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.

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