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. 2000 May;20(9):3086–3096. doi: 10.1128/mcb.20.9.3086-3096.2000

FIG. 5.

FIG. 5

Associations of Cdc45p with chromatin, Mcm2p, and ARS1 are affected in cdc7 and dbf4 mutants. (A) Association of Cdc45p with chromatin is reduced in cdc7 and dbf4 mutants. Wild-type (YB0469) (WT), cdc7-1 (YB0472), cdc7-4 (YB0547), and dbf4-1 (YB0548) cells expressing Cdc45HA3p were synchronized with α-factor and released at either 25 or 35°C. The cells were collected at the α-factor block or 30 min after release. Lysates were prepared and subjected to chromatin fractionation. Immunoblots of Cdc45p and Orc3p in the chromatin sediments (P) and Cdc45p in the supernatants (S) are shown. (B) Cdc45p-Mcm2p interaction is reduced in cdc7 and dbf4 mutants at the nonpermissive temperature. Cells were arrested as in panel A and released at 37°C. Whole-cell extracts (WCE) were prepared from the cells collected at the α-factor block or 30 min after release and subjected to immunoprecipitations (IP) using an anti-Mcm2p antibody. (C) Association of Cdc45p with ARS1 is undetectable in cdc7-1, cdc7-4, and dbf4-1 cells. Wild-type and mutant cells were synchronized with α-factor as in panel A and released at 35°C. The cells were collected at the indicated time points and analyzed by Cdc45p CHIP. (D) DNA content of the samples used in panel C.