SUPPLEMENTARY INFORMATION

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Walter Green
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1 DOI:.38/n3343 DIC/Ht Ht DAPI Anphse/Telophse (% of ells) Time from HU relese (min) untrete Nsg1-GFP Myo1-mCherry untrete HU pulse 5 15 Onset of ring ontrtion to NE segregtion (min) untrete 6 mm HU Onset of ring ontrtion to DNA segregtion (min) +HU TEL1 Net1 Sp4 8 1 rdna - HU tel1 + HU rdna tel Time fter nphse onset (min) e Asission (umultive %) HU + HU Time from memrne f sml1 + HU 8 6 Time from memrne g h1 + HU 8 6 Time from memrne h Mein sission time (min) (n=) (n=48) sml1 (n=33) h1 (n=5) - HU + HU Supplementry Figure 1. Chromosome segregtion n sission ynmis uring replition stress. () Wil type ells expressing Ht-mCherry were trete with mm HU for hours, n trnsferre to fresh mei. The Ht-mCherry signl ws use to estimte the frequeny of nphse (with elongte nulei) or telophse (with seprte nulei) ells t the inite times (left). Two inepenent experiments were performe with similr results; results from one experiment re shown. The following numer of ells n ws sore for eh time point: n=17 ( min), 146 ( min), 16 (6 min), 156 (9 min), 164 (1 min), 14 (15 min), 149 (18 min). The mirogrphs show ells 1 min fter HU wshout stine with DAPI to visulize DNA (right). The rrows point to hromtin riges. () Kinetis of nuler ivision (visulize with Nsg1) reltive to tomyosin ring ontrtion (Myo1) in wil type ells in log-phse or fter exposure to HU t ºC, s in Fig.. Asterisk mrks the time of nuler ivision. Numers inite time in minutes. The grph shows the time of nuler ivision reltive to the onset of ring ontrtion (time ). Boxes inlue 5% of t points, whiskers 9%; meins re shown s lines n mens s rosses. The kinetis etween the two onitions were onsiere to e not sttistilly signifint (p >.5, Mnn-Whitney test). n = 3 untrete ells n HU-trete ells, poole from two inepenent experiments. () The time of hromosome segregtion (Ht-mCherry) reltive to tomyosin ring ontrtion (Myo1-GFP) in wiltype ells in the sene or presene of 6 mm HU. HU-trete ells were imge for hours immeitely fter HU ition. The grph shows the time of hromosome segregtion reltive to the onset of ring ontrtion (time ). Mein (lines) n men (rosses) re shown (n = 8 untrete ells n 66 HU-trete ells, poole from 3 inepenent experiments). () Segregtion of the rdna (Net1-GFP) n TEL1R (TetR-YFP) in representtive wiltype ell in the presene of 6 mm HU, s in (). Arrowhes mrk the spinle poles, n sterisk mrks the nuleolus, n rrows mrk TEL1R. Time is shown in minutes reltive to nphse onset; nuleolr segregtion ours t min n TEL1 segregtion follows t 1 min. The grph shows the time of segregtion of the rdna n TEL1R in the sene n presene of HU. n = 5 untrete n 4 HU-trete ells, poole from inepenent experiments (p >.5, Stuent's t-test). (e-h) Asission ynmis of untrete n HU-trete wil type ells (), sml1 (e) n h1 (f) mutnt ells, imge s in (). The grphs in (e-f) show the frtion of ells ompleting sission reltive to the time of memrne ingression; the mein sission times re shown in (h). n +HU from () re represente s she lines for omprison in (f-g). n is the totl numer of ells in eh tegory, poole from two inepenent experiments, n is shown in rkets in (h). Asterisk mrks signifint ifferenes etween trete n untrete ells (p <.1, Mnn-Whitney test). In (-), oxes inlue 5% of t points, whiskers 9%; meins re shown s lines n mens s rosses. Sle rs in (,, ): µm Mmilln Pulishers Limite. All rights reserve

2 % of ells MRE11-GFP foi in presene of HU 5 yk3 h1 h1 yk3 % of ells 5 MRE11-GFP foi uring mitosis untrete 15-1 yk3 h1 h1 yk3 HU pulse MRE11-GFP MRE11-GFP DIC DIC MRE11-GFP DIC yk Supplementry Figure Cytokinesis-epenent DNA mge fter DNA replition stress. () Cells of the inite strins were trete with mm HU for hours n imge for minutes. The perentge of ells with Mre11-GFP foi is shown. n = 41 (wil type); 46 (); 35 ( yk3 ); (h1 ); (h1 yk3 ) where n is the totl numer of ells from one experiment eh. () After HU wshout, ells trete s in () were imge uring nuler elongtion. n = 98 ( Untrete). For HU-trete ells: n=19 (wil type); 176 (); 134 ( 15-1); 1 ( yk3 ); 18 (h1 ); 116 (h1 yk3 ), where n is the totl numer of ells, poole from two experiments, exept for HU-trete n ells, whih were poole from 3 experiments eh. () Timelpse imges of representtive wil type, n yk3 ells expressing Mre11-GFP, fter ytokinesis following HU pulse. The rrows point to nuler Mre11 foi. Numers inite time in minutes where time is the frme efore ytokinesis. Frmes were selete to show Mre11- GFP foi in the mutnt. No foi were etete etween 18 n 4 minutes. Sle rs: µm. For omplete imge series of eh ell, see Vieos Mmilln Pulishers Limite. All rights reserve

MRE11-GFP foi fter nphse onset Cells with us 8 8 yg1-6 yg1-6 yg1-4 6 8 DNA elongtion to Durtion of Memrne ingression ring ontrtion (min) ring ontrtion (min) to reuing (min) -4 - % of ells 4 6 8 yg1-

() Anphse progression in ells with fluoresently tgge histone (Ht) n myosin (Myo1). Numers inite time in minutes. Sle r is µm.

n = 5 (wil type), 3 (yg1-), 34 (), where n = numer of ells poole from two experiments.

n = 98 ells (wil type), 46 (yg1-), (), where n = numer of ells poole from two experiments. () Time of pperne of the next u fter ytokinesis (reuing) reltive to the time of memrne losure (GFP-CAAX).

3 MRE11-GFP foi fter nphse onset Cells with us 8 8 yg1-6 yg1-6 yg DNA elongtion to Durtion of Memrne ingression ring ontrtion (min) ring ontrtion (min) to reuing (min) -4 - % of ells yg1- umultive % e SS PS SS yg1- SS PS SS Supplementry Figure 3 Chromosome segregtion n ytokinesis in yg1- n ells. () Anphse progression in ells with fluoresently tgge histone (Ht) n myosin (Myo1). Numers inite time in minutes. Sle r is µm. The grphs show the time of tomyosin ring ontrtion reltive to nuler elongtion (left) n the urtion of ontrtion (right), in the inite ell types. Line represents the men. n = 5 (wil type), 3 (yg1-), 34 (), where n = numer of ells poole from two experiments. () Time-lpse imging ws use to etermine the perentge of ells with Mre11-GFP foi fter nuler elongtion in the inite strins. n = 98 ells (wil type), 46 (yg1-), (), where n = numer of ells poole from two experiments. () Time of pperne of the next u fter ytokinesis (reuing) reltive to the time of memrne losure (GFP-CAAX). Numer of ells (poole from two inepenent experiments): n = 35 (wil type), 38 (yg1-), 5 (). The reuing kinetis of wil type n mutnt ells were onsiere signifintly ifferent (p <.1, Mnn- Whitney test). () Slies from tomogrm of the septum in ell. Arrowhes point to nuler memrne entering the hnnel (left); Arrows point to plsm memrne unerlying the hnnel (right). Sle rs re. µm. (e) Seril ultr-thin setions (6-7 nm) from wil type n yg1- ells 3 hours fter relese from G 1 lok. Septum hnnels were present in ll exmine yg1- mutnt ells (9), wheres ll wil-type ells showe intt sept (13). Continuous septum eposition might onstrit these hnnels n le to their elye losure. Arrows point to lune trversing the septum of the mutnt. PS, primry septum, SS, seonry septum. Sle r is.5 µm Mmilln Pulishers Limite. All rights reserve

4 Asission (umultive %) 8 6 Time from memrne yg1- yg1- h1 Asission (umultive %) 8 6 Time from memrne h Normlize intensity Normlize intensity yg Normlize intensity Distne (px) Onset of ring ontrtion to DNA segregtion (min) yg1- yg1- yg1- slk19 Supplementry Figure 4 The role of Ah1 n Ipl1 in sission inhiition in yg1- n mutnts. () Frtion of GFP-CAAX ells of the inite strins ompleting sission reltive to the time of memrne ingression. Wil-type n yg1- t from Fig. 3 re shown for omprison. n = 53 (yg1- h1 ); 5 (); 5 ( h1 ), where n = totl numer of ells poole from two inepenent experiments. p =.31 (yg1- vs. yg1- h1 ) n p <.5 ( vs. h1 ) (Mnn-Whitney test). () Time series of memrne ingression n resolution of the inite strins. Sle r is µm. The grphs show the GFP fluoresene intensity ross the levge plne in the mile optil setion, s in Fig.. () Quntifition of the time of hromosome segregtion in the inite strins. Boxes inlue 5% of t points, whiskers 9%; meins re shown s lines n mens s rosses. yg1- n from Fig. 3 re shown for omprison. Time ws efine s the frme efore the onset of ontrtion of the myosin ring. n = 3 ells (yg1- ); 38 (yg1- slk19 ); 6 ( ). Dt were poole from two inepenent experiments. Asterisk mrks signifint ifferenes (p <.1, Mnn Whitney test) Mmilln Pulishers Limite. All rights reserve

5 LC(IV:XII) Gl Glu No riges With riges - - Full ring ontrtion to DNA segregtion (min) SPB istne (µm) Time from nphse onset (min) Mx SPB istne (µm) Dientri, no riges Dientri, with riges LC(IV:XII) Dientris, no riges Dientris, with riges LC(IV:XII) time (h) Dientri Other Chromosomes time (h) 3 GAL GAL 3 GLUC EtBr GLUC 3 Chromosome rekge ( ) reltive to unroken Dientri GAL 3 GLUC 3 RDN1 Southern GAL GLUC 3 Time fter G1 relese (h) e Chromtin Brige Intensity t the u nek (A. U.),,, 1, ontrol Dientris, with riges yg1- Supplementry Figure 5 Chrteriztion of the onitionl ientri LC(IV:XII). () Growth test of seril ilutions of the inite ell types in rih meium pltes with gltose (Gl) or gluose (Glu). () Time of hromosome segregtion for ells with the onitionl ientri hromosome in gluose, reltive to full ontrtion of the myosin ring. Boxes inlue 5% of t points, whiskers 9%; meins re shown s lines n mens s rosses. n = ells (ientri, no riges); 19 (ientri, with riges), from one experiment. () Spinle trks (left) n mximl spinle length (right) of ells with wil type hromosomes () n the two tegories of ells with ientris hromosomes (LC(IV:XII)). SPB istne is the liner istne etween the two spinle pole oies (SPBs) t eh time point (Sp4-GFP). Both grphs show the men n SD. Anphse onset ws efine when SPB istne ws ove.5 µm. n = 14 ells, poole from two inepenent experiments, for eh ell type or tegory. () PFGE nlysis of ientri hromosomes. Cells growing in gltose were rreste in G 1 with lph ftor. The ulture ws split in two n gluose e to one hlf. Cells were relese in gluose or gltose s inite, n smples tken t times, 1 n 18 min for FACS, PFGE n Southern lot nlysis. Asterisk mrks position of roken ientri moleules. The grph shows the mount of roken moleules in Southern lot, reltive to intt ientris n ritrrily set to 1 t time. (e) Asission ifferenes in the presene of istint types of hromtin riges re not ue to ifferenes in the mount of hromtin present t the ytokinesis site. The intensity of fluoresently lele histones t the u nek ws low in onensin-efiient riges (whih inhiit sission), wheres it ws pproximtely -fol higher in oth sissionompetent ientri ells n in sission-efetive mutnts. The grph shows fluoresene intensities of Ht-mCherry in the u nek region t the onset of myosin ring ontrtion (Myo1-GFP), for the inite ell types n onitions. Vlues re in ritrry units (A. U.) n kgrounsutrte. Lines represent the men. n = 15 ells (); 15 (Dientris, with riges); 15 (yg1-); 15 (); where n = numer of ells per strin, poole from two inepenent experiments Mmilln Pulishers Limite. All rights reserve

6 Asission (umultive %) 8 6 mre11 tel1 r9 HU + HU Asission (umultive %) 8 6 mr1 Time from memrne hk1 Time from memrne r53-1 Time from memrne Asission (umultive %) Mein sission time (min) 8 6 (n=) mre11 (n=) tel1 (n=39) r9 (n=36) mr1 (n=4) r53-1 (n=3) (n=48) - HU (n=37) (n=8) r53-1 (n=39) mre11 (n=) ns tel1 (n=4) mre11 (n=33) r9 (n=) mr1 (n=) r53-1 (n=) + HU 8 6 tel1 (n=35) sml1 me1 (n=5) sml1 me1 tel1 (n=31) Asission (umultive %) 8 6 Time from memrne (n=37) yg1- (n=51) yg1- r53-1 (n=78) yg1- mre11 (n=51) 8 6 Time from memrne tel1 (n=3) yg1- tel1 (n=3) yg1- sml1 (n=3) yg1- sml1 me1 (n=) yg1- sml1 me1 tel1 (n=37) Supplementry Figure 6 The role of DNA mge n replition hekpoint in sission ynmis in HU-trete, yg1- n ells. (-) Muttion of MRE11, TEL1, RAD9, MRC1, CHK1 n RAD53 le to sission elys even in the sene of HU, perhps ue to enogenous replition stress. However, no signifint ifferenes in sission re foun etween wil type n hekpoint-efiient HU-trete ells. Asission ynmis of untrete n HU-trete ells of the inite strins t ºC re shown. Cells were trete s in Fig. S1-. Open inverte tringles represent untrete ells n fille inverte tringles represent HU-trete ells. n +HU from Fig. S1e re represente s lue n green she lines for omprison. The mein sission times re represente for the inite strins n onitions. n = totl numer of ells poole from two inepenent experiments is represente in rkets. Asterisk represents signifint ifferenes reltive to HU (p <.5, Mnn-Whitney test). ns, non signifint. (-) Intivtion of the upstrem hekpoint kinses Me1 n Tel1 (homologues of mmmlin ATM n ATR) oes not restore sission in or yg1- ells. Asission ynmis in the inite strins t 37 ºC. GFP-CAAX ells were imge n nlyze s in Fig. 3,. SML1 ws elete to llow viility of the me1 mutnt. n = totl numer of ells poole from two inepenent experiments (exept for yg1- r53-1, whih is from 3 experiments) is represente in rkets. yg1- n t from Fig. 3 re represente for omprison. Signifint ifferenes were foun only for yg1- r53-1 n yg1- mre11 reltive to yg1- (p <.1, Mnn-Whitney test) Mmilln Pulishers Limite. All rights reserve

7 Myo1-GFP Tu1-GFP HU pulse Myo1-GFP Tu1-mCherry SPB istne (µm) Time from nphse onset (min) yg1- Sp4-mCherry Cin8-GFP yg1- Sp4-mCherry Iqg1-GFP yg1- Time (min) Time (min) Supplementry Figure 7 Spinle ynmis in HU-trete, yg1- n ells. () Anphse spinle isssemly visulize through tuulin (Tu1) n myosin (Myo1) ynmis in ell t 37 ºC (top) n in wil type ell fter HU exposure t ºC (ottom). Asterisk mrks full ontrtion of the myosin ring. Arrow points to epolymerizing spinle. Numers inite time in minutes. Sle rs re µm. () Spinle trks of wil type n yg1- ells. SPB istne is the liner istne etween the two spinle pole oies (SPBs) t eh time point. Men n SD re shown. Anphse onset ws efine when SPB istne ws ove.5 µm. Arrows mrk the time of spinle isssemly. n = 7 ells (wil type); 6 (yg1-). (-) Visuliztion of Cin8-GFP n Iqg-GFP ynmis uring spinle elongtion (Sp4-mCherry) in wil type n yg1- ells; time intervl is 1.5 minutes. All sle rs: µm Mmilln Pulishers Limite. All rights reserve

8 h1 Myo1-GFP Tu1-GFP Asission (umultive %) 8 6 Dientris, no riges Time from memrne h1 h1 h1 Asission (umultive %) 8 6 Dientris, with riges Time from memrne h1 h1 h1 Supplementry Figure 8 Role of Ch1 n Ah1 in spinle stility n ientri rige etetion. () Anphse spinle isssemly visulize through tuulin (Tu1-GFP) n myosin (Myo1-GFP) ynmis in representtive h1 ell. An sterisk mrks full ontrtion of the myosin ring. 85% (6/) of h1 ells isssemle the spinle fter ring ontrtion. Sle r: µm. () Cumultive perentge of ells with resolve memrnes (sission) for ells of the inite strins n tegories. Wil-type n h1 ientri ells (from Fig. 5 n Fig. 7,, respetively) re shown for omprison. h1 h1, no riges n = 34 ells; h1 h1, with riges n = (poole from two inepenent experiments). Asission kinetis of h1, with riges n h1 h1, with riges were signifintly ifferent (p <.5, Mnn-Whitney test) Mmilln Pulishers Limite. All rights reserve

9 Supplementry Vieo Legens Supplementry Vieo 1 Cytokinesis n DNA mge fter replition stress. Time-lpse of representtive wil type ell expressing Mre11-GFP to visulize DNA mge, fter ytokinesis following HU pulse. DIC n GFP hnnels re shown. Numers inite time in minutes, time is the frme efore ytokinesis. This vieo is ssoite with Supplementry Fig.. Supplementry Vieo Cytokinesis n DNA mge fter replition stress. Time-lpse of representtive ell expressing Mre11-GFP to visulize DNA mge, fter ytokinesis following HU pulse. DIC n GFP hnnels re shown. Numers inite time in minutes, time is the frme efore ytokinesis. This vieo is ssoite with Supplementry Fig.. Supplementry Vieo 3 Cytokinesis n DNA mge fter replition stress. Time-lpse of representtive yk3 ell expressing Mre11-GFP to visulize DNA mge, fter ytokinesis following HU pulse. DIC n GFP hnnels re shown. Numers inite time in minutes, time is the frme efore ytokinesis. This vieo is ssoite with Supplementry Fig.. Supplementry Vieo 4 Intivtion of onensin inhiits sission. EM tomogrm n 3D moel of the memrne orgniztion t the meil septum of yg1- ell. Plsm memrne is in purple, nuler envelope in light gry, mirotuules in green n vesiles in yellow. This vieo is ssoite with Fig. 3. Supplementry Vieo 5 Intivtion of topoisomerse II inhiits sission. EM tomogrm showing the memrne orgniztion t the meil septum of ell. This vieo is ssoite with Supplementry Fig Mmilln Pulishers Limite. All rights reserve

supplementary information

supplementary information DOI: 1.138/n2131 Protein levels (% of ) e Full-length protein remining (%) 1 5 1 5 1 1 5 5 Hs7 Syt1 Syt2 β-atin CSP +tsyn Hs7 Syt1 Syt2 P4 rin.1.1.1 1 [Trypsin] (g/l) f +tsyn SNARE-omplexes remining (%)