rad26 (B), rad17-w (C) and hus1-4 (C). Wild-type and the respective mutant strains carrying either pREP41-HA (p182) or pREP41-HA-rad4+ (p247) were assayed for survival following exposure to 20 µg/ml bleomycin sulphate, 10 mM HU or UV radiation (0-150 J/m2). Comparison of B and C with D shows that the presence of the HA-Rad4-expressing plasmid suppresses sensitivity to all three agents in rad26 and rad17-w but not hus1-4 cells when compared with control cells carrying the vector alone.

View larger version (49K): [in a new window]   Fig. 4. Multi-copy HA-rad4+-mediated suppression of rad3-56, rad26 and rad17-w correlates with restoration of a checkpoint. Checkpoint-deficient strains rad3-56 (A), rad26 (B), rad17-w (C) and hus1-4 (D) carrying either pREP41-HA (p182) or pREP41-HA-rad4+ (p247) were synchronized in G2, incubated in the absence () or presence () of 20 µg/ml bleomycin and the septation index scored at 20-minute intervals to quantify the number of cells passing through mitosis. A mitotic delay can be seen in rad3-56, rad26 and rad17-w cells expressing multi-copy HA-rad4+ (p247) following exposure to bleomycin, whereas hus1-4 cells undergo mitosis with similar kinetics regardless of the presence or absence of multi-copy HA-rad4+ and in the presence or absence of damage. Microscopic analysis of the same cells (stained with DAPI) shows that all mutants exhibit cut cells in the presence of damage (top-right panel), whereas those mutants in which suppression was observed exhibit an elongated cell phenotype (lower-right panel) indicative of a checkpoint delay.

View larger version (97K): [in a new window]   Fig. 5. HA-Rad4p-mediated suppression restores Cds1 kinase activity in response to the inhibition of DNA replication and correlates with nuclear localization of Rad9 in rad17-w mutants. (A) Cds1 kinase activity was assayed in asynchronous cultures of wild-type and rad3-56, rad26, rad17-w and hus1-4 cells carrying either pREP41-HA (p182) or pREP41-HA-rad4+ (p247) following growth in the absence (-) or presence (+) of 10 mM HU for 3 hours. As shown, Cds1p kinase activity can be detected in all strains in which suppression was observed. (B-F) Nuclear localization of Rad9p in rad17-w mutants. The effects of multi-copy Rad4p and HA-Rad4p on Rad9p localization are compared in wild-type and rad17-w cells. Nuclei were visualized with 4',6-diamidino-2-phenylindole (DAPI), whereas Rad9p was visualized using an anti-myc monoclonal antibody. (B) Wild-type cells (SpSc 529) and Rad9p residing in the nucleus. In contrast, Rad9p locates to the cytoplasm in rad17-w cells (SpSc 530) carrying pREP41-HA. (C) The presence of the HARad4p plasmid in rad17-w cells causes Rad9p to relocate to the nucleus (D), however, the same was true for rad17-w cells expressing the untagged version of rad4+, which remain checkpoint deficient (E). (F) Rad9p nuclear staining is absent in rad17-w cells containing the HA-Rad4p plasmid and deleted for hus1+, implying that nuclear localization of Rad9p is Hus1p-dependent.

View larger version (29K): [in a new window]   Fig. 6. (A) In response to DNA damage (or replication inhibition - not shown), Rad3-Rad26 is loaded onto the DNA. The 9-1-1 complex is loaded independently of Rad3-Rad26 by Rad17, which is itself constitutively DNA associated. Rad4, which may be constitutively chromatin associated or loaded in response to DNA damage, makes contact with Rad3-Rad26 (possibly by associating with both proteins) and Rad17. Rad4 anchors Crb2 and, indirectly, Chk1 to the region. It is anticipated that Rad4 plays a similar role in tethering Cds1 to the checkpoint complex in the event of an inhibition of DNA replication. (B) In the absence of Rad4, Rad3 and 9-1-1 are loaded normally, but Chk1 (or Cds1 in the case of replication inhibition) is not available for phosphorylation or activation, whereas Rad9 and Hus1 are.