Is Cdk7/cyclin H/MAT1 the genuine cdk activating kinase in cycling xenopus egg extracts?

Transcription

1 Oncogene (1997) 15, 1303 ± Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Is Cdk7/cyclin H/MAT1 the genuine cdk ctivting kinse in cycling xenopus egg extrcts? Didier Fesquet, Nthlie Morin, Mrcel Doree nd Alin Devult Centre de Recherches de Biochimie McromoleÂculire, CNRS ERS 155, BP 5051, Montpellier cedex, Frnce Formtion of ctive cdk (cyclin dependent kinse)/ cyclin kinses involves phosphoryltion of conserved threonine residue in the T loop of the cdk ctlytic suunit y CAK (Cdk Activting Kinse). CAK ws rst puri ed iochemiclly from higher eukryotes nd identi ed s trimeric complex contining cdk7 ctlytic suunit, cyclin H nd MAT1 (Me nge Á trois), memer of the RING nger fmily. The sme trimeric complex is lso prt of sl trnscription fctor TFIIH. In udding yest, the closest homologs of cdk7 nd cyclin H, KIN28 nd CCL1, respectively, lso ssocite with TFIIH. However, the KIN28/CCL1 complex does not disply CAK ctivity nd distinct protein kinse le to phosphorylte monomeric CDC28 nd GST-cdk2 ws recently identi ed, chllenging the identi ction of cdk7 s the physiologicl CAK in higher eukryotes. Here we demonstrte tht immunodepletion of cdk7 suppresses CAK ctivity from cycling Xenopus egg extrcts, nd rrest them efore M-phse. We lso show tht speci c trnsltion of mrnas encoding Xenopus cdk7 nd its ssocited suunits restores CAK ctivity in cdk7-immunodepleted Xenopus egg extrcts. Hence, the cdk7 complex is necessry nd su cient for ctivtion of cdk-cyclin complexes in cycling Xenopus egg extrcts. Keywords: cdk; cdk7; CAK; phosphoryltion; cell cycle; Xenopus Introduction Cdk-cyclin kinses complexes re key regultors of cell cycle progression. Their tightly regulted ctivtion ensure the ordered nd proper execution of cell cycle events (DNA repliction, mitosis). The rst step of this ctivtion process occurs t speci c points of the cell cycle, when memer of the cyclin fmily ecomes ville for inding to prticulr cdk (cyclin dependent kinse) ctlytic suunit. Second, cdk inhiitory/ctivtory phosphoryltions, some of which re involved in cell cycle checkpoints, control the ctlytic ctivity of the complex. Third, the recently descried cyclin kinse inhiitors my rke the cell cycle y interfering with the complex formtion nd/or y inhiiting the kinse ctivity. These events ensure Correspondence: A Devult This work is dedicted to Jen-Clude Cvdore who pssed wy on the 19th of Mrch 1997 Received 30 Jnury 1997; revised 27 My 1997; ccepted 27 My 1997 wves of ctivtion nd inctivtion of vrious cdkcyclin complexes. All cdk-cyclin complexes re phosphorylted in vivo on cdk threonine residue (Thr161 in cdk1), which in cdk2 is locted in domin de ned s the T loop (Morgn, 1995). This phosphoryltion event, rought out y CAK (Cdk Activting Kinse), not only confers high kinse ctivity to the complex, ut lso strengthens the inding etween the cdk nd cyclin prtners. Although CAK ctivity is pprently invrint during the cell cycle, nd might not e t rst sight, mjor regultor of CAK ctivity, the determintion of suunit composition of CAK hs rised intriguing questions s to how this ctivity is in turn ctivted. Indeed, CAK is itself memer of the cdk fmily. Its ctlytic suunit cdk7 forms complex with two proteins: cyclin H nd MAT1 (Me nge Á trois), memer of the RING nger fmily (Poon nd Hunter, 1995). The in vitro reconstitution of CAK ctivity hs depicted two ctivtory pthwys. First, stiliztion nd ctivtion of cdk7-cyclin H heterodimers re dependent on the phosphoryltion of threonine 176 ( threonine in the ctivtion domin of cdk7) y CAK ctivting kinse (CAKAK) (Fisher et l., 1995). Second, MAT1 cting s n ssemly fctor promotes stle ssocition nd ctivtion of unphosphorylted cdk7 nd cyclin H (Devult et l., 1995; Fisher et l., 1995; Tssn et l., 1995). Recent dt suggest tht n uto regultory loop involving cdc2-cyclin B nd cdk2-cyclin A could ct s CAKAK in vitro (Fisher et l., 1995). An unexpected nding rising from the studies of trnscription nd DNA repir hs lso led to the identi ction of cdk7 nd its ssocited suunits s the RNA polymerse II C-terminl domin (CTD) kinse component of sl trnscription fctor TFIIH (Svejstrup et l., 1996). In udding yest, the closest homologs of cdk7 nd cyclin H re KIN28 nd CCL1, respectively (Fever et l., 1994; Roy et l., 1994). These proteins were lso found to e components of TFIIH nd to hve CTD kinse ctivity. In contrst, the Kin28p/Ccl1p complex does not disply CAK ctivity (Cismowski et l., 1995). Recently, new monomeric protein kinse termed CIV1 (CAK in vivo) or CAK1 le to ssocite to nd phosphorylte CDC28/GST-cdk2 ws cloned in S. cerevisie. (Thuret et l., 1996; Kldis et l., 1996; Espinoz et l., 1996). It ppers, therefore, tht in the udding yest two di erent protein kinses re involved in the regultion of trnscription nd cell cycle control. These dts chllenge the identi ction of cdk7 s the physiologicl CAK in higher eukryotes. In the present work we give strong evidence tht cdk7 complexes is the mjor if not only CAK, t lest in the frog erly emryo.

2 1304 Results nd discussion Although the iochemicl puri ction of CAK ctivity in higher eukryotes resulted in the identi ction y di erent groups of only one kinse, the cdk7/cych/ MAT1 complex, the possiility existed tht nother kinse might hve een lost during preprtion of extrcts or chromtogrphic frctiontion. We therefore sked wht e ect depletion of cdk7 would hve on CAK ctivity in Xenopus extrcts. The reson for choosing these extrcts is tht they cn e prepred in such wy tht they perform two to three rounds of ordered cell cycle events including DNA repliction, chromosome condenstion, nucler envelope rekdown, mitotic spindle formtion nd chromosome segregtion when sperm nuclei re dded (Hollowy et l., 1993 nd references herein). Hence, they must contin the relevnt CAK ctivity necessry for the ccomplishment of these physiologicl events. Depletion ws crried out using polyclonl ntiody directed ginst the lst 16 C-terminl mino cids of Xenopus cdk7. As shown in Figure 1, this ntiody depleted extrcts of more thn 95% of cdk7. In control experiments, we veri ed tht this tretment did not chnge cdc2 levels in the extrct (not shown). E ect of cdk7 depletion on CAK ctivity ws qunti ed y n indirect ssy. Recominnt cyclin A ws dded to the depleted extrct nd its ility to generte H1 histone kinse ctivity ws mesured. Cyclin A ssocites with cdc2 rther thn cdk2 in Xenopus egg extrcts s they contin pool of free monomeric cdc2, whilst cdk2, which is present in mounts tenfold lower thn cdc2, is mostly if not entirely ound to cyclin E nd not ville for inding cyclin A (Strusfeld et l., 1994; Jckson et l., 1995). We found tht the depletion of cdk7 drsticlly reduced CAK ctivity to ckground levels (Figure 1). We lso veri ed tht the mterils immunoprecipitted y the nti-cdk7 ntiodies contined signi cnt mounts of CAK ctivity (Figure 1c). It is well estlished tht ssemly of complexes etween mitotic cyclins nd cdc2, s well s their ctivtion through phosphoryltion of threonine 161 in cdc2, re oth required to drive extrcts prepred from Xenopus eggs into mitosis (Murry nd Kirschner, 1989; Solomon et l., 1990, 1992). Though cdk7- depletion resulted in n extrct with very low if ny ility to ctivte its endogenous cdc2 kinse, minor, yet physiologiclly importnt CAK could possily e su cient to drive cell cycle in the sence of cdk7. To ddress this question, we monitored the fith of sperm chromtin in cdk7-depleted extrcts. As shown in Figure 2, control extcts, fter performing S-phse, eventully entered M-phse, s reveled y the condensed ppernce of the chromtin nd the sence of nucler envelope. In shrp contrst, cdk7- depleted extrcts never entered mitosis. They did, however, replicte DNA, due to ctive cdk2/cyce kinse which is known to e lredy present in unfertilized eggs nd does not need to e ctivted through phosphoryltion of cdk2 in its T-loop for the ccomplishment of the rst emryonic S-phse (Strusfeld et l., 1994; Jckson et l., 1995). We emphsize, rstly tht controls shown in the ove experiments were mde, using pre-immune serum from the sme rit s tht used to deplete cdk7, secondly tht preincution of the nti-cdk7 serum with 1 mg/ ml of the 16 C-terminl mino cid peptide of Xenopus cdk7 restored oth cycling nd CAK ctivity (dt not shown). We thus conclude tht our cdk7 ntiody speci clly depletes Xenopus cycling extrcts of its mjor physiologicl CAK. At rst sight, no other ctivity is le to sustitute for cdk7 complexes nd to drive cycling extrcts into mitosis. ctl α-cdk7 c ctl α-cdk7 SN pellet Figure 1 Depletion of cdk7 results in depletion of CAK ctivity in Xenopus egg extrcts. Egg extrcts were treted with protein A-Sephrose eds pre-loded with pre-immune (ctl) or nti-cdk7 (-cdk7) serums. Superntnts were nlysed for the presence of cdk7 y Western lotting (). H1 histone kinse ctivities were mesured on superntnts fter ddition of cyclin A () nd on - cdk7-protein A eds fter ddition of cyclin A nd GST-cdk2 (c) ctl ctl α-cdk7 α-cdk2 α-cdk7 Figure 2 Depletion of cdk7 inhiits entry into M-phse in Xenopus egg extrcts. () Sperm nuclei were dded to cycling extrcts pre-treted with pre-immune (ctl) or nti-cdk7 (-cdk7) nd followed y Hoechst uorescence. Microgrphs were tken 75 min fter nuclei ddition. Appernce of the nuclei in the nti-cdk7 extrct did not evolve, even fter prolonged times (t lest 2 h). () Autordiogrm showing the incorportion of [ 32 P]-dCTP in DNA of in vitro ssemled nuclei. Smples were tken from control cycling extrct (ctl), cdk7-depleted extrct (cdk7) or cdk2-depleted extrct (-cdk2) nd incuted for 1 h with [ 32 P]-dCTP following sperm heds ddition

3 Although these results re highly suggestive of oth the identity etween cdk7-contining complex nd CAK nd the sence of ny other signi cnt CAK ctivity, we were concerned tht mterils with CAK ctivity other thn cdk7 could still hve een trpped on the eds, perhps speci clly ttched to cdk7 complexes. This would e reminiscent of the cse of CIV1 (CAK in vivo) (Thuret et l., 1996), which ws shown recently to co-purify with CDC28 in the udding yest. In the sme wy, n ntiody directed ginst cdc2 ws reported to co-immunoprecipitte from DU249 cells n unidenti ed ctivity le to phosphorylte cdc2 on threonine 161 (Krek nd Niegg, 1992). To eliminte such possiility, we wnted to verify tht the re-introduction of pure cdk7 complex could rescue CAK ctivity from immune-depleted extrcts. To produce these cdk7 complexes, we voided using ny exogenous eukryotic source such s culovirus infected cells since these could result in ctive contminnts. Becuse it ws found di cult to produce in E. coli ll three cdk7, cyclin H nd MAT1 suunits in solule ctive form, we took dvntge of the cpctity of Xenopus egg extrcts to synthesise proteins. Extrcts mde ccording to Mtthews et l. (1994) were tested for their ility to trnslte cdk7, cyclin H nd MAT1 from in vitro trnscried mrnas. These extrcts were RNAse A-treted so tht no endogenous protein could e synthesized de novo. Figure 3 shows tht Xenopus cdk7, cyclin H nd MAT1 could ech e synthesized in these extrcts from the corresponding mrnas (+), in mounts 4 ± 5-fold tht of endogenous levels (7). 35 S-methionine lelling experiments lso showed tht ech suunit ws synthesized without ny detectle contminnt cdk7 cyc H MAT (Figure 3, lnes 1 ± 3). When extrcts contining ech one of the three newly synthesized suunits were mixed together, heterotrimeric complex could e immunoprecipitted with nti cdk7 ntiodies (Figure 3, lne 4), s previously descried with proteins produced in reticulocyte lystes or in the culovirus system (Devult et l., 1995; Fisher et l., 1995; Tssn et l., 1995). Since Xenopus cyclin H nd MAT1 re not resolved y SDS ± PAGE, we veri ed their presence in the complex y introducing them lterntively s unlelled species (dt not shown). We voided dding ll thee mrnas in the sme trnsltion rection mixture ecuse this ws found to result in the production of uneven mounts of ech suunits, possily due to some competition etween mrnas for recruitment into polysomes. We then sked if the in situ reconstituted cdk7 complex could rescue CAK ctivity. Extrcts immunodepleted of cdk7 were supplemented with mrnas encoding either cdk7, cyclin H or MAT1. After 2 h of synthesis, these extrcts were pooled nd incuted further to llow suunit ssemly. Cdk7 complexes were then recovered y immunoprecipittion nd nlysed for CAK ctivity in depleted extrcts. Figure 4 shows tht synthesis of ll three cdk7 complex suunits generted CAK ctivity, s reveled y direct phosphoryltion of GST-cdk2 (upper pnel) nd n nti-cdk7 ip cdk7 + mrna GST-cdk2 ( 32 P) H1 ( 32 P) cdk7 MAT1 cyc H Figure 3 In vitro trnsltion of cdk7, cyclin H nd MAT1 mrnas in Xenopus egg extrcts. () Extrcts were complemented (+) or not (7) with mrnas encoding either cdk7, cyclin H or MAT1 nd incuted for 2 h. Aliquots (1 to 2 ml) were nlysed y Western lotting with the corresponding ntiodies. () mrnas encoding cdk7 (lne 1), cyclin H (lne 2) or MAT1 (lne 3) were trnslted in extrcts in the presence of [ 35 S]methionine. After synthesis (2 h), liquots of ech trnsltion rection were either nlysed directly y SDS ± PAGE (lnes 1, 2 nd 3) or mixed together in equivlent mounts, incuted for further 45 min period, nd immunoprecipitted with n nti-cdk7 ntiody (lne 4). Mterils dsored on the protein A-Sephrsoe eds were eluted in Lemmli u er nd nlysed y SDS ± PAGE nd uorogrphy nti-cyca ip cdk cyc A + mrna cdc2 (lot) H1 ( 32 P) Figure 4 Synthesis of cdk7, cyclin H nd MAT1 rescues CAK ctivity in cdk7-depleted extrcts. () Extrcts were treted with protein A-Sephrose pre-loded with pre-immune (lne 1) or nticdk7 (lnes 2 nd 3) ser. In lne 3, the extrct ws then split in three to synthesize cdk7, cyclin H or MAT1 mrnas nd pooled to llow cdk7-complex formtion (45 min; see Figure 3). In ll cses, extrcts were nlly immunoprecipitted with n nti-cdk7 ntiody. CAK ctivities of immunoprecipitted mterils were monitored either through phosphoryltion of GST-cdk2, or indirectly y mesuring H1 histone kinse ctivity of ctivted GST-cdk2/cyclinA complexes (see Mterils nd methods). () Extrcts were treted with control (lnes 1 nd 2) or cdk7-speci c (lnes 3 nd 4) ntiodies nd complemented (lne 4) or not (lnes 1, 2 nd 3) with mrnas to produce trimeric cdk7/cyclin H/MAT1 complex, s descried ove, except tht cyclin A ws lso dded (lnes 2, 3 nd 4) during the 45 min complex formtion period. At the end, ll extrcts were immunoprecipitted with n nti-cyclin A ntiody. Immunoprecipitted mterils were nlysed for the presence of cdc2 nd H1 histone kinse ctivities

4 1306 indirect H1 histone kinse ssy (lower pnel). As trnsltion of mrnas should e llowed for out 2 h to restore CAK ctivity to its originl level, it ws not esy to restrt cycling in depleted nd rrested extrcts. However, we veri ed tht these newly synthesized cdk7 complexes could direct the ctivtion of endogenous cdc2 present in the depleted extrct (Figure 4). Cyclin A ws dded to the extrct during the ssemly of the cdk7 trimeric complex, nd its ility to form ctive complexes with cdc2 ws monitored. The reson for choosing cyclin A rther thn cyclin B ws tht, in contrst to cyclin B, cyclin A does not ind stly to cdc2 unless cdc2 ecomes phosphorylted on threonine 161 (Desi et l., 1995), nd tht cyclin A-ssocited cdc2 escpes inhiition due to inhiitory phosphoryltion on threonine 14 nd tyrosine 15 (Devult et l., 1992; Clrke et l., 1992). This llowed us to monitor CAK ctivity oth y the formtion of cyclin A/cdc2 complex nd the resulting H1 histone kinse ctivity. As shown y Western lotting, no cdc2 could e immunoprecipitted with cyclin A ntiodies in the cdk7-depleted extrct (Figure 4, upper pnel, lne 3). However, synthesis of ll suunits of the cdk7 trimeric complex rescued ssemly of ctive cyclin A/cdc2 complexes (Figure 4, lnes 4). This could e correlted with phosphoryltion of cdc2, s reveled y its increse in electrophoretic moility (not shown). We lso tested the ility of di erent comintions of cdk7, cyclin H nd MAT1 to rescue CAK ctivity. We found tht esides the trimeric complex cyclin H/cdk7 dimers could generte CAK ctivity, lthough to lesser extent thn tht of the trimeric complex. In contrst, no ctivity ws ssocited with the synthesis of ny one single suunit (dt not shown). These results re consistent with reconstitution experiments of cdk7 complexes in other systems (Devult et l., 1995; Fisher et l., 1995; Tssn et l., 1995). The ove experiments do not completely rule out the possiility tht cdk7 kinse cted s positive regultor of n unidenti ed CAK (possily CAK1/CIV1), not depleted from the extrcts, ut whose ctivity would require the continuous presence of cdk7. This is however unlikely, ecuse ddition of phosphtse inhiitors, including okdic cid, during immuno-depletion did not rescue CAK ctivity, nd recominnt yest Ck1/Civ1 protein redily restored CAK ctivity even in cdk7- depleted extrcts (dt not shown). Tken together these experiments show tht, t lest in the erly Xenopus emryo, cdk7-contining complexes re necessry nd su cient for ctive complexes of cdc2 with mitotic cyclin, nd presumly other cyclin-cdk complexes, to e formed therey llowing cell cycle progression under physiologicl conditions. It ppers, therefore, tht in contrst to the sitution found in S. cerevisie, higher eukryotes use single kinse, cdk7/cyclin H/MAT1, to execute di erent fundmentl cell functions. The function this kinse opertes depends proly on its ssocition sttus: in its free, trimeric form, it would ct s cell cycle ctivtor, wheres s component of TFIIH, it could modulte trnscription nd/or DNA repir. Of course, one must consider the possiility of the existence of CIV1 homolog in higher eukryotes. However, it hs een shown tht in S. pome, kinse homologous to cdk7/cyclin H, mop1(crk1)/mcs2 hs CAK ctivity (Dmgnez et l., 1995; Buck et l., 1995). Moreover, cdk7 does rescue mutnts defective for the ctlytic suunit mop1 (crk1). Therefore, this orgnism, like higher eukryotes might not need CIV1 homolog, t lest to perform cdk ctivtion. Finlly, it seems tht CIV1 itself my hve more thn one function in S. cerevisie s ck1(civ1) mutnt lleles were found to cuse defect in spore wll morphogenesis without ecting the CAK ctivity needed to progress through meiosis I nd II (Wgner et l., 1997). Mterils nd methods Xenopus egg extrcts Cycling extrcts were freshly prepred ccording to Murry nd Kirschner (1989). Extrcts for synthesis of exogenous mrnas were mde ccording to Mtthews' procedure (Mtthews, 1994), including 0.2 mg/ml RNAseA tretment nd frozen t 7708C. Nucler ssemly/disssemly nd DNA repliction Dememrnted frog sperm nuclei, prepred s descried previously (Lohk nd Msui, 1984), were dded (200 ml) were dded to cycling extrcts. Nucler formtion, nucler envelope rekdown were followed y oservtion with uorescent microscope, using Hoechst to stin chromosomes. DNA repliction ws ssyed y mesuring the incorportion of rdiolelled 32 P--dCTP into DNA (Morin et l., 1994). Recominnt proteins nd cdna Humn cyclin A nd GST-cdk2 were prepred s descried (Devult et l., 1992; Clrke et l., 1992). mrnas encoding Xenopus cdk7, cyclin H nd MAT1 were in vitro trnscried from their corresponding cdnas inserted in psp64t-ri plsmid. Recominnt yest Civ1 protein ws kindly provided y Crl Mnn, CEA, Frnce. Trnsltion in Xenopus levis extrcts Trnsltion of in vitro trnscried mrnas in RNAse A- treted Xenopus levis extrcts were mde essentilly ccording to Mtthews (1994). Upon thwing, 50 ml liquots of extrcts were supplemented with 1 ml of 500 mm cretine phosphte, 0.5 ml of 100 mm spermidine nd 50 mci [ 35 S]methionine. Trnsltion ws initited y dding mrna to nl concentrtion of 0.1 mg/ml nd llowedtoproceedfor2ht218c. In some experiments, efore trnsltion, extrcts were rst treted for immunodepletion. Immunologicl methods Antiser for Xenopus cyclin H nd MAT1 nd humn cyclin A were rised in rits ginst the entire proteins mde in E. coli. Antiser speci c for Xenopus cdc2, cdk2 nd cdk7 were prepred ginst peptides corresponding to their lst 16 C-terminl nimo cids (Devult et l., 1992; Fesquet et l., 1993). For immunodepletion, tches (50 ml) of undiluted Xenopus extrct were mixed with 7 ml of protein A- Sephrose eds predsored with 15 ml of nti-cdk7, cdk2 (Figure 2) or pre-immune serum nd incuted t 48C for 1 h with gentle shking. For immunoprecipittion, extrcts were rst diluted t lest 20-fold in RIPA u er (Devult et l., 1992). Immune ser nd protein A-Sephrose eds were dded nd the mixture incuted for 1 h t 48C. After severl wshes in the sme RIPA u er, the eds were either

5 treted with Lemmli smple u er for gel nlysis or sumitted to CAK ssys. CAK ctivities We ssyed either directly the phosphoryltion of humn GST-cdk2 or indirectly the phosphoryltion of H1 histone fter ddition of GST-cdk2 nd humn cyclin A. In the former cse, immunoprecipittes on protein A eds were incuted for 30 min with shking in 20 ml mixture contining 0.2 mg GST-cdk2, 50 mm Tris-HCl ph 7.5, 10 mm MgCl 2 nd 5 mci [ 32 P]g-ATP (90 mci/mmol). In the ltter cse, either liquots of crude extrcts (1 to 2 ml) or ed pellets were rst incuted in 20 ml ofcocktil contining 0.1 mg GST-cdk2, 0.1 mg cyclin A, 0.33 mm ATP, 10 mm Tris-HCl, ph 7.4, 16.6 mm MgCl 2. After 30 min t 258C, one volume of mixture contining 2 mg/ml histone H1 (Boehringer Mnnheim), 20 mm HEPES, ph 7.4, 10 mm MgCl 2 nd 250 mm [ 32 P]g-ATP (90 mci/mmol) ws dded nd further incuted for 15 min. All rections were stopped y dding Lemmli smple u er References Buck V, Russell P nd Millr JB. (1995). EMBO J., 4, 6173 ± Cismowski MJ, L GM, Solomon MJ nd Reed SI. (1995). Mol. Cell. Biol., 15, 2983 ± Clrke PR, Leiss D, Pgno M nd Krsenti E. (1992). EMBO J., 11, 1751 ± Dmgnez V, Mkel TP nd Cottrel G. (1995). EMBO J., 14, 6164 ± Desi D, Wessling HC, Fisher RP nd Morgn DO. (1995). Mol. Cell. Biol., 15, 345 ± 350. Devult A, Fesquet D, Cvdore JC, Grrigues AM, Le JC, Lorc T, Picrd A, Philippe M nd Doree M. (1992). J. Cell. Biol., 118, 1109 ± Devult A, Mrtinez AM, Fesquet D, Le JC, Morin N, Tssn JP, Nigg EA, Cvdore JC nd Doree M. (1995). EMBO J., 14, 5027 ± Espinoz FH, Frrell A, Erdjument-Bromge H, Tempst P nd Morgn DO. (1996). Science, 273, 1714 ± Fever WJ, Svejstrup JQ, Henry NL nd Kornerg RD. (1994). Cell, 79, 1103 ± Fesquet D, Le JC, Derncourt J, Cpony JP, Gls S, Girrd F, Lorc T, Shuttleworth J, Doree M nd Cvdore JC. (1993). EMBO J., 12, 3111 ± Fisher RP, Jin P, Chmerlin HM nd Morgn DO. (1995). Cell, 83, 47 ± 57. Hollowy SL, Glotzer M, King RW nd Murry AW. (1993). Cell, 73, 1393 ± Jckson PK, Chevlier S, Philippe M nd Kirschner MW. (1995). J. Cell. Biol., 130, 755 ± 769. Kldis P, Sutton A nd Solomon MJ. (1996). Cell, 86, 553 ± 564. Krek W nd Nigg EA. (1992). New Biol., 4, 323 ± 329. Lohk MJ nd Msui Y. (1984). Dev. Biol., 103, 434 ± 442. Mtthews G. (1994). Cell Biology: Lortory Hndook. Celis JE (ed). Acdemic Press: Sn Diego. pp. 131 ± 139. Morgn DO. (1995). Nture, 374, 131 ± 134. Morin N, Arieu A, Lorc T, Mrtin F nd Doree M. (1994). EMBO J., 13, 4343 ± Murry AW nd Kirschner MW. (1989). Nture, 339, 275 ± 280. Poon RY nd Hunter T. (1995). Curr. Biol., 5, 1243 ± Roy R, Admczewski JP, Seroz T, Vermeulen W, Tssn JP, Sche er L, Nigg EA, Hoeijmkers JH nd Egly JM. (1994). Cell, 79, 1093 ± Solomon MJ, Glotzer M, Lee TH, Philippe M nd Kirschner MW. (1990). Cell, 63, 1013 ± Solomon MJ, Lee T nd Kirschner MW. (1992). Mol. Biol. Cell., 3, 13 ± 27. StrusfeldUP,HowellM,RempelR,MllerJL,HuntTnd Blow JJ. (1994). Curr. Biol., 4, 876 ± 883. Svejstrup JQ, Vichi P nd Egly JM. (1996). Trends Biochem., 21, 346 ± 350. Tssn JP, Jquenoud M, Fry AM, Frutiger S, Hughes GJ nd Nigg EA. (1995). EMBO J., 14, 5608 ± Thuret JY, Vly JG, Fye G nd Mnn C. (1996). Cell, 86, 565 ± 576. Wgner M, Pierce M nd Winter E. (1997). EMBO J., 16, 1305 ± 1317.