SUPPLEMENTARY INFORMATION

Transcription

1 doi:.8/nature979 Financial support: CS is a Royal Swedish Academy of Sciences Research Fellow supported by Knut and Alice Wallenbergs Foundation. This work was financed European research council (ERC starting grant), Swedish Research Council, Swedish Cancer Society, Cornell s and Karolinska Institute s research foundations (CS), Vinnova, Swedish foundation for Strategic research (SSF), Japan Science and Technology Agency (JST) (CS/KS), Ministry of Education, Culture, Sports, Science and technology (KS/TI/YK), and Japan Society for promotion of Science (KS/TI/YK). Supplementary Table. Yeast strain genotypes Changes to original genotypes of W (ade-, trp-, can-, leu-, leu, his-,, ura, RAD), YPH9 (leu-del, trp-del6, ura-, ade-, his-del, lys-8, cyh R, kar-del, cys) and R7 (HO HML/HMRalpha, MATa, cry, ura, ade, leu) (from Prof J. Haber) are listed. Strain Origin Modification CB67 W TetR-GFP-HIS, TetOs-URA CB W SMC-6HIS-xFLAG-KAN CB8 W SMC6-6HIS-xFLAG-KAN, ade::galho, HO cutsite at chr V (4kb from left telomere) CB W SMC-6HIS-xFLAG-KAN CB67 W GDP-TK::URA, ADH-hent-AURIC CB77 W GDP-TK::URA, ADH-hent-AURIC, smc6::leu, smc6-6 on YCPLac (TRP) CB8 W GDP-TK::URA, ADH-hent-AURIC, top-4

2 RESEARCH SUPPLEMENTARY INFORMATION CB84 W GDP-TK::URA, ADH-hent-AURIC, smc-9 CB8 W GDP-TK::URA, ADH-hent-AURIC,top sgs CB869 W GDP-TK::URA, ADH-hent-AURIC, sgs::kan CB889 W GDP-TK::URA, ADH-hent-AURIC, top::kan CB96 W GDP-TK::URA, ADH-hent-AURIC, scc-4 CB94 W GDP-TK::URA, ADH-hent-AURIC, smc6::leu, smc6-6 on YCPLac (TRP), rad::his CB9 W GDP-TK::URA, ADH-hent-AURIC, rad::his CB944 W GDP-TK::URA, ADH-hent-AURIC, srs::his CB96 W SMC6::LEU, smc6-6 on YCPLac (TRP), TetR-GFP-HIS, TetOs-URA CB967 W SMC6::LEU, smc6-6 on YCPLac (TRP), rad::kan, TetR-GFP-HIS, TetOs-URA CB974 W prs6-ura CB987 W top-4, prs6-ura CB W GDP-TK::URA, ADH-hent-AURIC, mms-ch::his CB W SMC6::LEU, smc6-6 on YCPLac (TRP), prs6-ura CB7 W SMC6::LEU, smc6-6 on YCPLac (TRP), top-4, prs6- URA CB674 R7 SMC6-6HIS-xFLAG-KAN CB7 R7 NSE4-6HIS-xFLAG-KAN CB768 R7 SMC-6HIS-xFLAG-KAN CB77 R7 SMC-6HIS-xFLAG-KAN CB778 R7 SMC6-6HIS-xFLAG-KAN, mre::hph CB799 YPH9 SMC6-6HIS-xFLAG-KAN, chr I fragmented, fragments: and 4 kb CB8 YPH9 SMC6-6HIS-xFLAG-KAN, chr IV fragmented, fragments: and 4 kb CB7 W SMC6-6HIS-xFLAG-KAN CB86 W SCC-6HIS-xFLAG-KAN CB W SMC6-6HIS-xFLAG-KAN, cdc6::hisg, GAL-ubiR-CDC6 CB4 R7 SCC-6HIS-xFLAG-KAN CB67 W SMC6-6HIS-xFLAG-KAN, top-4

3 RESEARCH Supplementary Table. Statistics for ChIp- sequencing experiments

4 RESEARCH SUPPLEMENTARY INFORMATION a Wild type Top fork rotation Top (+) Sc Top I Smc/6 normal levels of (+) Sc replication normal DNA SCI II top mutant Smc/6 Top fork rotation (+) Sc (+) Sc Top (+) Sc higher levels of (+) Sc DNA replication delay on long chromosomes SCI Top smc6 mutant (+) Sc III no fork rotation (+) Sc (+) Sc Top higher levels of (+) Sc DNA replication delay on long chromosomes b long chromosome SCI fork rotation SCI + fork movement + (+) Sc short chromosome Smc/6 fork rotation chromosomal rotation Figure S. Chromosome length influences replication-induced topological stress. ai, During DNA replication the positive supercoils which accumulate ahead of the moving replication fork have to be removed to allow full replication of chromosomes. This process has been shown to depend on topoisomerases (Top, Top) which travel with the replication fork and release the tension by creating transient breaks in the DNA. 4

5 RESEARCH Superhelical tension can also be reduced if the fork rotates with the turn of the DNA helix. This rotation will not only prevent accumulation of super helical tension ahead of the fork, it also creates sister chromatid intertwinings (SCIs) behind. These have to be removed by Top before chromosome segregation. aii, Here we show that budding yeast type topoisomerases are crucial for timely completion of replication of long, but not short chromosomes, suggesting that superhelical tension increases with chromosome length. aiii, This phenotype is shared by mutants of the Smc/6 complex, and we show that this complex accumulates on chromosomes with high levels of SCIs. It also reduces the accumulation of SCIs between the two copies of a replicated plasmids in a top mutant. This leads to a model in which the Smc/6 sequesters nascent SCIs behind the fork, thereby driving fork rotation and removal of superhelical tension ahead of the replication machinery. If Smc/6 is mutated, rotation is inhibited and this leads to an accumulation of supercoils ahead of fork and a subsequent replication delay. b, The chromosome length-dependent nature of the replication delay in top, top and smc/6 mutants can be explained if rotation occurs on a chromosome scale (large red arrow), as shorter chromosomes are expected to be easier to rotate than longer ones. If so, resolution of topological tension is not restricted to closed chromosomal domains of defined sizes. Alternatively, replication blocks occur with a fixed probability of less than one per replicon, thereby taking place more frequently on longer chromosomes.

6 RESEARCH SUPPLEMENTARY INFORMATION Sjogren et al., Figure S a chr III circle enrichment area/kb chr III cc=,88 chr IV Chromosome length (kb) b c Smc6, circular chr III Smc6, linear chr III ARS6 ARS ARS7 ARS8 CEN ARS9 ARS7 ARS8 ARS9 CEN ARS ARS ARS4 ARS ARS ARS d Smc6, mtdna 4 9 of topological tension is not restricted to closed chromosomal domains of defined sizes Alternatively, replication blocks occur with a fixed probability of less than one per replicon, thereby taking place more frequently on longer chromosomes. 6 Figure S. ChIp-sequencing analysis confirms the increased association of Smc6 to longer chromosomes and to a short circular chromosome. a, Enrichment of Smc6-Flag per kb and chromosome length in benomyl-arrested G/M cells (CB7) at ⁰C. Each black diamond represents one chromosome, enrichment on ChrIII circ is indicated by the red square. cc: correlation coefficient for a linear equation. b-c, Smc6-Flag distribution on linear and circular ChrIII determined by Chip

7 RESEARCH cells (CB7) at ⁰C. Each black diamond represents one chromosome, enrichment on ChrIII circ is indicated by the red square. cc: correlation coefficient for a linear equation. b-c, Smc6-Flag distribution on linear and circular ChrIII determined by Chip sequencing. Top row; Binding ratio (ChIp/) of Smc6 within running bp windows (bp step size). Significantly enriched regions (p-value < -8 ) in Ip fraction are orange, grey colored regions were not significantly enriched. The vertical axis indicates fold enrichment in linear scale. The horizontal axis represents positions in kb from left telomere of the chromosome and positions of ORFs are marked with blue bars. Second row; Number of sequence reads from ChIp fraction for each bp window is plotted. Third row; Number of sequence reads from fraction for each bp window is plotted. d, Smc6-Flag distribution on mitochondrial DNA. As Smc6-Flag should not bind to mitochondrial DNA, background levels can be estimated by calculating enrichment ratio for mitochondrial DNA in the ChIp fraction. As expected, there is almost no accumulation of Smc6-Flag on mitochondrial DNA (enrichment value:.), suggesting that almost all peaks in the ChIp-sequencing analysis can be considered as significant binding sites. For details, see b-c. 7

8 RESEARCH SUPPLEMENTARY INFORMATION a 4 min wt topδ top-4 topδ sgsδ sgsδ 4 min wt smc6-6 mms-ch scc-4 smc-9 b 7 min 6 4 wt top-4 smc6-6 top-4 smc6-6 Figure S. Representative FACS analyses corresponding to figures and 4. a, Representative FACS analysis corresponding to figure. Cell samples were collected after release from an alpha factor induced G arrest at indicated time points. b, Representative FACS analysis corresponding to figure 4. See a, for details. 8

9 RESEARCH p-values smc6-6 topδ topδ IV,9,,8 XV,4,4, VII,4,4, XVI,6,, XIII,6,, Chromosomes II XIV X XI V,8,,,6,88,,,,96,8,74,8,8,976,97 VIII IX,66,897,9,67,7,86 III,846,98,69 VI,79,647,49 I,,4, Chromosome length (kb) chromosome cut-off length in a smc6-6 mutant chromosome cut-off length in topδ and topδ mutants Figure S4. Smc6, Top and Top are needed for full replication of long chromosomes. Deletion of TOP or TOP significantly delays replication of chromosomes which are ~94 kb and longer. The smc6-6 mutation inhibits replication of chromosomes that are ~746 kb or longer. Diagram to the left shows the length of each chromosome. Green bar: cut-off chromosome length for a significant difference in replication between wild type and type I topoisomerase mutants. Red bar: cut-off chromosome length for a significant difference in replication between wild type and smc6-6 cells. The P-values for the difference in chromosome BrdU signal between mutant and wild type cells are displayed to the right. P-values (t-test, P<.) are based on results from n=7 (wild type) and n= (mutant cells) experiments. See text, figure and methods section for details on the experiment. 9

10 RESEARCH SUPPLEMENTARY INFORMATION a Ratio of BrdU incorp. after bulk replication,8,6,4, - CTP + μ M CPT IV III Chromosomes b 4 min CPT - + Figure S. Inhibition of Top by CPT delays replication of long chromosomes. a, Quantification of BrdU incorporation into ChrIV and III after completion of bulk replication as in figure. Wild type W cells were either left untreated or grown in the presence of µm CPT during S-phase progression. The experiment was performed twice, error bars represent standard deviations. b, A representative FACS analysis of cell cycle progression

11 RESEARCH a,7 wt radδ b Ratio of BrdU incorp. after bulk replication,, radδ smc6-6 smc6-6 4 min, IV Chromosomes III wt radδ radδ smc6-6 smc6-6 c.7 d wt Ratio of BrdU incorp. after bulk replication,,, IV Chromosomes III sgsδ srsδ wt sgsδ srsδ 4 min Figure S6. The chromosome length-dependent phenotype in the smc6-6 mutant is not caused by defective homologous recombination and neither Sgs nor Srs influences replication in a chromosome length-dependent manner. a and c, Quantification of BrdU labelled chromosomes in wild type (CB67), rad (CB9), rad smc6-6 (CB94), smc6-6 (CB77), sgs (CB869), and srs (CB944) cells as in figure. The results are based on n=7 (wild type), n= (rad and rad smc6-6), n= (smc6-6), n= (sgs ), and n= (srs ) experiments, error bars represent standard deviations. b and d, FACS profiles of cell cycle progression from representative experiments are shown.

12 RESEARCH SUPPLEMENTARY INFORMATION a % Budded cells wt smc6-6 smc6-6 radδ time after G release (min) b % Nuclear division wild type time after G release (min) nuclear mass streched nuclear masses (min) c % Nuclear division smc time after G release (min) (min) d % Nuclear division smc6-6 radδ time after G release (min) (min) Figure S7. A nuclear division delay in the smc6-6 mutant is not rescued by deletion of RAD. a d, Wild type (CB67), smc6-6 (CB96), or smc6-6 rad (CB967) cells were arrested in G at C using alpha factor. The temperature was then raised to C for 4 minutes before release into the cell cycle at the restrictive temperature. Samples for (a)

13 RESEARCH budding and (b d) nuclear division were withdrawn every minutes after release. Nuclear division was scored in DAPI-stained cells fixed with.7% formaldehyde. At least cells per sample were scored, and the experiment was repeated twice with similar result. FACS analysis was performed before G synchronization, hours after alpha factor addition, just before temperature increase (.h after alpha factor addition), and then every minutes after release. Red FACS profiles indicate the, and hours time points after G release.

14 RESEARCH SUPPLEMENTARY INFORMATION Sjogren et al., Figure S8 a Raff / Gal Raff G 4 8 G 4 8 G 6 G 6 Raff / Gal Raff 4 8 G G 6 G G Raff / Gal Raff G 4 8 G 4 8 G 6 G 6 well b Raff Cdc6 off Chr IV Raff / Gal Cdc6 on G 4 6 Asc.G PFGE Southern Chr IV Southern Chr III Chr III G 4 6 Asc.G c Chromosome IV and III in well (%) Cdc6 on Chr IV Cdc6 off Chr IV Cdc6 on Chr III Cdc6 off Chr III G time after G release (min) d Raff Cdc6 off ARS44 CEN4 ARS46 ARS Raff / Gal Cdc6 on ARS44 CEN4 ARS46 ARS e Raff Cdc6 off ARS ARS48 ARS Raff / Gal Cdc6 on ARS ARS48 ARS alpha factor addition, just before temperature increase (.h after alpha factor addition), and then every minutes after release. Red FACS profiles indicate the, and hours time points after G release. 4 Figure S8. Recruitment of Smc6 to chromosome arms is reduced when cells pass through an S-phase with low levels of replication initiation. Cells with Flag-tagged Smc6 and the CDC6 gene under the control of a galactoseinducible promoter (CB) were treated as indicated in the methods section to allow progression through an S-phase with reduced levels Cdc6 and replication initiation. As shown before, and as indicated by the FACS analysis, the Cdc6-depleted cells went

15 RESEARCH progression through an S-phase with reduced levels Cdc6 and replication initiation. As shown before, and as indicated by the FACS analysis, the Cdc6-depleted cells went through S-phase without any sign of DNA duplication. However, Southern blot analysis of the PFGE gel using probes against ChrIV and III showed that at the end of the experiment, -4% of these chromosomes had initiated replication as indicated from the retention of the signal in the well. This is likely due to residual amounts of Cdc6 present in these cells. Despite this partial replication, the genome-wide number of significant Smc6 interaction sites in G/M was decreased by ~6% in the Cdc6-depleted cells as compared to the control (data not shown). The remaining Smc6 association was concentrated around centromeres, which replicate early during budding yeast S-phase. a, PFGE gel stained with ethidium bromide (left panel), Southern blot of this gel using probes against ChrIV (middle panel) and ChrIII (right panel). b, FACS analysis. c, Quantification of Southern blots in a). The signal in the gel as compared to the total signal (well and gel) is plotted. d, Smc6 binding to the centromeric region of ChrIV in Cdc6-depleted (Raff) or un-depleted cells (Raff/Gal). e, Smc6 binding to a region of ChrIV arm (8-, kb from left telomere) in Cdc6-depleted (Raff) or un-depleted cells (Raff/Gal). The experiment was performed twice with similar results. In the ChIp on chip maps, blue horizontal lines are indicating open reading frames, CEN denoting the position of the centromere and the vertical red lines and numbers specifying the positions of the autonomously replicating sequence (ARS). The y-axis indicates log of the signal strength, and the x-axis the chromosomal coordinates in kb.

16 RESEARCH SUPPLEMENTARY INFORMATION a Nse4, circular chr III b Smc6/mreΔ, circular chr III c Smc, linear chr III d Smc, linear chr III Smc, circular chr III Smc, circular chr III e Scc, linear chr III ARS ARS ARS ARS ARS ARS4 ARS ARS6 - CEN ARS7ARS8 ARS ARS4 ARS ARS - ARS Scc, circular chr III ARS6 ARS8 ARS7 ChrIV arm (8-, kb from left telomere) in Cdc6-depleted (Raff) or un-depleted 8 cells (Raff/Gal). The experiment was performed twice with similar results. In the ChIp on ARS ARS ARS ARS ARS ARS4 ARS ARS6 chip maps, blue horizontal lines are indicating open reading frames, CEN denoting the CEN ARS7ARS8 position ARS9 of the centromere ARS and the vertical ARS red lines and numbers specifying the positions ARS4 - of the autonomously replicating sequence (ARS). The y-axis indicates log of the signal ARS8 ARS ARS6 ARS7 strength, and the x-axis the chromosomal coordinates in kb Figure S9. Circularization of ChrIII increases the number of Nse4 interaction sites, but leaves the binding pattern of Smc, Smc and Scc unchanged. a e, Cells with Flag-tagged: (a) Nse4 and circular ChrIII (CB7), (b) Smc6, mre and circular ChrIII (CB778), (c) Smc and either linear (CB) or circular ChrIII (CB768), (d) Smc and either linear (CB) or circular ChrIII (CB77), (e) Scc and either linear (CB86) or circular ChrIII (CB4) were arrested in G/M at C. Samples for analysis by ChIp on chip on whole genome arrays were withdrawn from the

17 RESEARCH (CB768), (d) Smc and either linear (CB) or circular ChrIII (CB77), (e) Scc and either linear (CB86) or circular ChrIII (CB4) were arrested in G/M at C. Samples for analysis by ChIp on chip on whole genome arrays were withdrawn from the synchronized cultures. The orange peaks indicate the significant binding sites for Nse4/Smc6/Smc/Smc/Scc on chromosomes. Blue horizontal lines indicate open reading frames, CEN denotes the position of the centromere and the vertical red lines and numbers specify the positions of the autonomously replicating sequence (ARS). The y- axis indicates log of the signal strength, and the x-axis the chromosomal coordinates in kb. 7

18 RESEARCH SUPPLEMENTARY INFORMATION Sjogren et al., Figure S a Scc, circular chr III b Scc, linear chr III ARS6 ARS ARS7 ARS8 ARS9 CEN ARS7 ARS8 CEN ARS ARS ARS ARS4 ARS ARS ARS c,, cc (wild:all chr)=, peaks/kb,,, circle chr III chr IV, chr III, Chromosome length (kb) d, enrichment area/kb 4,,,, circle chr III chr III cc (wild:all chr)=, chr IV Chromosome length (kb) Figure S. ChIp-sequencing analysis show that the amount of Scc on 8 chromosomes is uncorrelated to chromosome length, and confirms that circularization of ChrIII leaves Scc binding pattern largely unchanged. Cells with Flag-tagged Scc, with either linear (CB86) or circular ChrIII (CB4) were arrested in G/M at C and processed for ChIp-sequencing as in Figure S. a-c, Scc- Flag distribution on linear (a) and circular ChrIII (b) as determined by ChIp sequencing. Top; Binding ratio (ChIp/) of Smc6 within running bp windows (bp step

19 RESEARCH arrested in G/M at C and processed for ChIp-sequencing as in Figure S. a-c, Scc- Flag distribution on linear (a) and circular ChrIII (b) as determined by ChIp sequencing. Top; Binding ratio (ChIp/) of Smc6 within running bp windows (bp step size). Significantly enriched regions in fraction are orange, grey colored regions were not significantly enriched. The vertical axis indicates fold enrichment in linear scale. The horizontal axis represents positions in kb from left telomere of chromosomes along chromosome with position of ORFs (blue bars). Second row; Number of sequence reads from ChIp fraction for each bp window is plotted. Third row; Number of sequence reads from fraction for each bp window is plotted. Scc-Flag association to mitochondrial DNA was as low as that of Smc6-Flag, showing that almost all Scc peaks in the ChIp-sequencing analysis can be considered as significant binding sites (data not shown, see Figure S for detailed explanation) 9

20 RESEARCH SUPPLEMENTARY INFORMATION a top-4 prs6 prs6 prs6 + HindIII prs6 + HindIII prs6 top-4 prs6 catenated dimer relaxed linearized monomer catenated dimer supercoiled monomer no EtBr + EtBr b Nb.BtsI - + top-4 prs6 prs6 prs6 + HindIII decrease No. of catenanes increase ladder of nicked catenated dimer relaxed supercoiled catenated dimer linearized monomer supercoiled monomer Figure S. Replication of an episomal plasmid produces closed catenated molecules in top-4 strains. a and b, Southern blots of prs6 isolated from G arrested top-4 cells grown at nonpermissive temperature (lanes top-4 prs6). Plasmid prs6 prepared from E. coli served as control and was either left untreated (lanes prs6) or cleaved by HindIII (lanes prs6 + HindIII).

21 RESEARCH In a, plasmid DNA prepared from top-4 cells was resolved on a gel with (+ EtBr) or without (no EtBr) ethidium bromide. Confirming the observation of Baxter and Diffley, resolution of closed, catenated prs6 dimers on a gel lacking EtBr results in plasmid variants which generally migrates above the relaxed monomer. When EtBr is present in the gel, its intercalation into the plasmid will result in DNA supercoiling. This will compact the dimer structure so that it migrates below the relaxed monomer. In b, plasmid DNA isolated form top-4 cells were either left untreated or incubated with the Nb.BtsI enzyme prior to electrophoresis in an EtBr-containing gel. The Nb.BtsI endonuclease creates a single nick in prs6. The Nb.BtsI-induced resolution of the plasmid forms migrating below the relaxed monomer into a ladder of nicked catenated dimers which migrate above, identify the fast migrating form of prs6 as closed, supercoiled catenated dimers. Supplementary references. Hamer, L., Johnston, M. & Green, E., D. Isolation of yeast artificial chromosomes free of endogenous yeast chromsomes: Construction f alternate hosts with defined karyotypic alterations. Proc Natl Acad Sci 9, 76-7 (99).. Piatti, S., Bohm, T., Cocker, J.H., Diffley, J.F. & Nasmyth, K. Activation of S- phase-promoting CDKs in late G defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. Genes Dev, 6- (996).. Baxter, J. & Diffley, J.F. Topoisomerase II inactivation prevents the completion of DNA replication in budding yeast. Mol Cell, 79-8 (8).