Figure 1.

Replication-independent chromatin assembly is cell cycle regulated in extracts from synchronized wild-type cells. (A Upper) FACScan analysis of α-factor synchronized cells. (Lower) Assembly-driven supercoiling of a relaxed plasmid (lane 1) is compared in extracts from unsynchronized cells (Unsync., lanes 2–4), and extracts from cells synchronized in G₁ (lanes 5–7), S (lanes 8–10), G₂ (lanes 11–13), and M (lanes 14–16) by release from α-factor arrest. The time of harvest after arrest is indicated. Reactions were performed for 30 min at 30°C by using 25, 50, and 100 μg of protein for each extract. (B) Assembly in extracts from cells arrested in S phase by culture in the presence of hydroxyurea (HU, lanes 1–3) and in G₂/M phase by exposure to nocodazole (Nocod., lanes 7–9). Assembly in unsynchronized extract is also shown (Unsync., lanes 4–6). Reactions were performed as in A. The migration of open circular/relaxed DNA (linking number 0) [O/R], highly supercoiled species (SC), and intermediate topoisomers (Int.) is indicated.