Regulation of replication timing in fission yeast

Abstract

Here we report the first characterization of replication timing and its regulation in the fission yeast Schizosaccharomyces pombe. We used three different synchronization methods: centrifugal elutriation, cdc10 temperature-shift and release, and starvation for deoxyribonucleoside triphosphates (dNTPs) by treatment with hydroxyurea (HU) followed by removal of HU, to study the times when specific autonomously replicating sequence elements (ARS elements; potential replication origins) replicate during S phase. We found that individual ARS elements replicate at characteristic times, some early and some late, independently of synchronization method. In wild-type cells treated with HU, early ARS elements replicated but late ones did not. However, in HU-treated mutant cells lacking the Rad3 (similar to human ATR and ATM) or Cds1 (similar to human CHK2) checkpoint kinase, both early and late ARS elements were able to replicate. Thus under conditions of dNTP starvation the Rad3 and Cds1 kinases are needed to suppress the replication of normally late-replicating regions.

Fig. 1. Synchronization of S.pombe cells by centrifugal elutriation. (A) Diagram illustrating changes in number of nuclei per cell and in cell size during the cell cycle. (B) Percentage of cells with a septum at various times after elutriation. (C–E) Frequencies of various DNA contents per nucleus, determined by quantitative microscopy of Sytox Green fluorescence, in uninucleate and binucleate cells at 90, 150 and 180 min after elutriation. Note that the scale of fluorescence intensity is not linearly proportional to DNA content; the G₂ peak occurs at less than twice the intensity of the G₁ peak.

Fig. 2. 2D gel analyses of replication intermediates (RIs) from three regions of the S.pombe genome in cdc10-M17 cells synchronized to early S or later S phase by centrifugal elutriation. In each diagram, the white boxes represent the sequences of interest and the black bars represent the hybridization probes utilized. (A) ars2-1 is replicated both actively (bubble arc) and passively (Y arc) in both the early S (90 min after elutriation; Figure 1C) and later S (180 min after elutriation; Figure 1E) samples. The 6.2 kb genomic HindIII fragment was detected by a 5 kb HindIII–PstI probe. (B) ars727 is located in S.pombe cosmid c3B8 on chromosome II (DDBJ/EMBL/GenBank accession number AL022244). The probe detects both a 572 bp fragment and a 4.9 kb fragment. Only the latter is seen in the autoradiograms. In (C), several telomeric HindIII restriction fragments of 5–6 kb are detected by the telomere probe, which corresponds to the 800 bp of telomere-associated sequence in the plasmid pEN42 (Nimmo et al., 1994). The white box represents telomere-associated sequences and the grey box represents telomeric simple repeat sequences. The location of the HindIII sites with respect to the telomeres varies from telomere to telomere and strain to strain. The faint signal seen in early S phase has the shape expected for a ‘double-Y’ line (Brewer and Fangman, 1987), but alternative explanations are not excluded. See the text for additional details.

Fig. 3. FACS characterization of the cdc10-M17 strain synchronized by temperature block and release. Cells were incubated at 37°C for 4.5 h, then spun down and resuspended in fresh medium at 25°C. FACS profiles at various times after shift to 25°C are shown. See the main text and Materials and methods for additional details.

Fig. 4. 2D gel analyses of various ARS elements in cdc10-M17 cells synchronized by temperature block and release. (A) ars2-1 replicates in early and middle S phase. In some autoradiograms the RIs are obscured by background noise. (B) The K-repeat ARS replicates in early S phase. The diagram of cen2 sequences is based on Smith et al. (1995). Each type of box represents a different type of repeat sequence. (C) The rDNA ARS also replicates in early S phase. The diagram is based on Sanchez et al. (1998). (D) Diagram showing the position of the FACS peak (as in Figure 3) as a function of time after release. The diagram is spread out so that the times correspond to those of the autoradiograms above.

Fig. 5. 2D gel analyses of late-replicating regions in cdc10-M17 cells synchronized by temperature block and release. (A) Isolated DNA was digested with HindIII, subjected to 2D gel electrophoresis and then probed with ars2-2. (B) EcoRI-digested DNA was subjected to 2D gel electrophoresis and then probed with ars727. (C) DNA samples digested with EcoRI or with HindIII were probed with the 800 bp telomere probe.

Fig. 6. FACS characterization of the cdc10-M17 strain synchronized by hydroxyurea block and release at 25°C. Cells were incubated in 12 mM HU for 5 h, then washed and resuspended in fresh medium lacking HU. FACS profiles at various times after removal of HU are shown. See the main text and Materials and methods for additional details.

Fig. 7. 2D gel analyses of early- and late-replicating regions in cdc10-M17 cells synchronized by HU block and release at 25°C. DNA samples were taken at the indicated times, digested with HindIII, and subjected to 2D gel analysis. (A) RIs accumulate near the K-repeat ARS during the HU block. (B–D) RIs accumulate near ars2-2, ars727 and telomeres after the HU block. (E) Diagram showing the position of the FACS peak (as in Figure 6) as a function of time after removal of HU.

Fig. 8. 2D gel analyses of early- and late-replicating regions synchronized by HU block and release at 30°C. A non-temperature-sensitive strain was employed (ura4-D18 leu1-32 ade6-704 h^–). DNA samples were taken at the indicated times, digested with HindIII, and subjected to 2D gel analysis. (A) RIs accumulate near the K-repeat ARS during the HU block. (B) RIs accumulate near telomeres 20–30 min after the HU block. (C) Diagram showing the position of the FACS peak as a function of time after removal of HU.

Fig. 9. Effects of the rad3Δ and cds1Δ mutations on accumulation of RIs when replication is slowed by HU treatment. Log phase wild-type (wt; 972 h^–), cds1Δ and rad3Δ strains were incubated with 12 mM HU for the indicated times at 30°C. For an unknown reason, in this experiment, 12 mM HU failed to completely block DNA synthesis, as indicated by the rightward movement of the FACS peaks (especially obvious at 4.5 h). The 0 h FACS peaks show the profiles for the log phase cells. In our experience, the 2N position for log phase cells is usually slightly to the left of the 2N position for synchronized cells, as in this case. Aliquots of the cell cultures were taken at the indicated times and DNA was purified, then digested with HindIII, for 2D gel analyses.