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.