Inhibitors of topoisomerases I and II arrest DNA replication, but do not prevent nucleosome assembly in vivo

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1 Inhiitors of topoisomerses I n II rrest DNA replition, ut o not prevent nuleosome ssemly in vivo ANTHONY T. ANNUNZIATO Deprtment of Biology, Boston College, Chestnut Hill, MA 02167, USA Summry Speifi inhiitors of eukryoti DNA topoisomerses I n II (mptothein n VM-26, respetively) were use to exmine the involvement of topoisomerses in DNA replition n hromtin ssemly in vivo. When use singly, either mptothein or VM-26 inhiite DNA synthesis in HeL ells y more thn 80 %; when use simultneously, the inhiitors effetively stoppe replition, emonstrting tht t lest one lss of topoisomerse must e tive for fork propgtion in vivo. To stuy nuleosome ssemly uring topoisomerse inhiition, three experimentl strtegies were employe: (1) pulse-hse experiments; (2) nlyses of hromtin synthesize uring resiul replition in the presene of either mptothein or VM-26; n (3) the ssemly of previously replite, unssemle DNA, generte in the presene of protein synthesis inhiitors. Using sensitivity to mirool nulese n the mturtion of nonnuleosoml replition intermeites s riteri, neither mptothein nor VM-26, lone or in onert, inhiite nuleosome ssemly uner ny experimentl protool teste. These t provie eviene tht, lthough topoisomerse tivity is essentil for DNA replition, neither ontinuous fork propgtion nor topoisomerse tivity is require for hromtin ssemly on new DNA. Key wors: topoisomerse, DNA replition, hromtin ssemly, DNA superoiling. Introution Among the unertinties surrouning the erliest esription of the DNA oule helix ws the question of how suh struture oul 'unoil' for replition to proee (Wtson & Crik, 1953). It is now known tht speifi enzymes, DNA topoisomerses I n II, filitte the unwining neessry for fork migrtion uring DNA synthesis, s well s for etention of ughter hromosomes (reviewe y Wng, 1985, 1987). Speifilly, in eukryotes it ppers tht either topoisomerse I or II n t s swivel for fork propgtion (Yng et l. 1987; Brills l. 1987; Snpk, 1986), while topoisomerse II is require for hromosome segregtion (DiNro et l. 1984; Holm et l. 1985; Snpk et l. 1988). In oth prokryotes n eukryotes DNA is negtively superoile, with the level of in vivo superoiling mintine in prokryotes through the intertion of DNA gyrse (whih n introue negtive superoils) n DNA topoisomerse I (whih relxes DNA) (Drli, 1984). In eukryotes, negtive superoils re restrine in nuleosomes (Germon et l. 1975). Although no eukryoti enzyme with gyrse tivity hs s yet een purifie, the presene of gyrse hs een inferre through nlyses of hromtin trnsription n nuleosome ssemly (Kmie & Worel, 1985; Kmie et l. Journl of Cell Siene 93, (1989) Printe in Gret Britin The Compny of Biologists Limite ; Villeponteu et l. 1984; Hn et l. 1985; Ryoji & Worel, 1984; Glikin et l. 1984; Ruerti & Worel, 1986). Eukryoti topoisomerse II hs een iniretly implite in DNA gyrtion (Villeponteu et l. 1984; Hn etl. 1985; Ryoji & Worel, 1984; Glikin et l. 1984; Ruerti & Worel, 1986), preominntly through the use of the rug novoioin (n inhiitor of topoisomerse II n teril gyrse; Drli & Frno, 1988). It shoul e note, however, tht eukryoti topoisomerse II lone nnot introue negtive superoils into lose irulr DNA (Wng, 1985; Hirose & Suzuki, 1988). The use of novoioin to investigte the role of topoisomerse II in hromtin metolism hs reently ome into question, prtiulrly s regrs nuleosome ssemly. Novoioin my intert non-speifilly with histones (Cotten et l. 1986), n thus my prevent hromtin ssemly y isrupting histone-histone or histone-dna ssoitions, rther thn through inhiition of gyrse tivity (Sely et l. 1986). The epipoophyllotoxin VM-26 (teniposie) hs, however, een shown to e speifi inhiitor of topoisomerse II, interfering with the DNA rejoining step tht follows strn pssge (Wng, 1987; Drli & Frno, 1988). Similrly, the lkloi mptothein inhiits the rejoining step tlyze y eukryoti DNA topoisomerse I (Wng, 1985; Drli & Frno, 1988). 593

2 Histone synthesis is for the most prt temporlly ouple to DNA replition, n thus the norml sustrte for e novo nuleosome ssemly is newly synthesize DNA (for reviews, see DePmphilis & Wssermn, 1980; Lskey & Ernshw, 1980; Annunzito & Sele, 1983). Although numer of ell-free extrts hve een evelope tht will promote hromtin ssemly with or without onurrent DNA synthesis (Glikin et l. 1984; Ruerti & Worel, 1986; Dilworth et l. 1987; Cusik et l. 1984; Stillmn, 1986; Almouzni & Mehli, 1988,), the eviene tht topoisomerses re require for nuleosome formtion in vitro hs thus fr een erive from non-repliting systems (Germon et l. 1979; Glikin et l. 1984; Ruerti & Worel, 1986; Almouzni & Mehli, 19886). Beuse the topologil properties of repliting ellulr DNA re expete to e rilly ifferent from those of lose irulr (nonrepliting) plsmis use in suh stuies, the role of topoisomerses in nuleosome ssemly onto nsent DNA in vivo hs een exmine in the following report. By using the topoisomerse I n II inhiitors mptothein n VM-26, n three istintly ifferent experimentl protools, eviene is provie tht, while topoisomerse tivity is neessry for DNA replition, topoisomerse inhiitors o not prevent nuleosome ssemly onto newly replite DNA. Mterils n methos Cell ulture n leling HeL S3 ells were mintine in spinner ulture t 37 C in Egle's miniml essentil meium (Joklik moifie) supplemente with 9% lf serum n 1 % fetl lf serum. Long-term leling of ells with [ 14 C]thymiine (SOmCi mmoll" 1, New Engln Nuler) ws performe t 0'02/iCiml" 1 of ell ulture for one genertion (24h). For pulse leling, ells (~4 X 10 5 ml" ) were hrveste y entrifugtion (250g; 1-5 min), n onentrte 25-fol (0-5-1 min pulse) or 10-fol (20-40 min lel) in prewrme whole meium, followe y equilirtion t 37 C for 5 min. For min leling, ells were inute with [me</iy/- 3 H]thymiine (60Cimmol~', New Engln Nuler) t 50/iCiml~. Leling in the presene of either 131 mm-ncl or 200/igmF 1 yloheximie ws performe t 5-10/iCiml" 1 ; leling in the presene of 100/iM-VM-26 or 100/iM-mptothein ws performe t 25 nc\ ml"'. Cmptothein n VM-26 were generous gifts from The Division of Cner Tretment, NIH, n Bristol Meyers Co., respetively. Cmptothein n VM-26 were store s 10 mm n 50 mm stok solutions, respetively, in imethylsulfoxie. For leling in the sene of protein synthesis, ells were pre-inute for 10 min in either 200 (is ml~ yloheximie or 131 mm-ncl, efore the ition of [%]thymiine. Leling ws then performe in the ontinuous presene of either yloheximie or NCl. As require, mptothein n VM-26 were e to terminte leling, n the ells were wshe twie in pre-wrme meium ontining /iM eh of mptothein n VM-26. Cells were then resuspene n 'hse' in whole meium ontining either 100 or 200/iM of mptothein n VM-26. Hyroxyure ws e s require to finl onentrtion of 5 10 mm, from sterile 100 mm stok (in ulture meium). To mesure DNA synthesis, ells were inute with [ 3 H]thymiine for the times inite (text). Leling with [ 3 H]thymiine ws performe t 4/iCi ml~ in whole meium, n ws hlte y iluting smple of ells 10-fol with ie-ol uffer A (lomm-tris-hcl, ph7-6, 3mM-MgCl 2> 2mM-2- merptoethnol) ontining 0-1% soium zie. Cells were pellete, resuspene in wter, n preipitte with 10% trihloroeti i. Nuler isoltion: igestion with mirool nulese Cells were wshe twie with uffer A, resuspene in uffer A, n lyse with Doune homogenizer. Nulei were ollete y entrifugtion (800g-; 1-25 min), n wshe twie with uffer A. For igestions with mirool nulese, nulei with resuspene in uffer A t 40 units of A^ ml" (A26O mesure in 1 % SDS), n CCl2 ws e to finl onentrtion of 0-5 mm. Mirool nulese (Sigm) ws e to finl onentrtion of 1 to 4unitsml~, n igestions were performe t 37 C for the times given in the figure legens. To prepre hromtin frtions for gel eletrophoresis, nulese retions were terminte y the ition of EGTA (ph 7-6) to finl onentrtion of 1 mm, n ooling on ie. Nulei were then pellete, yieling the first superntnt (SI), ontining preominntly mononuleosomes. Nulei were then lyse y resuspension in 2mM-EDTA, ph7-4, n insolule hromtin ws remove y entrifugtion (12500if, lomin), yieling the seon superntnt frtion (S2), ontining mononuleosomes n nuleosoml oligomers. The remining pellet ws resuspene in 2 mm-edta, 0-6 M-NCl, ph 7-4; this ioni strength removes histone HI n most non-histone proteins from the nuleus, further soluilizing high moleulr weight hromtin. More importntly, nsent non-nuleosoml (unssemle) hromtin is relese from the nuleus y this tretment. Suh unssemle newly replite hromtin is typilly insolule uner onitions of low ioni strength (for etils, see Annunzito et l. 1981; Annunzito & Sele, 1982). Insolule hromtin ws gin remove y entrifugtion, yieling the thir superntnt (S3) n finl insolule pellet (P). Approximtely % of the ulk hromtin DNA ws soluilize y these proeures. Chromtin frtions were either juste to 10 mm-mgnesium ette n preipitte immeitely, or preipitte following inution with 0-5% SDS n 150/igml~ proteinse K overnight; oth tretments yiele ientil results in DNA/SDS-ontining gels. Chromtin frtions were preipitte with 2 volumes of ethnol in soli COy^ethnol slurry, rie, soluilize in eletrophoresis uffer (40 mm-trisette, ph 7-2, 20mM-soium ette, 1 mm-edta) me 5 % in glyerol n 1 % in SDS. Polyrylmie sl gels (3-9 or 7%) were prepre oring to Loening (1967), n run t 110 V in the presene of 01 % SDS, s esrie (Annunzito et l. 1981). DNA gels were stine with ethiium romie, photogrphe, impregnte with 2,5-iphenyloxzole (Bonner & Lskey, 1974; Lskey & Mills, 1975), rie, n expose to Kok X-Omt AR film. To mesure i-preipitle riotivity, ontrol nulei pre-lele with [ 14 C]thymiine for 24 h in the sene of ny inhiitors were mixe with [ 3 H]thymiine-lele nulei n igeste with mirool nulese until t lest 50% of the [ C]DNA ws renere i-solule. Smples of the totl igest were remove, ple into 20mM-EDTA, n preipitte with 10% trihloroeti i. The reltive nulese sensitivity of newly replite hromtin ws then expresse s 3 H/ 14 C. Riotivity etermintions Smples of hromtin frtions were preipitte, together with 594 A. T. Annunzito

3 50 fig eh of DNA n lumin rrier, with 10% trihloroeti i, ollete on glss-fier filters (Reeve Angel 934-AH, Whtmn), n wshe with trihloroeti i n then with 95 % ethnol. Drie filters were inute with 0-2ml of NCS (Amershm Corp.) ontining 5% wter, n ounte. The frtion of 14 C ounts registere in the 3 H hnnel ws sutrte utomtilly, using the H numer metho (Bekmn Instruments). Results Effet of topoisomerse inhiitors on DNA replition In the following experiments, speifi inhiitors of eukryoti DNA topoisomerses I n II (mptothein n VM-26, respetively) were use to ssess the role of topoisomerse tivity in hromtin ssemly. To ensure tht these inhiitors were tive uner our experimentl onitions. The effets of mptothein n VM-26, lone or in onert, on DNA replition were ssye. HeL ells were lele in the presene or sene of mptothein, VM-26, or oth inhiitors simultneously (Fig. 1). When use lone, either mptothein or VM-26. inhiite thymiine inorportion y more thn 80%. The prtil inhiition of DNA replition y either mptothein or VM-26 hs een oserve (Misr & Roerts, 1975; Rihter et l. 1987; Yng et l. 1987), onsistent with the oservtion tht oth types of topoisomerse re lote t or ner replition forks in eukryoti ells (Nelson et l. 1986; Snpk, 1986; Yng Inorportion time (min) Fig. 1. Effets of mptothein n VM-26 on DNA replition. HeL ells were prelele for one genertion with [ 14 C]thymiine, n then inute for the times inite with [ 3 H]thymiine. After 7-5 min, the ells were ivie, n further inute in the sene ( ) or presene of 100jiM-VM-26 (O), 100/iM-mptothein ( ), or 100 J/M eh of VM-26 n mptothein ( ). DNA synthesis is expresse s 3 H/ H C. et l. 1987; Brill et l. 1987). Similrly, it hs een shown tht either topoisomerse I or topoisomerse II n promote the replition of simin virus 40 (SV40) DNA in vitro (Yng et l. 1987). Thus, it is most likely tht the ontinue low level of replition in the presene of mptothein is ue to topoisomerse II sustituting for topoisomerse I, s hs een emonstrte in yest topi mutnts (Uemur & Yngi, 1986; Brill et l. 1987). However, the erese in DNA synthesis oserve with either inhiitor suggests tht topoisomerses I n II t oorintely uring norml ell replition. Although topoisomerse I is instrumentl in fork migrtion, the persistene of DNA synthesis in the presene of mptothein unersores the non-essentil nture of this enzyme (Yng et l. 1987). This hs een most keenly illustrte in yest topi mutnts, whih remin ompletely vile (with slowe growth) in the sene of topoisomerse I tivity, ue to the ility to topoisomerse II to t in sustitution (Uemur & Yngi, 1984, 1986; Goto & Wng, 1985). For this reson, there is no iret metho of seletively mesuring topoisomerse I tivity (or inhiition thereof) in living ells. However, the simultneous inhiition of oth enzymes in vivo (s in yest topl-top2 oule mutnts) hs een shown to reue DNA synthesis to insignifint levels (Uemur & Yngi, 1986; Brill et l. 1987), emonstrting the epenene of replition on the ontinue funtion of t lest one lss of topoisomerse. Thus, the nlysis of replition is not only highly effetive mens of ssessing the simultneous inhiition of topoisomerses I n II, ut it is lso the most relile wy of oing so in living ells. To test further the effetiveness of mptothein n VM-26 in vivo, the level of DNA synthesis in the presene of oth rugs ws exmine. When mptothein n VM-26 were e to ells simultneously, thymiine uptke ws essentilly eliminte (Fig. 1). The itive ffet of mptothein n VM-26 is onsistent with the tivities of the two inhiitors eing ifferent (Drli & Frno, 1988). These results lso emonstrte tht the persistene of replition when the rugs re use singly (ove) is not ue to suoptiml inhiitor levels. None of the inhiition ws ue to the Me2SO (imethyl sulfoxie) use to soluilize the rugs: ition of Me 2 SO to 1-2 % (the onentrtion use in Fig. 1) h no effet on DNA synthesis (t not presente). The egree of suppression of DNA synthesis y mptothein/vm-26 ws equivlent to tht oserve in yest topl-top2 oule mutnts (Brill et l. 1987), initing tht virtully no topoisomerse tivity persists t these rug onentrtions. The ility of these gents to inhiit topoisomerses seletively in vivo therefore provies mens of exmining topoisomerse requirements uring hromtin ssemly. Effet of topoisomerse inhiitors on mirool nulese sensitivity of newly replite hromtin Newly replite hromtin is more sensitive to igestion to i soluility with mirool nulese thn ulk hromtin, ut hieves norml nulese resistne min fter pssge of the replition fork (Levy & Topoisomerses n hromtin ssemly in vivo 595

4 Jo, 1978; Annunzito & Sele, 1983). The quisition of nulese resistne hs een strongly orrelte with the ssemly of DNA into nuleosomes (Levy & Jo, 1978; DePmphilis & Wssermn, 1980; Annunzito & Sele, 1983,6), n n thus e use s one riterion for ssessing the ssemly proess. Beuse inhiition of topoisomerse tivity versely ffets DNA replition, it ws importnt to etermine if newly replite DNA oul e ssemle into hromtin in the sene of ongoing DNA synthesis, prior to the exmintion of topoisomerse funtion. HeL ells were lele for 30s in vivo with [ 3 H]thymiine, therey prouing lele nsent DNA tht ws highly nulese sensitive (Fig. 2A). When the pulse ells were hse for 30 min in the presene or sene of 5 mm-hyroxyure (whih immeitely n totlly hlts DNA replition in HeL ells; Sele & Simpson, 1975), nsent hromtin ompletely ttine norml nulese resistne in oth ses (Fig. 2A). Thus, ongoing DNA replition is not require for the mturtion of newly replite hromtin DNA. The experiment ws then repete, with the exeption tht 100 ^im eh of mptothein n VM-26 ws sustitute for hyroxyure (Fig. 2B). The presene of the topoisomerse inhiitors i not prevent the mturtion of pulse-lele hromtin DNA: fter 30 min the preferentil nulese sensitivity of nsent hromtin ws entirely reverse, suggesting tht ontinuous topoisomerse tivity is not require for the ssemly of nuleosomes onto newly replite DNA in vivo. Aquisition of norml nuleosoml perioiity in the presene of mptothein n VM-26 In series of previous reports (Annunzito et l. 1981; Annunzito & Sele, 1982, 1984) two lsses of newly replite hromtin (lele for s in vivo) were esrie tht iffer in struture, soluility properties, n requirements for mturtion. One lss is nuleosoml, solule t low to intermeite ioni strengths, n quires mture nuleosoml omposition n norml repet length in the sene of onurrent protein synthesis. In ontrst, the other is leve irregulrly y mirool nulese (ppering s non-nuleosoml smer in), is insolule t low to intermeite ioni strengths, n requires protein synthesis to gin norml suunit struture. The non-nuleosoml omponent onsists of unssemle DNA, n thus omprises the sites of e novo nuleosome ssemly. The pperne of typil suunit orgniztion on this omponent of nsent hromtin extly prllels the quisition of norml mirool nulese resistne, n thus n e use s ignosti feture of nuleosome ssemly (Annunzito & Sele, 19836). To monitor the quisition of nuleosoml perioiity, ells were lele with [ 3 H]thymiine for 60s, isolte nulei were igeste with mirool nulese, n hromtin ws frtionte s esrie in Mterils n Methos. Chromtin DNA ws then nlyze y gel eletrophoresis, ethiium romie stining, n fluorogrphy (Fig. 3A,B, lnes -). As seen in the ethiium romie-stine gel, ulk u 10. \ B Digestion time (min) Digestion time (min) Fig. 2. Topoisomerse inhiitors o not prevent the mturtion of newly replite hromtin, s mesure y mirool nulee sensitivity. A. Cells were lele with [ 3 H]thymiine for 30s ( ), or lele for 30s, hrveste, resuspene in prewrme ulture meium in the sene (O) or presene ( ) of 5 mm-hyroxyure, n inute t 37 C for n itionl 30min (O, ). (Note: ells hse in hyroxyure were rought to 10 mm-hyroxyure immeitely fter the 30-s leling perio.) Isolte nulei were mixe with nulei prelele with [ H C]thymiine for one genertion, igeste with mirool nulese t 4 units ml" 1 for the times inite (verge 66% of [ 14 C]DNA renere i-solule fter 30min), n i preipitte. The nulese sensitivity of newly replite hromtin, reltive to ulk hromtin, is expresse s 3 H/ 14 C; nuler rtios efore igestion hve een normlize to 10. B. Cells were lele with [ 3 H]thymiine for 30s ( ), or lele for 30s, rought to 100[iM eh of mptothein n VM-26, hrveste, resuspene in prewrme ulture meium ontining 100 ^M eh of mptothein n VM-26, n inute t 37 C for n itionl 30 min (O). Isolte nulei were mixe with nulei prelele with [ C]thymiine, igeste with mirool nulese t 4 units ml" 1 for the times inite (finl verge 61 % of [ 14 C]DNA renere i-solule), n i preipitte. Reltive nulese sensitivity is expresse s 3 H/ 14 C. hromtin in the S2 n S3 frtions onsiste of nuleosoml monomers n oligomers (Fig. 3A, lnes n ); the pellet ontine high moleulr weight oligonuleosomes, s well s resiul monomers, imers, et. (Fig. 3A, lne ). New hromtin DNA in the S A. T. Annunzito

5 A e f B e e f Fig. 3. Aquisition of norml nuleosoml perioiity in the sene of DNA replition. Cells were lele with [3H]thymiine for 60s (lnes -), or lele for 60s, rought to 5 mm-hyroxyure, wshe in pre-wrme ulture meium ontining hyroxyure, n inute t 37 C in the presene of 5 mm-hyroxyure for n itionl 90min (lnes -f). Isolte nulei were igeste with mirool nulese (1-2 units ml~ ) for 2min, n solule hromtin ws relese with 2mM-EDTA (S2, lnes n ), or 2mM-EDTA, 0-6M-NCl (S3, lnes n e), leving finl insolule pellet (P) (lnes n f) (see Mterils n methos). Chromtin DNA ws sujete to eletrophoresis, stine with ethiium romie (A), n nlyze y fluorogrphy (B). The position of mononuleosoml DNA is inite (M). onsiste minly of monomers n imers (Fig. 3B, lne ), while non-nuleosoml new DNA ws present in the S3 n P frtions, ppering s n unstruture smer (Fig. 3B, lnes n ; lso, Fig. 4A). When ells were lele for 60s with [3H]thymiine n then hse in the presene of 5 mm-hyroxyure, ll the hromtin frtions isplye norml suunit struture n nulese resistne, n the fluorogrph ws inistinguishle from the stine gel (Fig. 3A,B, lne -f). This is ientil to the mturtion of nsent hromtin uner ontrol onitions (Annunzito et l. 1981). Thus, ontinue DNA synthesis is not require for nuleosome ssemly s etermine y gel eletrophoresis, onsistent with the onlusions se on i preipitility (Fig. 2A). When ells were pulse-lele for 30 s with [ 3 H]thymiine, n then hse for 30min in the presene of oth mptothein n VM-26, typil nuleosoml perioiity n nulese resistne were gin onferre on the new DNA (Fig. 4A,B). This provies further eviene tht one DNA hs een replite, the ontinue tion of either topoisomerse I or topoisomerse II is not neessry for nuleosome ssemly. However, it remine possile tht 'ltent' nuleosomes, prtilly ssemle uring the 30-s pulse, were lrey present on new DNA prior to the inhiition of topoisomerse tivity. To ress this importnt point, two further experimentl strtegies were employe. Struture of hromtin replite in the presene of either mptothein or VM-26 Although either mptothein or VM-26 lone reues DNA replition more thn fivefol, mesurele level of thymiine inorportion persists in the presene of either iniviul inhiitor (Fig. 1). As seon pproh to ssessing the role of topoisomerse tivity in hromtin iosynthesis, n in orer to etermine the iniviul ontriutions of topoisomerses I n II, the properties of hromtin replite uring the inhiition of either topoisomerse I or topoisomerse II were exmine. HeL ells were therefore lele with [3H] thymiine in the ontinuous presene of either mptothein or VMTopoisomerses n hromtin ssemly in vivo 597

6 A e B -M 26, n isolte nulei were igeste with mirool nulese for nlysis in gels (Fig. 5). All hromtin frtions generte y mirool nulese igestion (Fig. 5), inluing the S3 (lne ), showe norml nuleosoml orgniztion; there ws no eviene of unssemle DNA, n no umultion of 100 se-pir sunuleosomes, s ws previously reporte when novoioin ws use to inhiit topoisomerse II (Ruerti & Worel, 1986). Further, hromtin replite in either mptothein or VM-26 possesse nulese resistne inistinguishle from tht of ontrol ulk hromtin, s mesure y igestion to i soluility (t not presente). Thus, lthough replition is versely ffete uner these onitions, DNA ssemle uring single topoisomerse inhiition possesses typil nuleosome orgniztion. Assemly of previously replite, non-nuleosoml DNA in the sene of topoisomerse tivity When protein synthesis is loke in HeL ells y yloheximie, NCl, or other gents, replition ontinues t reue rte for min (Roins et l. 1970; Weintru, 1974; Sele & Simpson, 1975). Chromtin replite uner suh onitions is nuleosome-efiient, ontining histone otmers of prentl origin only (Sele, 1976; Weintru, 1976), n thus is pproximtely twie s sensitive s ontrol hromtin to nulese igestion (Sele & Simpson, 1975; Weintru, 598 A. T. Annunzito Fig. 4. Aquisition of norml nuleosoml perioiity in the presene of mptothein n VM-26. Cells were lele with [ 3 H]thymiine for 30s (A), or lele for 30 s, rought to 100 f.im eh of mptothein n VM-26, hrveste, n inute t 37 CC in ulture meium ontining 100 IMA eh of mptothein/vm-26 for n itionl 30min (B). Isolte nulei were igeste with mirool nulese (1-2 units ml" 1 ) for 2min, prouing SI (lne ), S2 (lne ), S3 (lne ) n P (lne ) hromtin frtions (see Mterils n methos). Chromtin DNA ws sujete to eletrophoresis, n nlyze y fluorogrphy (only the fluorogrphs re shown). The position of mononuleosoml DNA is inite (M). 1974). As expete, oth nuleosoml n non-nuleosoml regions n e etete on hromtin so synthesize (Weintru, 1976; Sele, 1976; Annunzito & Sele, 1982). When ells re returne to norml meium, histone synthesis resumes (Weintru & Holtzer, 1972), n histone-eplete hromtin eomes ssemle into nuleosomes (Weintru, 1974; Sele & Simpson, 1975). As the thir pproh to etermining the role of topoisomerses in nuleosome ssemly, the ility of nonnuleosoml DNA to e ssemle in the presene of mptothein n VM-26 ws exmine. Cells were lele with [ 3 H]thymiine for 20 min in the presene of either 131 mm-ncl or ^ g m P 1 yloheximie, onitions tht ompletely n immeitely hlt protein synthesis in HeL ells (Sele & Simpson, 1975; lso, Annunzito, unpulishe oservtions). In greement with previous finings, oth lsses of newly replite hromtin exhiite n pproximtely twofol greter sensitivity to mirool nulese, reltive to ulk hromtin (Fig. 6). Susequent to leling, some of the ells were returne to norml meium ontining either 5 mm-hyroxyure plus 1-2% Me 2 SO (Fig. 6A), or 100 (XM. eh of mptothein n VM-26 (Fig. 6A,B), n then further inute for min. Neither hyroxyure nor mptothein/vm-26 prevente the quisition of norml nulese resistne. Further, inresing the rug onentrtions twofol, to 200 (UM, yiele results ientil to those otine uner

7 A B M- -M Fig. 5. Nuleosoml perioiity of hromtin replite in the presene of mptothein or VM-26. Cells were lele with [3H]thymiine for 40min in the ontinuous presene of 100 fjmmptothein (A) or IOO/IM-VM-26 (B). Isolte nulei were igeste with mirool nulese (1 units ml" 1 ) for 2min, prouing SI (lne ), S2 (lne ), S3 (lne ) n P (lne ) hromtin frtions. Chromtin DNA ws sujete to eletrophoresis, n nlyze y fluorogrphy (only the fluorogrphs re shown). The position of mononuleosoml DNA is inite (M). stnr onitions (Fig. 6B), n no erese in hromtin ssemly ws oserve. Consistent with these finings, mptothein n VM-26 i not prevent nuleosome ssemly s etermine y gel eletrophoresis, following replition in NCl (Fig. 7A,B, lne ), or yloheximie (Fig. 7C); inresing the inhiitor onentrtions to 200 ^M i not lter these results (Fig. 7C, lne ). Beuse hromtin replite in the presene of yloheximie or NCl is lerly 50% nuleosomeefiient (ue to the lk of new histones ville for ssemly), histone eposition n nuleosome ssemly must hve ourre uring topoisomerse inhiition, following the resumption of protein synthesis. It is therefore onlue tht neither onurrent DNA replition nor topoisomerse tivity is require for nuleosome ssemly on previously replite, histone-efiient DNA. Disussion Numerous lines of eviene hve emonstrte tht mptothein n VM-26 re potent n seletive inhiitors of topoisomerses I n II (Hsing et l. 1985; Chen etl. 1984; Uvry et l. 1986; Rihter et l. 1987). In omplete greement with the preite effets of topoisomerse inhiition, loo^m-mptothein n VM-26 together rreste replition, eliminting the possiility tht topoisomerse tivities were in lrge exess, reltive to inhiitor onentrtion. Nevertheless, experiments were performe in whih inhiitor levels were oule (to 200 fm), with ientil results: no iminution of hromtin ssemly ws etete. The experimentl strtegies presente in this stuy hve me it possile to test the requirements for topoisomerses uner three ifferent moes of nuleosome ssemly: (1) hromtin DNA replite normlly, ut ssemle in the presene of mptothein n VM26; (2) DNA replite n ssemle uring the ontinuous inhiition of either topoisomerse I or topoisomerse II; n (3) histone-efiient hromtin DNA, umulte uring protein synthesis inhiition, n ssemle susequently. Uner no irumstnes teste i mpothein or VM-26 prevent or retr nuleosome formtion, proviing eviene tht topoisomerse tivity is inessentil for the ssemly of newly replite DNA. As with ny stuy employing inhiitors, the question of possile sie effets must e resse. Both mptothein n VM-26 intert with topoisomerses, stitopoisomerses n hromtin ssemly in vivo 599

8 10 20 Digestion time (min) Digestion time (min) Fig. 6. Topoisomerse inhiitors o not prevent the mturtion of previously replite, non-nuleosoml hromtin DNA. A. Cells were lele with [ 3 H]thymiine for 20 min in the presene of 131 mm-ncl ( ), or lele in NCl for 20 min, rought to either 5 mm-hyroxyure or 100/iM eh of mptothein/vm-26, hrveste, wshe twie with prewrme ulture meium ontining either hyroxyure or mptothein/vm-26 (to remove the NCl) n then inute in ulture meium ontining 5 mmhyroxyure for 75 min ( ), or ontining 100 /JM eh of mptothein/vm-26 (O, ), for 30min (O), or 60min ( ). Isolte nulei were mixe with nulei prelele for one genertion with [ 14 C]thymiine, igeste with mirool nulese for the times inite (finl verge 70 % of [ 14 C]DNA renere i-solule), n i preipitte. B. Cells were lele with [ 3 H]thymiine in the presene of 200/(g ml" 1 yloheximie for 20 min (0), or lele in yloheximie for 20 min, wshe twie with prewrme ulture meium ontining either 100/iM or 200/iMmptothein/VM-26 (to remove the yloheximie), n then inute for n itionl 60 min in ulture meium ontining either 100;<M (O) or 200^M (A) eh of mptothein/vm-26. Isolte nulei were mixe with nulei prelele for one genertion with [ 4 C]thymiine, igeste with mirool nulese for the times inite (verge 58% ([ 14 C]DNA i-solule), i preipitte, n nlyze s in Fig. 2. Reltive nulese sensitivity is expresse s 3 H/ l4 C. lizing 'levle omplexes' tht introue either singlestrne (mptothein) or oule-strne (VM-26) reks into DNA, when expose to strong protein enturnts (suh s lkli or SDS) (Horwitz & Horwitz, ; Trsk et l. 1984; Hsing et l. 1985; Ross et l. 1984; Cheney l. 1984; Roweef /. 1986). However, the stiliztion of levle omplexes shoul not e viewe s eing similr to the utting of superoile DNA y DNses, therey permitting the DNA to relx. Cmptothein n VM-26 inhiit the relxtion of superoile DNA y topoisomerses I n II (Horwitz & Horwitz, 1971; Rihter et l. 1987; Uvry et l. 1986), using superhelilly oile plsmis to remin s suh. Thus, the stilize levle omplexes o not llow for free rottion of superoile DNA, n nnot t s swivels to relieve tortionl stress. During nuleosome ssemly, DNA eomes negtively superoile in onjuntion with its ssoition with histones (Glikin et l. 1984; Dilworth et l. 1987; Stillmn, 1986). The pprent isrepny etween the results presente in this report, n those supporting onnetion etween tive gyrtion n ssemly (Glikin et l. 1984; Ruerti & Worel, 1986), my e ue to the non-speifi effets of novoioin (Cotten et l. 1986; Sely et l. 1986; Almouzni & Mehli, 19886). It shoul e note, however, tht the inhiitory effet of novoioin ws oserve uring nuleosome ssemly onto irulr DNA in the sene of DNA replition (Glikin et l. 1984; Ruerti & Worel, 1986). Uner suh irumstnes, topoisomerses my e instrumentl in relieving torsionl stresses tht umulte uring the ssemly of hromtin. Thus, topoisomerse I hs een foun to filitte ssemly in vitiv, in non-repliting systems (Germon et l. 1979; Almouzni & Mehli, 19886). Nevertheless, it must e stresse tht in the erliest esriptions of nuleoplsmin-meite hromtin ssemly in vitm (Lskey et l. 1978), topoisomerse tivity ws not require for the formtion of nuleosomes s etermine y seimenttion or eletron mirosopy (lthough topoisomerse ws of ourse neee for the superoiling ssy). The effiient ssemly of liner DNA in vitro lso rgues ginst role for tive gyrtion in the ssemly proess (Almouzni & Mehli, 19886). Normlly, replition n nuleosome ssemly re ouple, n histones re eposite within minutes of DNA synthesis (Hilern & Wlters, 1976; Worel et l. 1978; Annunzito et l. 1981; Jkson & Chlkley, 1985). Unique fetures of nsent DNA (for exmple, prtilly single-strne regions ner the replition fork) my ovite the neessity for ontinue topoisomerse tivity, provie tht the DNA hs lrey een replite. Suh oopertive effet of DNA synthesis on hromtin ssemly hs een note y others (Stillmn, 1986; Almouzni & Mehli, 1988), n my help to explin why nuleosome ssemly in vitro proees muh more rpily when newly replite DNA is the sustrte, reltive to ssemly in non-repliting system (Almouzni & Mehli, 1988«). However, the results presente in this report lso emonstrte tht rpily moving fork is not requirement for hromtin ssemly, sine hyroxyure lso file to prevent the formtion of nuleosomes on DNA replite prior to ssemly. After the experiments reporte herein were omplete, Sekiguhi & Kmie (1988) esrie the effets of severl 600 A. T. Annunzito

9 A B C M- Fig. 7. Assemly of previously replite, non-nuleosoml hromtin DNA in the presene of mptothein n VM-26. A. Cells were lele with [ 3 H]thymiine for 40 min in the presene of 131 mm-ncl. Isolte nulei were igeste with mirool nulese (1-2 units ml" 1 ) for 2 min, prouing SI (lne ), S2 (lne ), S3 (lne ), n P (lne ) hromtin frtions. B. Cells were lele with [ 3 H]thymiine in the presene of 131 mm-ncl s in (A), rought to 100fiM eh of mptothein n VM-26, wshe twie with prewrme ulture meium ontining 100 /1M eh of mptothein n VM-26, n further inute t 37 C in ulture meium ontining 100/iM eh of mptothein/vm-26 for n itionl 75 min. Isolte nulei were igeste with mirool nulese, prouing hromtin frtions s in A. Lnes re s in A. C. Cells were lele with [ 3 H]thymiine in the presene of 200^gml~' yloheximie, prouing non-nuleosoml hromtin DNA ientil to tht shown in A (see Annunzito & Sele, 1983, for etils). The ells were then wshe n further inute for 60min in ulture meium ontining 200/iM eh of mptothein n VM-26. Isolte nulei were igeste with mirool nulese; the S2 (lne ), S3 (lne ), n P (lne ) frtions re shown. For A, B, n C, hromtin DNA ws sujete to eletrophoresis, n nlyze y fluorogrphy. The position of mononuleosoml DNA is given (M). topoisomerse inhiitors on in vitro hromtin ssemly. Consistent with our finings, neither erenil (n inhiitor of mitohonril n virl topoisomerses) nor the quinolone nliixi i (whih loks the tion of teril DNA gyrse) prevente nuleosome ssemly in vitro, lthough some ltertions of nuleosome sping were oserve with nliixi i. Curiously, seon quinolone (oxolini i) i versely ffet hromtin formtion when use in high onentrtion. In light of the fts tht the quinolones: (1) in iretly to DNA (Shen & Pernet, 1985), n (2) re extremely poor inhiitors of eukryoti topoisomerses (Gellert, 1981; Drli & Frno, 1988), it woul pper tht the effets of these inhiitors on hromtin ssemly shoul e ssesse with ution. If neither topoisomerse nor gyrse tivities re require for hromtin ssemly, then it my e tht the negtive superheliity of eukryoti hromtin DNA is generte s iret onsequene of histone-dna intertions uring hromtin iosynthesis. Although there is some eviene for gyrtion in eukryotes (Hirose & Suzuki, 1988), geneti stuies hve shown tht in yest, gyrse-like tivities o not t upon ulk enogeneous 2jm\ plsmis in vivo (Sver & Huermn, 1986). Of ourse, it remins possile tht rypti topoisomerse, istint from topoisomerses I or II, is meiting nuleosome ssemly. This woul seem to e highly unlikely, however, in the light of the severe epression of replition oserve when the reognize topoisomerses re inhiite (this report), n of the umulte eviene (otine using oth inhiitors n top mutnts) tht strongly points to the sene of suh n enzyme. I thnk Ann Deroux for exellent tehnil ssistne. This work ws supporte y grnt from the Ntionl Topoisomerses n hromtin ssemly in vivo 601

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