Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding

Abstract

Using a cytological assay to monitor the successive chromatin association of replication proteins leading to replication initiation, we have investigated the function of fission yeast Cdc23/Mcm10 in DNA replication. Inactivation of Cdc23 before replication initiation using tight degron mutations has no effect on Mcm2 chromatin association, and thus pre-replicative complex (pre-RC) formation, although Cdc45 chromatin binding is blocked. Inactivating Cdc23 during an S phase block after Cdc45 has bound causes a small reduction in Cdc45 chromatin binding, and replication does not terminate in the absence of Mcm10 function. These observations show that Cdc23/Mcm10 function is conserved between fission yeast and Xenopus, where in vitro analysis has indicated a similar requirement for Cdc45 binding, but apparently not compared with Saccharomyces cerevisiae, where Mcm10 is needed for Mcm2 chromatin binding. However, unlike the situation in Xenopus, where Mcm10 chromatin binding is dependent on Mcm2-7, we show that the fission yeast protein is bound to chromatin throughout the cell cycle in growing cells, and only displaced from chromatin during quiescence. On return to growth, Cdc23 chromatin binding is rapidly reestablished independently from pre-RC formation, suggesting that chromatin association of Cdc23 provides a link between proliferation and competence to execute DNA replication.

Figure 1.

Cdc23 is chromatin bound throughout the cell cycle. (A) Cdc23 is constitutively located in the nucleus before and after detergent washing. Top and middle panels: an asynchronous log phase culture of strain P1082, permeabilized by zymolyase digestion and either directly fixed (–Triton) or detergent washed and then fixed (+Triton). Bottom panels: strain P1128 (Mcm2-YFP, Cdc23-CFP) permeabilized by zymolyase digestion and detergent washed; Mcm2 is chromatin associated in only binucleate cells (in late M/G1/S), whereas Cdc23 is refractory to detergent extraction in all stages of the cell cycle. Bar, 10 μm. (B) Quantitation of data shown in A. Bin, binucleate (G1/S phase) cells; Unin, uninucleate (G2 phase) cells. (C) Digestion of DNA with micrococcal nuclease releases Cdc23, implying that Cdc23 that is refractory to detergent extraction is bound to chromatin. Cells prepared as in A were either detergent washed only (–MNase), treated with detergent and micrococcal nuclease (+MNase), or treated with detergent and micrococcal nuclease in the presence of EGTA (+MNase, +EGTA). Graphs show the percentage of cells with nuclear Cdc23 or DNA. (D) Cdc23 is chromatin associated in cells arrested at different cell cycle stages. Strains mutant in cdc10 (P1362), cdc22 (P1358), cdc25 (1357), or nda3 (1360) genes were shifted to the restrictive temperature (20°C for nda3, otherwise 36°C) for 3 h, and chromatin binding of Cdc23 was assessed by detergent washing as in A. The wild-type control strain used was P1082. (E) Cdc23 is not chromatin associated in cells arrested in G1 by nitrogen starvation. Log phase cells of strain P1122 was transferred to EMM-nitrogen medium for 16 h at 25°C after which cells were processed as in A and fixed either without (–T) or after detergent washing (+T). Bar, 10 μm. (F) Comparison of Cdc23-CFP levels with other CFP-tagged replication proteins by Western blotting. Strains used were P1122 (Cdc23), P1134 (Orc6), P1051 (Mcm2), and P1054 (Mcm7). Also shown are yellow fluorescent protein (YFP)-tagged Cdc45 (P1083) and Mcm2 (“Mcm2-Y,” P1046) and a wild-type strain (“no tag”). α-Tubulin is shown as a loading control. From quantitative analysis of Western blots with diluted Orc6, Mcm2, and Mcm7 samples, the relative abundance of the proteins is estimated at 1:2:15:15 for Cdc23:Orc6:Mcm2:Mcm7 (unpublished data).

Figure 2.

Cdc23 is displaced from chromatin after nitrogen starvation, but re-association with chromatin on re-entry to the cell cycle does not require pre-RC formation. (A) Experimental procedure; strains used were P1266 (degron mcm4tstd) and P1122 (mcm4⁺). (B) Nuclear localization (–Triton) and chromatin association (+Triton) of Cdc23 after nitrogen starvation (–N, 16 h) and re-entry to the cell cycle (+N, 2 h) in wild-type (wt) and mcm4 degron strains (mcm4ts-td). Only data for the mcm4ts-td strain are shown after nitrogen starvation (–N, 16 h), but the mcm4⁺ strain showed a similar result. (C) Quantitation of data from the experiment shown in B. (D) Flow cytometry, showing that S phase is blocked in the degron mcm4tstd strain and timing of S phase in the wild-type strain at 37°C.

Figure 3.

Sequential chromatin association of Mcm7 and Cdc45 during the fission yeast cell cycle. (A) Cdc45 is chromatin associated only during S phase. An asynchronous culture of strain P1083 was fixed directly (–T) or permeabilized, detergent-washed, and fixed (+T). Cdc45 is constitutively located in the nucleus during the cell cycle in directly fixed cells, confirming a previous result obtained in cells overexpressing Cdc45 (Miyake and Yamashita, 1998), but chromatin association is only seen in some binucleate cells (arrow). Bar, 10 μm. (B) An asynchronous culture of strain P1156, containing Mcm7-CFP, Cdc45-YFP, and α-tubulin-GFP, was processed by detergent extraction to reveal chromatin association of Mcm7 (left panels) and Cdc45 (middle panels). Cells are staged into a cell cycle sequence according to spindle length and nuclear separation: (1) metaphase/early anaphase (spindle <3 μm); (2) mid to late anaphase (spindle >3 μm); (3) G1/S (binucleate, no spindle); (4) G1/S (binucleate, no spindle, septum visible); and (5) G2 (uninucleate). Although GFP (tubulin) fluorescence leaks into the YFP channel in this experiment, tubulin can be unambiguously distinguished from Cdc45 because nuclear fluorescence is not seen in a strain only containing α-tubulin–GFP after detergent extraction and a strain only containing Cdc45-YFP does not show spindle fluorescence. Bar, 10 μm. (C) Quantitation of experiment shown in B, showing percentage of cells positive for Cdc45 or Mcm7 after detergent extraction. Numbers on the x-axis refer to cell cycle stages shown in B. Mcm7 chromatin association occurs from mid-anaphase (stage 2), but Cdc45 chromatin binding is only seen in binucleate cells lacking spindles (stages 3 and 4). Because a proportion of binucleate cells without spindles (stage 3) are positive for Mcm7 and not Cdc45, these may represent G1 cells. Binucleate cells with both Cdc45 and Mcm7 binding are likely to represent cells in S phase.

Figure 4.

Chromatin binding of Cdc45 is dependent on pre-RC formation, CDK, and Hsk1. (A) Cdc45 chromatin association is dependent on Mcm4 function. Strain P1162 containing a degron mcm4tstd mutation, Mcm2-CFP, and Cdc45-YFP was shifted to 37°C, and the percentage of binucleate cells with Mcm2 and Cdc45 after detergent extraction was scored. (B) Flow cytometric analysis of experiment shown in A, showing arrest of DNA replication by degron mcm4tstd mutation. (C) Cdc45 chromatin association is dependent on Cdc10 and Hsk1 function. Strains containing YFP-tagged Cdc45 and temperature-sensitive cdc10 (P1166) or hsk1 (P1184) alleles were grown to log phase at 25°C and then shifted to the restrictive temperature (36°C for cdc10, 30°C for hsk1), and the chromatin binding of Cdc45 in binucleate cells was monitored using the in situ chromatin binding assay. The wild-type control strain (shifted to 36°C) was P1083. +T, cells were examined after detergent extraction; –T, cells were fixed directly. (D) Flow cytometric analysis of experiment shown in C. (E) Cdc45 chromatin association is dependent on CDK. Strain P1276, containing a triple cyclin B deletion and an nmt1-regulatable cdc13⁺ gene was transferred to thiamine-containing medium at t = 0 to inactivate CDK, and chromatin association of Cdc45 was monitored. This block to S phase entry (shown in flow cytometric analysis in F) also prevents Cdc45 chromatin association. In A, C, and E the percentage of binucleate cells with chromatin bound Cdc45 is shown as a percentage of total binucleate cells.

Figure 5.

Inactivation of Cdc23 prevents Cdc45 chromatin association during S phase. (A) Experimental procedure. Strains used were P1100 (cdc23tstd) and P1083 (wt), both of which contain Cdc45-YFP. HU was added to cultures after refeeding to block cells in S phase and thus prevent displacement of Cdc45 at the permissive temperature. (B) Levels of Cdc23 protein during the experiment. Even lanes: protein levels for experiment shown in C; odd lanes: protein levels for the Mcm2 experiment shown in Figure 6. α-Tubulin is shown as a loading control. (C) Cdc45-YFP fluorescence after detergent extraction. For further details see text. (D) Quantitation of data shown in C. (E) Flow cytometric analysis of cdc23tstd cells shown in C; also shown is –HU control. (F) Flow cytometric analysis of degron cdc23tstd cells released from nitrogen starvation in absence of HU, showing block to S phase at 37°C and execution of DNA replication at 25°C.

Figure 6.

Inactivation of Cdc23 has no effect on Mcm2 chromatin association. Experimental procedure is shown in Figure 5A; strains used were P1098 (cdc23tstd) and P1051 (wt), both of which contain Mcm2-CFP. As before, HU was added to cultures after refeeding to block cells in S phase and thus prevent displacement of Mcm2 at the permissive temperature. (A) Mcm2-CFP fluorescence after detergent extraction. For further details see text. (B) Quantitation of data shown in A. (C) Flow cytometric analysis of cdc23tstd cells shown in A; also shown is –HU control.

Figure 7.

Cdc23 inactivation does not affect Cdt1. Strains expressing Cdt1-CFP and either wild-type (P1214) and or degron cdc23 alleles (nmt-cdc23tstd, P1226) were grown at 25°C to log phase and then transferred to medium lacking nitrogen for 16 h at 25°C to arrest cells in G1. Thiamine was added after 12 h of nitrogen starvation to reduce expression of Cdc23 in the degron strain. At 16 h the cultures were split, refed with nitrogen, and incubated at either 25°C or 37°C and analyzed, at the times indicated, by flow cytometry and fluorescence microscopy to detect nuclear Cdt1. (A) Analysis of percentage of cells showing nuclear Cdt1 after refeeding. Data shown are for directly fixed cells but similar results were obtained for cells that were detergent extracted before fixation. (B) Flow cytometric analysis for data shown in A, showing arrest of DNA replication in the degron cdc23 strain at 37°C.

Figure 8.

Inactivation of Cdc23 during an S phase arrest blocks S phase completion (A) Experimental procedure. Strains used were P1051 (mcm2-CFP); P1220 (mcm2-CFP, nmt-cdc23tstd); P1083 (cdc45-YFP), and P1221 (cdc45-YFP, nmt-cdc23tstd). Numbers in parentheses indicate experimental stages, which are referred to in other parts of the figure. (B) Quantitation of chromatin binding data for Cdc45 for the stages in the experiment shown in A. Left panel: data for cells at 25°C; right panel: data for where cells were shifted to 37°C. (C) Quantitation of chromatin binding data for Mcm2 for the stages in the experiment shown in A. The Mcm2 experiments were terminated at stages 3 and 4. (D) Flow cytometry analysis for data shown in B. In this experiment the peaks drift to the right due to cell elongation at 37°C. The percentages of binucleate cells at stages (5–8) are also shown. (E) Western analysis of Cdc23 levels during experiment; α-tubulin is shown as a loading control.

Figure 9.

Model showing possible basis of Cdc23/Mcm10 chromatin association in fission yeast and vertebrate cell cycles. Arrows indicate hypothetical protein-protein interactions that may be important for establishing chromatin association of Cdc23/Mcm10. For details see text.