Figure 7.

Reduced association between Cdc45 and Sld3 in the presence of Sld7. (A) The Sld3–Flag protein (1 pmol) and the Sld3–Flag–Sld7 complex (1 pmol) were incubated with Cdc45–Flag (0.4 pmol)-bound beads at 4°C for the indicated times. Half of the proteins bound to the beads were subjected to SDS–polyacrylamide gel electrophoresis, followed by western blotting, probed with anti-Sld3 and anti-Cdc45 antibodies. Lanes 11 and 12 contain samples of Sld3 (0.05 pmol), Sld3–Sld7 (0.05 pmol), and Cdc45 (0.2 pmol). (B) Plasmid encoding a Gal-inducible SLD7 gene was introduced into the sld3 temperature-sensitive mutants, sld3-4, -5, -6, -7, and -8. The cells were incubated on glucose or galactose plates lacking uracil at the indicated temperatures for 2 days. (C, D) Suppression of the HU sensitivity of the sld7Δ mutant by the introduction of JET1, an S-CDK bypass allele of the CDC45 gene (C), or DPB11 on a high-copy plasmids (D). YTT1 (sld7Δ) cells were transformed with the indicated plasmids. Tenfold dilutions of the cells were spotted onto plates and incubated at 25°C for 3 days.