Figure 5.

Cdc6 proteins bind to chromatin but Cdc6^K114Ep fails to promote loading of Mcm proteins. Western blots of chromatin fractions from cells (K4055, YB502, YB504, YB505) blocked in G2/M with nocodazole and released to an α-factor (G1) block for 30′, 60′, or 90′ expressing the indicated CDC6 alleles. Lanes 1, 6, 11, and 16 are the chromatin fractions at the nocodazole arrest. Lanes 2, 7, 12, and 17 are the chromatin fractions released to α-factor for 60′ in cells expressing wild-type CDC6 from the MET3 promoter (i.e., no methionine). The next three lanes of each set (e.g., 3–5) represent chromatin fractions released to an α-factor block for 30′, 60′, and 90′ expressing, from left to right, no CDC6, CDC6^WT, cdc6^K114E, or cdc6^{DE(223, 224)AA}. Blots were probed for Cdc6p, Mcm2p, Mcm3p, and Orc3p plus Orc6p.