FIGURE 5.

Cdc24 is required to recruit Dna2 to DSB sites. A, Cdc24 is required for Dna2 to localize to DSB sites. This assay was conducted as described in Fig. 1E except for modifications to the growth temperature. wt, cdc24^ts, or dna2^ts cells with an A₅₉₀ of ∼0.05 were each inoculated into an EGRL culture and incubated at 26 °C for 8.5 h. Next, the three cultures were shifted to 36.5 °C for an additional 8-h incubation followed by cell fixation with ethanol and analysis of fluorescent foci. B, a statistical measure shows a significant reduction of cdc24^ts cells having mCherry-Dna2 foci or dna2^ts cells having Cdc24-YFP foci. **, p < 0.01, statistically significant. C, the interaction between Cdc24^ts and Dna2 is significantly impaired at growth temperatures of 26 and 36.5 °C. The cdc24^ts and wt cells were grown to an A₅₉₀ of ∼0.3 at 26 °C and then were shifted to 36.5 °C for a 6-h incubation. Whole cell extracts were prepared and subjected to IP with monoclonal anti-HA-agarose beads against HA-Dna2. The pulled-down proteins were examined using the HA antibody to detect HA-Dna2 and the TAP antibody to detect Cdc24-TAP. D, the interaction between Dna2^ts and Cdc24 was undetected at 36.5 °C. The assay was performed as described in C except that IgG beads were used to pull down Cdc24-TAP. HA-Dna2 and Cdc24-TAP were detected by Western blotting using the HA and TAP antibodies.