Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication

Abstract

Chromosomes are replicated in characteristic, temporal patterns during S phase. We have compared the timing of association of replication proteins at early- and late-replicating origins of replication. Minichromosome maintenance proteins assemble simultaneously at early- and late-replicating origins. In contrast, Cdc45p association with late origins is delayed relative to early origins. DNA polymerase alpha association is similarly delayed at late origins and requires Cdc45p function. Activation of the S phase checkpoint inhibits association of Cdc45p with late-firing origins. These studies suggest that Cdc45p is poised to serve as a key regulatory target for both the temporal and checkpoint-mediated regulation of replication origins.

Figure 1

Temporally regulated origin associations of Mcm7p, Cdc45p, and DNA polymerases α and ɛ. (A–D) Cells of strains OAy534 [Mcm7p-HA (A)], OAy617 [Cdc45p-HA (B)], OAy644 [Polα-HA (C)], and OAy618 [Polɛ-HA (D)] were synchronized in G₁ phase with α factor and were released at 16°C (t = 0 min). At the indicated time points, chromatin-containing extracts were prepared from formaldehyde cross-linked cells and were immunoprecipitated with anti-HA monoclonal antibody. PCR with primers specific to the ARS305, ARS1, ARS501, and ARS603 chromosomal replication origins and the non-origin sequences 305+8kb and R11 amplified the precipitated DNA for analysis by PAGE and EtBr staining. (E) FACS analysis indicates the DNA content of cells throughout the time course in B. FACS profiles from the experiments in A, C, and D were very similar. (F) Budding index (% Unbudded) of cells throughout each time course in A–D was determined and plotted.

Figure 2

Quantification of the data for ARS305 and ARS603 in Fig. 1 A–D is plotted in A, B, C, and D, respectively. % Precipitated, determined as the densitometric value for each PCR product of an immunoprecipitated sample divided by the value for the PCR reaction of the corresponding input DNA sample multiplied by the appropriate dilution factor.

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.

Figure 4

Origin-loading of DNA polymerase α requires Cdc45p function. Cells of strains OAy644 (WT, Polα-HA) and OAy679 (cdc45–1, Polα-HA) were synchronized in G₁ phase with α factor and were released at 12°C (t = 0 min). Samples were collected at 30-min intervals (no samples collected at t = 30 or 60 min) for chromatin immunoprecipitation analysis, FACS analysis, and budding index determination (% Unbudded). PCR analysis was performed as in Fig. 1.

Figure 5

Rad53p regulates Cdc45p binding to late-firing origins. Cells of strains OAy617 [Wild-type, Cdc45p-HA (A)], OAy684 [rad53, Cdc45p-HA (B)], OAy644 [Wild-type, Polα-HA (C)], and OAy676 [rad53, Polα-HA (D)] were synchronized in G₁ phase with α factor and were released at 23°C into medium containing hydroxyurea (200 mM) (t = 0 min). Every 12 min, samples were removed for chromatin immunoprecipitation analysis, FACS analysis, and budding index determination (% Unbudded). PCR analysis was performed as in Fig. 1. The quantified data is presented. The original gels including all of the primer data are shown in Fig. 7, which is published as supplemental data at www.pnas.org. In addition, the FACS analyses of the experiments shown in A and B are available as supplemental data (Fig. 7 E and F, respectively). The failure to detect Cdc45p-origin association in G₁ cells treated with hydroxyurea was likely attributable to reaction of formaldehyde with the hydroxyurea amine groups, quenching the level of crosslinking below that necessary for detection of Cdc45p association with G₁ phase origins.