by the PCR-mediated method (Krawchuk and Wahls, 1999). The construction of Ams2-null and conditional ams2-shut-off strains was previously described

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1 Table S1. Fission yeast strains used in this study. Gene disruption was performed by the PCR-mediated method (Krawchuk and Wahls, 1999). The construction of Ams2-null and conditional ams2-shut-off strains was previously described (Takayama and Takahashi, 2007). Genomic Southern hybridisation was performed to confirm that the authentic genes were disrupted. wee1-50 (Russell and Nurse, 1987), cdc (Nurse et al., 1976), cdc22-c11 (Aono et al., 2002) or cdc25-22 (Russell and Nurse, 1986) were used as cell cycle mutants. Some strains were also generated by crossing. Figure S1. Wild-type (SP92, A) or Ams2-null (SP1698, B) cells expressing Cnp1-GFP were cultured in EMM2 at 33 C. A series of time-lapse images were taken at 2.5-minute intervals. The graphs show the corresponding intensity of a centromeric GFP-dot in the nucleus plotted as black diamonds. The intensities of the background GFP signals in the nuclei are also shown as grey circles. The asterisk indicates the period during which the centromeric GFP signals became weak. This period was presumed to be coincident with phase II (from prometaphase to anaphase A), at which centromere clustering becomes loose. The arrow indicates G1/S phase at which a short uptake of Cnp1 incorporation took place. Figure S2. Ams2-null cells expressing both Cnp1-GFP and Mrc1-GFP (SP1822) were cultured in EMM2 at 33 C. A series of time-lapse images were taken at 2.5-minute intervals. m and c indicate cells showing Mrc1-GFP signals on nuclear chromatin and Cnp1-GFP signals on centromeres, respectively. A representative S-phase Δams2 cell expressing only Cnp1-GFP (SP75, asterisk) is shown for comparison. Mrc1 protein was reported to be localized on nuclear chromatin at S phase (Tanaka and Russell, 2001). Bar, 10 μm. 1

2 Figure S3. Δams2 (Sp75) cells expressing a native promoter-driven Cnp1-GFP were cultured in EMM2 at 26 C in the absence or presence of 12mM HU for 6 h. The representative images (GFP; red, DAPI; blue) and the DNA contents estimated by flow cytometry are shown. The positions of 1C and 2C peaks are also indicated. Bar, 10 μm. Videos S1, S2 and S3. Wild-type (SP92, S1) or Δams2 (SP75, S2, S3) cells expressing Cnp1-GFP were cultured in EMM2 at 33 C. A series of time-lapse images were taken at 5-minute (S1, S2) or 1.5-minute (S3) intervals. Videos S4. Wild-type strain carrying the Sid4-mRFP gene (SP1055, arrowheads) and Δams2 (SP1698) strain, both of which express Cnp1-GFP, were mixed and cultured in EMM2 at 33 C. A series of time-lapse images were taken at 2.5-minute intervals. Before recording, we quickly observed mrfp signals to know wild-type cells from Δams2 cells. Videos S5 and S6. Wild-type cells expressing Cnp1 ts -GFP (SP1102) were cultured in EMM2 at 36 C, and then shifted to 22 C. Representative cells in which Cnp1 ts -GFP was incorporated at S phase (S5) or late-g2 (S6) are shown. Cnp1 ts -GFP protein showed accumulation into centromeres at approximately 8 10 min after septum formation, corresponding to S phase (S5) and the S-phase protein marker Mrc1 was localised in the nucleus (Tanaka and Russell, 2001), or at late-g2 (S6) after the cell length reached 10.5 μm (Mitchison, 1957). A series of time-lapse images were taken in 1.5-minute intervals. Video S7. Δams2 cells expressing Cnp1 ts -GFP (SP1205) were cultured in EMM2 at 36 C, and then shifted to 22 C. Shown is a representative cell in which Cnp1 ts -GFP failed to accumulate into centromeres at S and was reloaded at 2

3 late-g2. A series of time-lapse images were taken in 2.5-minute intervals. 3

4 Table S1. The fission yeast strains used in this study. Name Genotype Reference SP38 h - leu1-32 ura4-d18 lys1 + ::cnp1-gfp This study SP52 h + leu1-32 ade6-704 CN2 This study SP75 h - leu1-32 lys1 + ::cnp1-gfp Δams2::ura4 + This study SP91 h - leu1-32 ura4-d18 lys1 This study SP92 h - leu1-32 lys1 + ::cnp1-gfp This study SP143 h - leu1-32 ura4-d18 This study SP1055 h - lys1 + ::cnp1-gfp sid4-mrfp::kan R This study SP1102 h - leu1-32 ura4-d18lys1 + ::cnp1-1-gfp This study SP1205 h - ura4-d18 lys1 + ::cnp1-1-gfp Δams2::ura4 + This study SP1234 h + his2 lys1 + ::cnp1-gfp cdc25-22 This study SP1339 h - leu1-32 ura4-d18 Δbub1::ura4 + (Bernard et al., 1998) SP1468 h - leu1-32 ura4-d18 lys1 + ::GFP-cnp1 This study SP1628 h + ura4-d18 lys1 + ::cnp1-gfp cdc This study SP1633 h + ura4-d18 lys1 + ::cnp1-gfp cdc22-c11 This study SP1637 h + ura4-d18 lys1 + ::GFP-cnp1 Δams2::ura4 + This study SP1698 h - ura4-d18 lys1 + ::cnp1-gfp Δams2::ura4 + This study SP1699 h + ura4-d18 lys1 + ::cnp1-gfp cdc10-129δams2::ura4 + This study SP1704 h + ura4-d18 lys1 + ::cnp1-gfp cdc22-c11δams2::ura4 + This study SP1751 h + ura4-d18 lys1 + ::cnp1-gfp cdc25-22 Δams2::ura4 + This study SP1769 h - leu1-32 ura4-d18 lys1gfp-cnp1-nat This study SP1822 h - ura4-d18 lys1 + ::cnp1-gfp Δams2::ura4 + mrc1-gfp::kan R This study SP2959 h - leu1-32 ura4-d18 lys1 cdc This study SP2960 h - leu1-32 ura4-d18 lys1 Δams2::Kan R cdc This study SP2962 h - leu1-32 ura4-d18 lys1 cdc22-c11 This study SP2963 h - leu1-32 ura4-d18 lys1 Δams2::Kan R cdc22-c11 This study YTP155 h - leu1-32 ura4-d18 lys1 Δams2::Kan R (Takayama and Takahashi, 2007) YTP354 h - ura4-d18 lys1 his2 Δams2::Kan R cdc25-22 This study YTP355 h - leu1-32 ura4-d18 lys1 Δams2::Kan R cdc25-22 This study YTP366 h - leu1-32 ura4-d18 p nmt81 ams2-kan R This study YTP370 h + leu1-32 ura4-d18 p nmt81 ams2-kan R wee1-50 This study YTP374 h - leu1-32 ura4-d18 wee1-50 This study YTP379 h + leu1-32 ura4-d18 lys1 his2 cdc25-22 This study YTP389 h + p nmt81 ams2-kan R lys1 + ::cnp1-gfp This study YTP390 h + leu1-32 p nmt81 ams2-kan R wee1-50 lys1 + ::cnp1-gfp This study YTP393 h + wee1-50 lys1 + ::cnp1-gfp This study PHS10 h + ura4-d18 lys1 + ::cnp1-1-gfp Δhip1::Kan R This study PHS165 h+ leu1-32 ade6-704 CN2 GFP-cnp1-NAT This study 4

5 Supplementary References Aono, N., Sutani, T., Tomonaga, T., Mochida, S., and Yanagida, M. (2002). Cnd2 has dual roles in mitotic condensation and interphase. Nature 417, Bernard, P., Hardwick, K., and Javerzat, J.P. (1998). Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis. J Cell Biol 143, Krawchuk, M.D., and Wahls, W.P. (1999). High-efficiency gene targeting in Schizosaccharomyces pombe using a modular, PCR-based approach with long tracts of flanking homology. Yeast 15, Mitchison, J.M. (1957). The growth of single cells. I. Schizosaccharomyces pombe. Exp Cell Res 13, Nurse, P., Thuriaux, P., and Nasmyth, K. (1976). Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet 146, Russell, P., and Nurse, P. (1986). cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell 45, Russell, P., and Nurse, P. (1987). Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog. Cell 49, Takayama, Y., and Takahashi, K. (2007). Differential regulation of repeated histone genes during the fission yeast cell cycle. Nucleic Acids Res 35, Tanaka, K., and Russell, P. (2001). Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1. Nat Cell Biol 3,

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