Components and Dynamics of DNA Replication Complexes in S Components and Dynamics of DNA Replication Complexes in S. cerevisiae: Redistribution of MCM Proteins and Cdc45p during S Phase  Oscar M Aparicio, Deborah M Weinstein, Stephen P Bell  Cell  Volume 91, Issue 1, Pages 59-69 (October 1997) DOI: 10.1016/S0092-8674(01)80009-X

Figure 1 Sequence-Dependent Association of ORC with Replication Origins In Vivo (A) Lane 1: strain OAy470 (no Tag); lanes 2–6: strain OAy503 (Orc1p-HA), lane 2 was not treated with formaldehyde (no X-link), lanes 3–6 were treated with formaldehyde (with X-link), lanes 4–6 are 2.5-fold serial dilutions of the template DNA; lanes 7–11 are strains OAy576-OAy580, respectively, which harbor the indicated wild-type or mutant ARS1 allele at the native locus. (B) ORC mutations eliminate specific ORC-origin association. Strains OAy423 (wild-type, lanes 12 and 15), OAy422 (orc1–161, lanes 13 and 16), and OAy446 (orc2–1, lanes 14 and 17) were blocked in G2/M with nocodazole at 23°C. The arrested cultures were split and half was incubated at 37°C for 1 hr while the other half was maintained at 23°C. In each case, the nocodazole cell cycle block was maintained. Chromatin-containing extracts were prepared from formaldehyde cross-linked cells and immunoprecipitated with anti-HA monoclonal antibody (A) or with antibodies to Orc2p and Orc3p (B) (equivalent results were obtained with only anti-Orc3p monoclonal antibodies). Precipitated DNA was amplified by PCR with primers specific to ARS1, ARS305, and URA3 DNA and analyzed by PAGE and EtBr staining. PCR products from the corresponding input DNA samples are shown in the lower panels. Cell 1997 91, 59-69DOI: (10.1016/S0092-8674(01)80009-X)

Figure 2 ORC-Origin Association throughout the Cell Cycle Strain OAy503 (Orc1p-HA) was synchronized in late G1 with α factor and released from the block at 23°C (T = 0 min). Following the 84 min time point, α factor was added to block the cells in the next G1 phase. At 12 min intervals, samples were fixed for chromatin-immunoprecipitation analysis with anti-HA antibody, FACS analysis, and budding-index determination. The percentage of unbudded cells is shown to the right of the FACS profile. Cell 1997 91, 59-69DOI: (10.1016/S0092-8674(01)80009-X)

Figure 3 Genetic Analysis of ORC-Origin Interactions in the Pre-RC and the Post-RC Strains OAy423 (wild-type, lanes 1, 3, 9, and 11) and OAy561 (cdc6–1, lanes 2, 4, 10, and 12), OAy558 (wild-type, lanes 5, 7, 13, and 15) and OAy559 (cdc46–1, lanes 6, 8, 14, and 16) were blocked in late G1 with α factor or in G2/M with nocodazole at 23°C. The arrested cultures were split, and half was incubated at 37°C for 1 hr while the other half was maintained at 23°C. In all cases, the indicated cell cycle block was maintained. Chromatin immunoprecipitations were performed as described for Figure 1B. Cell 1997 91, 59-69DOI: (10.1016/S0092-8674(01)80009-X)

Figure 4 Cell Cycle–Dependent Association of Replication Proteins with Origin DNA (A) Strains OAy470 (no tag), OAy568 (Cdc6p-HA), OAy617 (Cdc45p-HA), OAy534 (Mcm7p-HA), and OAy535 (Mcm4p-HA) were arrested in late G1 with α factor (lanes 1–5) or in G2/M with nocodazole (lanes 6–10) at 23°C. (B) ORC1- and CDC6-dependent association of Mcm4p with origin DNA. OAy535 (Mcm4p-HA), OAy539 (Mcm4p-HA, orc1–161), and OAy554 (Mcm4p-HA, cdc6–1) were arrested with α factor at 23°C. Half of each culture was shifted to 37°C (lanes 14–16) while the other half was maintained at 23°C (lanes 11–13) and incubations were continued in the presence of α factor for 1 hr. These cells were fixed with formaldehyde and subjected to chromatin-immunoprecipitation analysis with anti-HA antibody. Cell 1997 91, 59-69DOI: (10.1016/S0092-8674(01)80009-X)

Figure 5 Mcm4p-DNA Association throughout the Cell Cycle (A) Strain OAy556 (cdc15–2, Mcm4p-HA) was synchronized in late M (telophase) by incubation at the cdc15–2 nonpermissive temperature and released by shifting to 23°C. (B) Strain OAy535 (Mcm4p-HA) was synchronized in late G1 with α factor and released at 23°C. At 12 min intervals, samples were fixed for chromatin-immunoprecipitation analysis with anti-HA antibody, FACS analysis, and budding-index determination. The percentage of unbudded cells is shown to the right of each FACS profile. Cells released from a cdc15–2 block are delayed in cytokinesis resulting in the 2N to 4N shift in the DNA content as a result of replication. For budding-index determination, large-budded cells at the cdc15–2 block and persisting after release were scored as two unbudded cells. Cell 1997 91, 59-69DOI: (10.1016/S0092-8674(01)80009-X)

Figure 6 Mcm4p, Cdc45p, and DNA Polε Associate with Origin and Nonorigin DNA during S Phase (A) Schematic of the ARS305–ARS306 region on the left arm of chromosome 3. The line represents a segment of the chromosome with the ovals indicating the sites of the origins. The thick bars show the relative positions of the five sequences analyzed. Arrows indicate the direction of replication fork movement. (B–D) Strains OAy535 (Mcm4p-HA, [B]), OAy618 (Polε-HA, [C]), and OAy617 (Cdc45p-HA, [D]) were synchronized in late G1 with α factor and released at 16°C (time = 0 min). Samples were collected and analyzed as described in Figure 5 except that primers specific to five unique sequences in the ARS305–ARS306 region were used. PCR reactions of the immunoprecipitated DNA are shown in the upper panels of (B)–(D). DNA from the 48 min sample (B) and the 60 min samples (C and D) and 2-fold serial dilutions of these were subjected to PCR amplification with the ARS305 primers (Titrations). The percentage of immunoprecipitated DNA (% Bound, see Experimental Procedures) is graphed as closed squares for each experimental sample and as open squares for control immunoprecipitations of the untagged strain (OAy470). The scale maximums for the % Bound are 0.40, 0.12, and 0.17, in (B), (C), and (D), respectively. Cell 1997 91, 59-69DOI: (10.1016/S0092-8674(01)80009-X)

Figure 7 Mcm4p-DNA and Polε-DNA Associations in S Phase Blocked Cells Strains OAy535 (Mcm4p-HA, [A]) and OAy618 (Polε-HA, [B]) were synchronized in late G1 with α factor and released into medium containing hydroxyurea at 23°C (time = 0 min). Samples were collected, analyzed, and quantified as in Figure 6. PCR reactions of the immunoprecipitated DNA are shown. The graphs are labeled as in Figure 6 except the % bound maximum is 0.14 for both experiments. Cell 1997 91, 59-69DOI: (10.1016/S0092-8674(01)80009-X)

Figure 8 A Model of DNA Replication Complexes during the Cell Cycle After binding the origin DNA, the ORC-origin complex is recognized by Cdc6p, which recruits MCMs to the origin forming the pre-RC. It is likely that there are other factors in the pre-RC (e.g., Dbf4p); however, they are not illustrated as it is not clear if they are stable members of the pre-RC. We hypothesize that activation of this complex leads to the formation of unwound DNA at the origin, and that this structure recruits the DNA replication enzymes (e.g., DNA Polε) and their accessory proteins to the origin forming the replicative complex (RC). It is possible, however, that the origin has yet to unwind when DNA Polε binds the origin. After initiation, we suggest that MCM proteins and Cdc45p become components of the resulting replicative complex at the replication fork. See Discussion for details. Cell 1997 91, 59-69DOI: (10.1016/S0092-8674(01)80009-X)