Figure 3.

Cdc45p associates with early-replicating origins in G₁ phase. Haploid cells of strains OAy556 [cdc15–2, Mcm4p-HA (A)] and OAy658 [cdc15–2, Cdc45p-HA (B and C)] were synchronized in late M phase by incubating at 37°C for 2 hours and were released at 23°C (t = 0 min). To block cells in G₁ phase, α factor was added 15 min before release from the M phase arrest in C. At 12-min (A and B) or 24-min (C) intervals, samples were fixed for chromatin immunoprecipitations with anti-HA antibody, FACS analysis, and budding index determination. In addition to the origin sequences analyzed in Fig. 1, ARS306, ARS607, and ARS606 were analyzed by PCR. Quantification of the ARS305 and ARS603 data in A and B and all of the data in C is plotted in Fig. 6, which is published as supplemental data on the PNAS web site, www.pnas.org. Because blocked-and-released cdc15 cells are delayed in cytokinesis, unseparated cells with new (small) buds were scored as two small-budded cells (or one small-budded cell and one unbudded cell when only one bud was visible on the unseparated pair of cells). The delay in cytokinesis also resulted in an apparent 2C-to-4C shift in DNA content as replication proceeded.