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Pre-RC assembly can occur in extracts of G1 cells but is blocked in extracts of G2/M cells. Whole-cell extracts prepared from Cdc6p-expressing cells (YGP82), arrested either in G1 phase with α-factor (α) or in G2/M with nocodazole (Noc), were incubated with wild-type (W) or mutant (M) ARS1 beads. The levels of ORC, Cdc6p, and Mcm2p associated with the beads (Bead) or present in the supernatants (Sup) were determined as above.
