ehrad acrIsiue(K-V) lsala 2,16 X Amsterdam, CX, 1066 121, Netherlands. Plesmanlaan The (NKI-AVL), Institute Cancer Netherlands id Clijsters Linda phase Cdc6 S of during re-accumulation prohibits degradation PIP-box-mediated ARTICLE RESEARCH ß 1336 2013 December 17 Accepted 2013; November 5 Received 1 the and chromosomes, condensed to translocates Cdc6 licensing: is the phase phase. during S G1 place of and take onset that mitosis the events preceding (Masai that regulatory the by means polymerases onto controlled this largely DNA-clamp Altogether, DNA loaded MCM2– 2010). of is homotrimeric al., by et recruitment (PCNA) out the enabling antigen carried chromatin, when nuclear is cell begins the that proliferating as facilitate Replication unwinding such subsequently DNA 7. factors, factors, of complex initiation process GINS replication licensed, complexes and DNA Once cyclin–Cdk2 Cdc45 kinase. by licensing. This Cdc7 activated replication. replication be and DNA hereafter can DNA of 7, origins called to origins replication 2 on is (numbered MCM2–7) which process proteins as MCM complex, to six referred (MCM) the of maintenance consists the mini-chromosome loads of Cdc6 factor Cdt1, the essential with an Together is (preRC). complex (Cdc6) pre-replication 6 protein control division Cell INTRODUCTION DNA cycle Cell of PIP-box, PCNA, Cdt1, licensing Cdc6, CRL4–Cdt2, control WORDS: erroneous KEY phase. different G2 and restrain S-to-G2 that S the G1, show Cdc6 during at replication results Cdc6 of our of that mechanisms summary, export propose nuclear We In cytoplasm. the transition. the to to Cdc6 and contributes limited absent phase, mostly Cdk1 is is G2 Cdc6 Cdt1 of time, reach this expression cells at but, When re-accumulates nuclear rapidly complex. Cdt1, Cdt2- the to and CRL4 PIP-box Similar N-terminal containing an phase. on S depends Cdc6 during of clearance and transition G1-S at re-accumulation the Cdc6 is nuclear APC/C–Cdh1-independent prevents novel Cdt1 that and a pathway Cdt1 as reveal degradation We of re- long cells. dynamics thus cycling as the in could investigated Cdc6 DNA Cdc6 carefully of phase, we Here, G1 re-licensing present. of the end risking the accumulate, at is again, APC/C–Cdh1 When off activity. exit APC/C–Cdh1 switched of cells anaphase result from when a degraded as efficiently licensing onwards is Cdc6 replication cells, cycling DNA In initiate mitosis. Cdt1 and Cdc6 ABSTRACT Ato o orsodne([email protected]) correspondence for *Author Netherlands. The de Amsterdam, van BT Faculty, 1081 Medical 7, VUmc Boechorststraat Oncogenesis, Program Research CCA/V-ICI iiino elBooy(5 n iiino oeua acngnss(7,The (B7), Carcinogenesis Molecular of Division and (B5) Biology Cell of Division 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,13–35doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. al nmtss el tr rprn o N replication DNA for preparing start cells mitosis, in Early 2 eto fOcgntc,Dprmn fCiia eeisand Genetics Clinical of Department Oncogenetics, of Section 1 n o Wolthuis Rob and 1,2, * erddtruhACCcnann d1(lokonas known is (also Cdc6 Cdh1 however, containing APC/C anaphase, license through to after degraded competence metaphase-to-anaphase Immediately the the This at transition. cells, 2013). increases licensing greatly al., proliferating replication et key DNA in (Clijsters when that, satisfied second APC/C–Cdc20 licensing is means by a checkpoint degraded key spindle also Geminin, the is a mammals, 2011). in Cdk1, inhibitor ligase Cdc20 (Pines, E3 inactivates the inhibitor containing through which 2004; degraded (APC/C–Cdc20), complex/cyclosome is al., checkpoint B1 spindle et cyclin the anaphase-promoting satisfy mitosis, stable (Ballabeni cells a exit When geminin of and 2013). formation al., inhibitor et to its Clijsters owing increase with greatly complex Cdt1 of levels fG,we d1lvl r tl ih ol oeatra to threat a through, progress pose and enter, could Sphase. they as high, re-licensing control prohibit cells still and cycling Cdc6 how are investigated we levels Here, end integrity. Cdt1 the genomic at when Cdc6 of G1, expression cells, of these activated In is licensed 2013). APC/C–Cdh1 al., already before et (Clijsters exit, probably, mitotic have, during origins cells their proliferating in found rapidly active we contrast, first that By highly 2005). Diffley, by the is and (Mailand degradation cells. APC/C–Cdh1 quiescent cycle, from though protected point, cell even is this phosphorylation, Cdc6 at the synthesized arise would newly re-enter replication when DNA In cells license 2004). Kirschner, to quiescent and opportunity Rape 1996; when al., et fact, Piatti 2002; 2005; Agami, al., newly and et is Cdc6 Duursma Hsu and 2014; off al., switches et APC/C (Bassermann the synthesized phase, However, G1 exit. of mitotic end during the al., Cdc6 by also of et is degradation re-replication the DNA (Liu of by SCF–Skp2 risk reduced The ligase 2006). al., E3 et and newly Nishitani 2004; the phosphorylation any via on that destruction rely chance subsequent might the mechanism destruction reducing Cdt1 phase, Another re-replicated. subsequently and S re-licensed is robustly of DNA formed coincides start clearance 2009; Cdt1 the Walter, Thereby, with and 2012). (Havens al., PCNA et interacts of Havens CRL4–Cdt2 form when DNA-loaded functional the Cdt1 with only This is (CRL4–Cdt2). cullin ligase motif Cdt2 E3 degradation containing the complex by (CRL)4 onto recognized typically loaded ring so-called is is a that PCNA requires degradation motif when Cdt1 PIP-box lost 2013). is al., et but (Clijsters phase DNA G1 in and exit that, 2013). mitotic of al., end suggests et the (Clijsters at events peaks time competence of of window licensing short when a schedule mitosis is there This cells, cycling human). rapidly in FZR1 d6i betfo al 1paeowrsad nms cells, strictly most in manner 2013). and, al., onwards a phase et G1 in early (Clijsters from 1A) absent anaphase (Fig. is Cdc6 in APC/C–Cdh1 on starts dependent degradation Cdc6 of Cdc6 degradation KEN-box-independent and APC/C–Cdh1- RESULTS d1 fe t ees rmgmnn ean rsn during present remains geminin, from release its after Cdt1,

Journal of Cell Science xrsigKNCc–eu eeiae yDCadfursec irsoy atrdiae fteidctdpae fmtssaeson E The to (E) compared shown. cells are all of mitosis fluorescence of average Right, phases cells. indicated individual the of curves of Left, images NEB. after Captured time microscopy. n against fluorescence plotted was and fluorescence DIC of by intensity imaged were KEN-Cdc6–Venus expressing EERHARTICLE RESEARCH uha uoaA(ljtr ta. 03.Hr,w investigated can that we exist Here, might 2013). mechanisms al., destruction et substrates, additional (Clijsters APC/C–Cdh1 whether A typical Aurora other than as cycle such appears cell Cdc6 S the cells, into cycling in hours in later several that means least this at Remarkably, are phase. cells until re-accumulate not does 10 with treated Cdc6. either of were degradation (mock)] siRNA KEN-box-independent control and APC/C–Cdh1- 1. Fig. xrsigCc–eu n eltdo d1wr imda nB h nest ffursec a lte gis ieatrNB et uvsof curves Left, NEB. after cells time U2OS against (C) plotted breakdown. was (control, envelope fluorescence controls. nuclear of with NEB, intensity compared shown. The cells microsc are B. all fluorescence mitosis in of and of as fluorescence (DIC) phases filmed average contrast indicated were Right, interference the Cdh1 cells. differential of of individual by Images depleted imaged si-CDH1. and were panel, Cdc6–Venus Cdc6–Venus Lower expressing expressing siRNA. cells no U2OS panel, (B) Upper proteins. indicated the for 5 1 mean 11, 6 ... KEN-Cdc6–Venus, s.e.m.; n 5 1 mean 11, m d1ihbtrR-36o etutetd xrcsfo hs el eete ujce owsenblotting western to subjected then were cells these from Extracts untreated. left or RO-3306 inhibitor Cdk1 M 6 s.e.m.). A ioi 2Scls[rnfce ihsRAagainst siRNA with [transfected cells U2OS Mitotic (A) loecnemcocp nclstetdwt iN.Cdc6– as well siRNA. as mitosis, with after and treated during stable cells largely remained Venus in microscopy fluorescence APC/ when onwards, active. transition not is G1-S C–Cdh1 the from Cdc6 destabilize isl,w olwdfursetCc–eu ytime-lapse by Cdc6–Venus fluorescent followed we Firstly, n 5 ora fCl cec 21)17 3614 doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal 1 mean 11, 6 ... si-CDH1, s.e.m.; n 5 ,mean 9, CDH1 6 ....()UO cells U2OS (D) s.e.m.). s-D1 ra or (si-CDH1) otos(control, controls opy. 1337

Journal of Cell Science ned E-d6Vnswssal taahs u suddenly but Ala. KEN-Cdc6– anaphase to named mutated at were stable Cdc6, Asn83 was and of KEN-Cdc6–Venus Glu82 Indeed, mutant Lys81, we which box in pathway, Venus, destruction degradation a Cdc6 used of existence apparent 90 APC/C–Cdh1-independent the 1B, investigate an (Fig. further To nucleus minutes). the 120 Cdc6 within and fluorescent from cells, specifically siRNA-treated hours declined several Cdh1 levels degraded In rapidly 1B,C). was (Fig. Cdc6 later However, phase. G1 in ARTICLE RESEARCH 1338 filmed APC/C-independent was the Cdc6–Venus activated, when is pathway precisely destruction more Cdc6 nucleus investigate the in To starts degradation around Cdc6 time Cdh1-independent a at destruction phase. scheduled G1 event of Cdc6 an end by the initiated APC/C-independent a is (supplementary that mechanism experiments 2000) unknown these from al., hypothesize previously live- et We our S1). (Petersen in Fig. Cdc6 material assay of pattern degradation degradation the A cell Leu59 to 1E). and contribute (Fig. Arg56 not fashion comprising did motif switch-like KEN-Cdc6– recognition a minutes). D-box 120 in suggested started 1D, (Fig. degradation nucleus Venus declining fluorescence the with in again, specifically phase, G1 in later disappeared fSpaeclsuo rtaoeihbto Fg 2D–F). (Fig. inhibition proteasome upon nucleus cells the in phase exclusively S accumulated fluorescently translation Cdc6, the of Furthermore, or with Cdt1, 2C). co-treatment S (Fig. tagged or by G1 cycloheximide MG-132-treated reversed in inhibitor was Cdc6 in as a of cells were increase to synthesis cells The phase led whether phase. protein S of MG-132 or in regardless inhibitor G1 Cdc6, reduction of proteasome a accumulation with rapid to cells Cdc6 of of related Decrease treatment loading. not destabilizes PCNA was of that timing suggested levels the to pathway This linked is 2A). the APC/C–Cdh1-independent Cdc6 (Fig. In the appeared minutes). degraded 90 that dots be to to Cherry–PCNA started minutes also when the 60 is KEN-Cdc6–Venus example at Cdh1, compare an of while 2B, presence 2A; Fig. nucleus, (Fig. in the formed to shown dots in began Cherry–PCNA started levels time, Cdc6 which cells phase, same process of G1 in of end a However, end the diminish, the until 2012). Following stable phase. al., remained replication G1 Cdc6–Venus et towards DNA degraded Cdh1, of (Burgess represent depleted was 2A) which re-distributed (Fig. (dots), Cdc6–Venus factories foci Cherry–PCNA cells, nuclear Cherry-tagged discrete control before phase, S most of substantially marker In fluorescent PCNA. a with together ora fCl cec 21)17 3614 doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal niiulcell. an individual indicates dot Each the cytoplasm. and nucleus the between compared were Levels Cdc6–Venus MG-132. of of addition after the time against plotted was of fluorescence intensity the S cells, In phase cells MG-132. phase with S treatment of after cytoplasm but the nucleus in the not in observed Cdc6– is (F) Venus MG-132. with cells phase of S treatment after Cdc6– accumulates (E) Venus MG-132. with cells phase of S treatment after accumulates Cdt1– Venus (D) treatment. MG-132 inhibited by was proteins of control). Degradation (loading actin and Cdt1, Cdc6 for probed of cells samples synchronized of S blots and Western G1 phase. in synthesized are and Cdt1 Cdc6 (C) (si-Cdh1). Cdh1- cells in depleted diminish Cdc6 of appear levels dots when PCNA (B) point. before this starts degradation control Cdc6 in cells, that Note of replication. beginning DNA the represents Time 0 cell. point individual an Each indicates dots. dot PCNA from of minutes appearance as the timed was Cdc6 degradation of of start The microscopy. fluorescence and (DIC) contrast interference differential by imaged were Cherry–PCNA and Cdc6– Venus coexpressing cells U2OS nucleus. (A) the in starts degradation Cdc6 Cdh1-independent 2. Fig.

Journal of Cell Science h iesn atrCt sdgae nSpaeb PCNA- a by CRL4– phase by S destruction in targeted degraded involving is CRL mechanism a Cdt1 a via dependent occurs factor transition by licensing G1-S the The low at in Cdc6 kept of initiated Degradation are is levels that pathway Cdc6 nucleus. degradation the phase, unknown onwards, S transition previously G1-S throughout the from and that conclude we Therefore, ARTICLE RESEARCH etblzn d1i hs;tu rvnigr-iesn of re-licensing preventing thus phase; S through mainly in function this Cdt1 perform to destabilizing over-replication. suggested of been inhibitor CRL4–Cdt2 has well-described via Cdt2 a occurs is transition Cdt2 that G1-S DCAF the The at conclude Cdc6 of Degradation we CRL. continuing a and via transition Therefore, occurs G1-S phase, the S at throughout 3B,C). starting (Fig. Cdc6, of degradation MLN- treatment by impaired strongly was 4924 transition KEN- G1-S a of the degradation increased to at Furthermore, Cdc6–Venus clearly 3A). accumulated (Fig. Cdc6 levels Cdt1 however, than Cdc6 extent MLN-4924; 2010). lesser of al., addition et the (Lin upon with CRLs treated of and inhibits phase activity which S MLN-4924, pathway in inhibitor synchronized NEDD8-activating-enzyme Cdc6-degradation were the the cells found, in we CRLs that the test of To 1C). involvement profile of (Fig. degradation potential blocked appearance the is the to APC/C–Cdh1 when similar with Cdc6–Venus 2013), of coincides al., et and cells (Clijsters transition dots live PCNA G1-S in Cdt1–Venus the DCAF of at by Destruction starts 2012). residues al., Cdt1 charged et PCNA of positively (Havens DNA-loaded Cdt2 adjacent Degradation to of PIP-box enzyme. recognition N-terminal E2 and an an called of binding with receptors requires interacting substrate ROC2, binding different damage or DNA ROC1 protein, binding ring adaptor a and (DDB1), the (DCAFs), Generally, DDB1-CUL4-associated factors and 2012). 1 4 al., et cullin protein Havens of 2009; consist Walter, CRL4s and (Havens Cdt2 h noeospoenlvl fbt d1adCc remained Cdc6 and Cdt1 these both of siRNA, pre-treat as levels Cdt2 well with protein as treated to endogenous control), cells positive the in MG-132, a wash-out, had (as MG-132 Cdt1 After inhibitor of we Cdc6. caused levels the MG-132 proteasome in of this increase addition an the The for re-accumulation. with Cdc6 kept phase, enabling normally cells are S levels occurred synchronized S2 Cdc6 during Fig. Because material profiles). Cdc6 low supplementary cycle see cell 4C; tried of after (Fig. for cells we Cdt2 S-phase-synchronized distribution, in of Cdc6 cycle stabilization depletion of cell stability the in follow change observed to investigate the To of CRL4–Cdt2. Cdc6, the of independently that target indicated important of further whether an This co-depletion is 4B). by Cdt1, (Fig. reversed like Cdt1 efficiently or is Cdc6 Cdt2 either of by in depletion depletion induced Cdt2 cycle. samples the re-replication that the the revealed cell Interestingly, 4B) of re-replication. the (Fig. distribution induces cytometry affect flow cycle by cell 4A could the Fig. Cdt2 of 4A). of (Fig. analysis Cdt1 Indeed, depletion of the levels after mentioned, by the substantially investigated of As of increased Cdc6 independently depleted Cdc6 Cdt2, of were of we of levels that depletion degradation The cells this, siRNA. in the by levels Cdt2 a for Cdc6 test in also Cdt2 changes is To measuring DCAF Cdc6 that of CRL4–Cdt2. hypothesized requirement We of 2006). al., target et (Jin origins oto el,Cc iapae tmttcei n i o re- not was Cdc6 did when phase, and S exit of end mitotic the In reached at cells in until microscopy. disappeared accumulate Cdt2 Cdc6 fluorescence could of cells, time-lapse Cdc6–Venus control role by co-express stability that Cherry–PCNA the cells and Cdc6 tested single we unperturbed on otherwise Therefore, underestimated. effects are Cdc6 be cells, and Cdt1 phase-synchronized both phase, S manner. Cdt2-dependent in a that, in quantification confirms degraded the 4D 4C); Fig. (Fig. controls in with compared as elevated, nti riiilstain fe G12teteto S- of treatment MG-132 after situation, artificial this In ora fCl cec 21)17 3614 doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal tbeatrtetetwt L-94(C). MLN-4924 with treatment after more is stable KEN-Cdc6–Venus however, B, point; in before this that starts Note KEN-Cdc6–Venus phase. of S degradation which of 0, start point time the at marks levels to relative are fluorescence levels The dots. from PCNA of time appearance the the against of plotted intensity was The fluorescence (C). or MLN-4924 (B) of absence presence the in microscopy or fluorescence (DIC) contrast by interference imaged differential were Cherry–PCNA and KEN-Cdc6– Venus coexpressing controls. cells loading U2OS as (B,C) Actin used hours. were 2 APC3 for and solvent or MLN-4924 inhibitor neddylation with (MG-132), treated inhibitor were proteasome cells S phase in S neddylation phase. of inhibition after CRL. stabilized G1-S a the via at occurs Cdc6 transition of Degradation 3. Fig. A d6is Cdc6 (A) 1339

Journal of Cell Science EERHARTICLE RESEARCH 1340 in Additionally, S3A). Fig. material supplementary panels, and lower 4E, 4F; (Fig. Fig. cells pathway degradation these phase of inability S the the activate with at combination to APC/C–Cdh1 in phase, of that G1 switch-off of conclude end the the We reflects onwards. pattern transition expression G1-S this the in from accumulated slowly nucleus then in first the but was presented exit, Cdc6–Venus scheme mitotic Cdt2, the at of normally, also depleted degraded cells see in panels; However, top 7). 4E, Fig. progressed (Fig. cells phase as disappeared S rapidly to signal end this but the phase, before G1 cells, nucleus Cdc6– of the of some in amounts In re-accumulated high fluorescence 1B). relatively Venus, (Fig. synthesized that cytoplasm cells the probably in detected exclusively hto d1 otiue oteihbtr fet fteE ligase E3 the of effects over-replication. inhibitory DNA the like on to Cdc6, CRL4–Cdt2 onwards. contributes of transition Cdt1, degradation G1-S of phase that S the that from conclude CRL we Furthermore, Cdt2-driven a important an of is Cdt1, target S4A). like Cdc6, as that Fig. indicate strongly interacted, results material These in (supplementary Cdc6 Cdt2 endogenous cells of and S-phase-synchronized assay immunoprecipitation Cdt2 an by Cdc6–Venus, detected expressing cells ocaiyti e erdto aha nmr eal we detail, control more in in mutants pathway Cdc6 degradation fluorescently-tagged new several followed this Cdc6 clarify in motif To PIP-box-like N-terminal conserved A ora fCl cec 21)17 3614 doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal ifrnilitreec otat(DIC) contrast interference differential by imaged were Cherry–PCNA and Cdc6–Venus U2OS coexpressing Live cells (E) plotted. and MG-132 treatment, and loading were for levels corrected Cdc6 and Cdt1 Relative upeetr aeilFg S3A. in Fig. shown material is supplementary control The of dots. appearance PCNA the after minutes and 60 before minutes 60 measured was siCDT2 with cells transfected in Cdc6–Venus of Relative fluorescence E. of Quantification (F) cells. control with appear, compared dots PCNA not after is degraded Cdc6 cells siCDT2, in with that treated S Note of shown. start are the phase from minutes indicated of the number at images (upper Still untreated panel). left or panel) against (lower siRNA an with treated and microscopy fluorescence and eet fteeprmn hw nC ( in shown shown is experiment the of repeats independent A three (D) of cells. quantification Cdt2-depleted in (w/o) wash- out MG-132 Cdt1 after and stable Cdc6 remained hours. 2 for MG- 132 inhibitor proteasome or the with without treated were cells S phase (siCTRL). siRNA control or against (siCDT2) siRNA an with cells treated U2OS (C) alone. depletion Cdt2 the after rescues observed Cdc6 re-replication and Cdt2 and or Cdt2 Cdt1, of control. co-depletion loading that a Note as used was Actin sorting. cell fluorescence-activated (B) and blotting for western processed (A) and BrdU with were pulsed cells unsynchronized After hours, pools. 48 siRNA indicated the with CRL4– Cdt2. via the occurs at transition Cdc6 G1-S of Degradation 4. Fig. AB 2Sclswr treated were cells U2OS (A,B) n 5 ;mean 3; 6 s.e.m.). CDT2 CDT2

Journal of Cell Science otiuet eonto yCL–d2(i ta. 2010; al., to et (Kim suggested CRL4–Cdt2 been and by hydrophobic have recognition by which to flanked residues, contribute a C-terminally charged includes is positively PIP- we This which motif 2007). al., Cdc6, motif, et box-like (Moldovan 5A,B) PIP-box-like of (Fig. core-element potential QxxI region In a substrate. N-terminal the discovered within evolutionary-conserved (PIP-box) motif the PCNA-interacting recognition a requires of typically degradation Cdt2-dependent cells. ARTICLE RESEARCH ots hsie,w is netgtdwehrtemotif the the either of peptides whether PCNA purified between with binding investigated direct interact reveal first could could Cdc6 we of vitro idea, PIP-box phase. the S this during resembling and test transition G1-S the To at Cdc6 of accumulation curve 5D, cell 5E). Fig. Fig. point and entire QIF-KEN- minutes; diamonds; 240 by the KEN-box, with and denoted 120 throughout the compare 5C, construct stable and (Fig. cycle fully PIP-box Cdc6 remained the a Cdc6–Venus, both in however, mutations Fig. Importantly material exit supplementary triangles; mitosis S3B). by at denoted pathway curve 5D, APC/C–Cdh1 was (Fig. the QIF-Cdc6–Venus, by to sequence, normally Phe14 PIP-box and core degraded the Ile12 in Gln9, mutant A Ala microscopy. were element, time-lapse Cdc6– PIP-box by two potential Next, followed the 2007). in al., mutated et constructs, Moldovan Venus 2011; al., et Michishita eraoe htti d2rcgiinmtfpoiistere- the prohibits motif Cdt2-recognition this that reasoned We hrfr,w sdafursec-oaiainasythat assay fluorescence-polarization a used we Therefore, . in n fG hs ol edt h eacmlto fCdc6, of re-accumulation the to lead would the at phase APC/C the G1 of inhibition of the motif, PCNA, this end from Without and phase. degradation CRL S of Cdt2-directed proteasome-dependent start a for PIP- by the Cdc6 recognized like targeting Cdc6, is of Cdt1, PIP-box depleted of the cells was box Apparently, in 4E). Cdc6 re-accumulation (Fig. wild-type Cdt2 for QIF-Cdc6–Venus of observed in that of presented to scheme similar timing very the The also see 7). 6A,B, Fig. at (Fig. whereas dots transition PCNA phase G1-S of Cdc6–Venus appearance the S the with of and wild-type coincided This end occurrence earlier. border earlier much the Indeed, G1-S occurred reached QIF-Cdc6–Venus the of cells phase. re-accumulation transit when they S only as appeared confirm through cells to live aimed progress in we concept Next, this unrecognized motif. previously a degradation as N- PCNA-directed functions the Cdc6 that of idea the the PIP-box of support terminal lengths results the these interaction of Therefore, was independent specific used. peptides than was the and affinity peptide, consistently Cdt1 found lower was a with wild- for PCNA though previously the with described Even to interacted S4B,C). Cdc6 PCNA Fig. type undetectable of material Cdc6 an to binding wild-type (supplementary reduced the level was however, Cdc6 that of PCNA; N-terminal found versions PIP-box-mutant to We Phe14) Cdc6. bound Ile12, or peptide Gln9, PCNA (at of regions Ala-mutated or wild-type eas,a es ntecl ie htw netgtd d6is Cdc6 investigated, we that lines cell the in least at because, ora fCl cec 21)17 3614 doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal hs cus1 or fe NEB after ( hours 10 occurs phase CAdt ( of dots appearance PCNA the after minutes 60 1ocr 0mntsatrNB( NEB mean after minutes 90 occurs (NEB). G1 breakdown envelope nuclear levels at to relative are levels The fluorescence indicated. as time, plotted against was fluorescence of intensity The microscopy. fluorescence DIC and by imaged were Cherry–PCNA and QIF-KEN-Cdc6–Venus coexpressing mean n loecneo niiulcls(wt- Cdc6–Venus, cells individual of average fluorescence the show Curves were imaged. plasmids cells indicated U2OS the Live expressing (D) shown. by are observed DIC, as time onset, indicated anaphase the after at mitosis in indicated phases the of Images microscopy. fluorescence and (DIC) interference contrast differential by imaged were Cherry–PCNA QIF- and coexpressing KEN-Cdc6–Venus cells U2OS Live indicated. (C) is core-element PIP-box species. The different from Cdc6 N- of the terminus of Alignment also (B) is indicated. PIP-box the of consensus sequence The indicated. ATPase are the domain and KEN-box the motif, PIP-box- like The Cdc6. of representation Cdc6. in PIP- motif N-terminal box-like conserved A 5. Fig. n 5 5 1 QIF-KEN-Cdc6–Venus, 11; 5 mean 15; 6 6 ...ErySpaeoccurs phase S Early s.d.). ....()Lv 2Scells U2OS Live (E) s.e.m.). n 6 5 n s.d.). 5 6 mean 16; 1 QIF-Cdc6–Venus, 11; A Schematic (A) 6 ...Lt S Late s.d.). n 5 n 30; 1341 5 23;

Journal of Cell Science ntecl yl,i 1pae n eas cyclin-A–Cdk2 because and phase, G1 in we a cycle, part, be cell In also Cdc6. the might of earlier much in onwards active export already phase is Cdk1 cyclin-E–Cdk2 the because this G2 triggering of conclude accumulation in of slow factor start 1999), significant the al., from et Petersen activity 1999; (Jiang onwards al., phase S et early from cytoplasm the to Cdc6 exporting mean EERHARTICLE RESEARCH 1342 the from hours 7.5 roughly minutes 446.6 6C,D; dots: (Fig. Cherry–PCNA phase S of from G2 transition appearance the to cytoplasmic around to cyclephase started nuclear suddenly rapid cell a that showed translocation the it Interestingly, during phase 5E). and S (Fig. stable of end begins, remained the at QIF-KEN-Cdc6–Venus replication Cdc6 controls activity when Cdk border, licensing G1-S is of phase. Cdt1 clearance the S efficient throughout cellular more at of much Cdt1 pool a factors of ensures entire degradation Cdc6 the and PIP-box-mediated before Concomitant fire, as removed. DNA forks re-replicating and first High re-licensing phase. the of replication S risk DNA in the before increase and but would phase licensing DNA G1 after of expression end Cdc6 the at synthesized rapidly 6 ...Ti ugssta,aatfo oefrCk in Cdk2 for role a from apart that, suggests This s.d.). 6 60.6; n 5 8; pt 9 to concentrationsup at RO-3306, 6E). that (Fig. found RO-3306 we reports, inhibitor other by Confirming Cdk1 prevented selective completely highly was the phase of addition S 1999). of late translocation al., cytoplasmic in Cdk1, et Cdc6 for (Lukas nondegradable role possible phase a S of of support In start the before long accumulates retdi 2pae hnCk sohrieatvtdt allow to activated 5 otherwise with is incubation prolonged Cdk1 After entry. when mitotic then phase, but G2 normally, relatively the in phase 5 arrested S with with through treated progressed agreement of cells hours function U2OS further 24 the that abrogate In found fully we shown). not Cdk2, does not RO-3306 (data that assumption 2006) Vassilev 2013; al., al., et Spencer 2009; et al., et (Ma Cdk2 block not does otat el rae ihahge oeo h rg 10 drug, the of dose higher a By inhibition. with Cdk1 treated of cells consequence contrast, typical to a re-replication DNA, of their rounds several multiply started phase G2 in arrested cells d1atvt aaye ula xoto d6a el progress cells phase. as G2 Cdc6 to of phase of basis S export the from nuclear of on accumulation catalyzes Cdk2, the activity for that conclusion role Cdk1 the a favor out partial we rule experiments, of formally Althoughour result not S5A). do a Fig. results material as our (supplementary possibly Cdk2 phase, of S inhibition in delayed became 3306, ora fCl cec 21)17 3614 doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal m ncl utr eim niisCk usatal but substantially Cdk1 inhibits medium, culture cell in M E-d6VnsadCherry–PCNA and KEN-Cdc6–Venus QIF- a coexpressing of cell images representative Still (E) of dots. appearance PCNA the after dots hours PCNA 10 diffusing phase by G2 marked of is start the and dots, PCNA starts export , Nuclear C. in cell losplmnaymtra i.S5A. Fig. material See supplementary RO-3306. also inhibitor treated Cdk1 cells the in with prolonged is phase phase. S G2 arrows entering phase; cells S towards of point end the at towards cells point Arrowheads phase. S of end the at of prevented export is nuclear QIF-KEN-Cdc6 the that Note phase. 5 with treated and n nidvda el(447 cell individual an indicates dot Each Cherry–PCNA. and QIF-KEN-Cdc6–Venus cells coexpressing U2OS live in measured was QIF-KEN-Cdc6–Venus of export nuclear and dots the PCNA between of Time appearance (C) (B). of dots appearance PCNA the after minutes and 60 before minutes 60 measured was Relative QIF-Cdc6–Venus (A). of shown fluorescence are start from phase minutes S indicated the of images Still- microscopy. fluorescence and (DIC) contrast interference differential by imaged were Cherry–PCNA and cells QIF-Cdc6–Venus U2OS coexpressing Live border. G1-S the at re-accumulates phase. QIF-Cdc6–Venus S of at end Cdc6 controls the activity Cdk 6. Fig. 5 or fe h perneof appearance the after hours 6 ;mean 8; 6 ...()Siliae fa of images Still (D) s.d.). m m m O30,the RO-3306, M O30 nG1 in RO-3306 M O30 for RO-3306 M AB Nuclear (A,B) 6 1minutes; 61 m MRO-

Journal of Cell Science EERHARTICLE RESEARCH el iutnosyrdc h eeso d1adCc yan by Cdc6 and that Cdt1 show of we levels Here, the degraded. S reduce fully when simultaneously is licensing trigger cells Cdt1 to before available the be starts, at could phase re-accumulates thus in and synthesized, G1 Cdt1 when of However, end Cdc6, and Cdk2. that and Cdc6 E implies cyclin coexpress this of independent from that is cycle cells licensing cell fully mitosis, the cycling might entering not In Cdc6 cells is of of quiescence. feature synthesis Cdc6 particular Rapid another when 2013). order be in al., even threshold et crucial licensing, (Spencer a stabilized replication above Cdc6 initiate of might level to synthesis the raise Cdc6 to being growth-stimulus-induced that help possible of is from burst it is strong phase, Cdc6 G1 Cdc6 a a during that degraded and observed protects and quiescence we synthesized both Because from activity APC/C–Cdh1. emerge by cyclin-E–Cdk2 they recognized as of cycle cell wave by the start Cdc6 phase that regulates G1 in which conditions 2013). CUL4B, is al., specific describe et and of under (Zou we levels CRL4, role Cdk2 with evolutionarily pathway a modulating cooperates from The the typically different Cdc6. which previously within Cdt2, new of a requires a on motif N-terminus depends describe pathway conserved PIP-box-like we This Here, transition. specifically unidentified on G1-S 2006). switched the is Pines, that at Cdc6 1996; for mechanism 1999; al., al., degradation et APC/C–Cdh1 Lukas et the 2002; al., when et in Piatti phase (Hsu synthesized S is However, that, before Emi1 2000). mitosis, inhibitor APC/C–Cdh1 expressed al., again et by concept in is Petersen catalyzed 2013; Cdc6 is al., the peaks et Cdc6 (Clijsters of competence supports activation destruction rapid licensing here before cells, described proliferating work The DISCUSSION to helps DNA, the phas onto S loaded of is beginning PCNA when the CRL4–Cdt2-dependent phase, at S before first, before (n but duplicated right The cytoplasm; off Cdc6, are Cdc6. (c), switches of that of nucleus; degradation APC/C DNA export that (n), the of propose nuclear integrity. when We regions Cdc6, triggers genomic phase removed. those of to activity G1 been re-licensing levels threat Cdk1 has of of nuclear Cdt1 phase, this end and risk the APC/C– G2 reduce the Cdc6 at controls phase, to at of are CRL4–Cdt2 G1 phase pool accumulates cells phase, In entire S Cdc6 such S the yellow). from WT Potentially, in Cdc6, transition cells, started. whereas, of cells some has KEN-box, mutant when in degradation Cdc6 double Next, that, the QIF-KEN, PIP-box. indicates upon blue; Cdc6 line dependent Cdc6, the dashed is of upon which mutant dependent Cdc6, cycle. PIP-box is QIF, cell of which green; the degradation Cdc6, the throughout of controls Cdc6 mutant Cdh1 of KEN-box regulation KEN, the red; of Cdc6, model Schematic 7. Fig. 1adtesato ioi Fg )(s ta. 02 ua tal., et Lukas 2002; al., et (Hsu 7) (Fig. mitosis 1999). of late start the the between and subsequent, to prohibited G1 yet exported distinct, remains these is replication-licensing By B1. Cdc6 pathways, cyclin phase, of synthesis resulting Cdt2 G2 the possibly Cdk1, from during inactive, of activity accumulating and is the by phase APC/C cytoplasm S whereas, the onwards, late transition When G1-S of in the re-appearance phase. from Cdc6 the of S levels prevent reduces at to Cdc6 mechanism a nuclear provides Cdc6 of hssqec feet ih ecuilydfeeti cells in different crucially be might events of sequence This loehr ecnld htPPbxdpnetdestabilization PIP-box-dependent that conclude we Altogether, R ) ula export. nuclear c), elcto n aeur eoi tblt.In stability. genomic safeguard and replication any re-license cells that risk DNA. the formed reduce newly greatly of will start This the phase. before S immediately pathway degradation overlapping fti rcsl ie d6dgaaincnrlptwyawaits pathway control investigations. follow-up degradation a elaborate Cdc6 by more of timed role context studied precisely exact the this the best into of within insight are DNA proper Therefore, to Cdc6 organism. developing alterations in of analyses motif detailed PIP-box-like of effects replication inactivation the detrimental mutations, of Cdc6 DNA mutating control the of additional accumulation the the of of in to lead existence control defects small the cycle Because or cell mechanisms. Cdc6 to of non-degradable related a form of be inactivation Cdc6, material of could localization supplementary intracellular This (see yet S5B). cells not causes human Fig. Cdc6 have in of we re-replication stabilization abnormal DNA However, extent 1997). what to al., determined 2004). et over- (Jallepalli causes al., CDC18 replication ortholog Cdc6 et the of mutants (Mimura mutant phosphorylation In re-replication phosphorylation in 1997). DNA Similarly, CDC6 Stillman, induces and of strongly (Liang overexpression replication addition, DNA of initiation cerevisiae 1(urm n gm,20)adwscoe notepVenus-N1 the into cloned was and R54,L59A-Cdc6–GFP was 2005) ( Agami, construct vector KEN-Cdc6-GFP- and The from KEN-Cdc6–Venus KEN-Cdc6 (Duursma of The N1 amplification 2013). PCR 2005). by Agami, synthesized described al., previously described and been been has (Duursma et construct have S1) Fig. constructs material (supplementary (Clijsters Cdt1–Venus and elsewhere Cdc6–Venus The siRNA and Plasmids were: 10 used (#217699, Sigma, RO-3306 Drugs (#C6255, MG-132 inhibitor 5 inhibitor as 2013). Chemicals, proteasome Cayman Cdk1 were (#13697, indicated), al., as nM), imaging concentration 830 et Calbiochem, time-lapse (Sigma, (Clijsters and Nocodazole medium synchronization, cytometry previously Cell Eagle’s antibiotics. flow modified described and serum blotting, Dulbecco’s calf western in fetal 8% cultured with (Gibco) were cells U2OS synchronization and culture Cell METHODS AND MATERIALS Mlenu hraetcl,1 Pharmaceuticals, (Millennium h rti eeso ifrn d6mtnsaeson(T wild-type (WT, shown are mutants Cdc6 different of levels protein The lal,Cc eesms ergltdt rvn nieyDNA untimely prevent to regulated be must levels Cdc6 Clearly, Nhe ora fCl cec 21)17 3614 doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal h d63luiezpe uatsossrn re- strong shows mutant zipper leucine cdc6-3 the , I/ Xho cioacaoye pombe Schizosaccharomyces ) h I-d6VnsadQIF-KEN-Cdc6–Venus and QIF-Cdc6–Venus The I). m )adndyainihbtrMLN-4924 inhibitor neddylation and M) m m ) rnlto niio cycloheximide inhibitor translation M), M). vrxrsino a of overexpression , Saccharomyces ,before e, 1343

Journal of Cell Science bandfo hrao ssto oridvda ON-TARGET-plus were individual (DTL) four Cdt2 of and set (FZR1) as oligonucleotides. Dharmacon Cdh1 from (GMNN), obtained Geminin Cdt1, Cdc6, 3 and where osrcswr yteie yPRapiiainwt h primer the with amplification PCR A by 5 synthesized FWD#1: were constructs ARTICLE RESEARCH nioyaantCt n 20 and 1344 2 Cdt2 with incubated against were lysates antibody immunoprecipitations, For Immunoprecipitation in room performed at was nm, 579 data of equilibrium formula: CWL Pherastar the the a using with of BMG Prism filters Graphpad analysis emission of a (CWL) S The wavelength on and temperature. center P measured a and with nm, was filter 531 excitation on fluorescence 96-well an incubation with in minutes reaction fluorimeter 10 dilutions After the 1:1 triplicate. serial mM ice, in 1 Elmer), by (Perkin and diluted Optiplates subsequently NaCl TAMRA-labeled were mM nM ( mixtures 125 the 2 concentration at possible 7.5, added with practically pH was maximum PCNA out HEPES/HCl were Purified (TCEP). mM carried tris(2-carboxyethyl)phosphine 50 sequences). was peptides in for carboxytetramethylrhodamine S1 reaction peptides, Table Cdc6 C-terminal size-exclusion binding material or supplementary The mutated and (see N- label an (TAMRA) and chromatography with synthesized Wild-type affinity (DE3) Bl21 chromatography. metal in expressed immobilized was PCNA Human assay anisotropy Fluorescence-polarization 0.5-2.0 with transfected 20 were or dishes plasmid Falcon 9-cm in Cells Transfection CATTTAGAGAGCTA-3 pLIB- plasmids 5 ( PCR pVenus-N1 FWD by primer synthesized GTCTCGGGCATTGAACAAAGC-3 the was S3B) with ( Fig. amplification material pVenus-N1 (supplementary into construct cloned and CGCATCGCGCCGCCCAAGCTG-3 ovre to converged nstoyraot and readout) anisotropy aybtendtst ae na xr u fsursFtest; F squares of sum extra an to on allowed based were datasets parameters between fitted vary three All analysis. regression nonlinear n eetdwt uoyi o 4husa niae,o ih40 with or indicated, as hours 24 for puromycin pBABE-Puro ug with 1 with selected co-transfected and were Cells protocol. transfection rnfce ihvrlpamd sn h tnadcalcium-phosphate standard were the cells using Phoenix plasmids (Invitrogen) protocol. Ecotropic transfection viral 2000 protocol. with Lipofectamine manufacturer’s transfected using to oligonucleotides according siRNA nM 3 ( pLIB-N1 into cloned were ( pLIB-Venus-N1 (primer into cloned amplification were PCR and by FWD#1) constructed were QIF-KEN-Cdc6-Venus ( pLIB-C1 into cloned F-1(urm n gm,20)adcoigit pLIB-Venus-N1 into cloning KEN-Cdc6- and from 2005) ( KEN-Cdc6 Agami, pLIB-KEN-Cdc6-Venus and of 2013). (Duursma amplification GFP-N1 PCR al., by et constructed was (Clijsters described previously Xho 9 9 GCGGCTACA h eune falpamd eevrfe.TesRA otarget to siRNAs The verified. were plasmids all of sequences The . T fCc n aetetre-eune d65 5 Cdc6#5: target-sequences the have and Cdc6 of UTR h Sprpamd SCc# n SCc# ohtre the target both pS-Cdc6#6 and pS-Cdc6#5 plasmids pSuper The Cherry–PCNA comprises pLIB-Cherry-PCNA plasmid viral The c I/ h CAconcentration; PCNA the Bam Pc ðÞ I.Tevrlpamd LBQFCc-eu n pLIB- and pLIB-QIF-Cdc6-Venus plasmids viral The HI). 9 steplrzto edn taPN ocnrto of concentration PCNA a at reading polarization the is -ATATCTCGAGACCATGCCTCAAACCCGATCCCAGGC- Xho . m .9fralcases. all for 0.99 Sprpamd sn h tnadcalcium-phosphate standard the using plasmids pSuper g D GCCAGT I/ -8Cc-eu n pLIB- and 2-18-Cdc6-Venus Bam HI). 9 Age d66 5 Cdc6#6: ; Pc GCTCCAAAAAGGAAGCCCGGGCCCCCC- K ðÞ Xho d I/ ~ bnigcntn)weeetmtdby estimated where constant) (binding P Bam Xho I/ 0 P Not and 0 z I.pI-d6Vnshsbeen has pLIB-Cdc6-Venus HI). I/ 9 9 I). Bam m 9 P mttdcdn r underlined) are codons (mutated K P -GCCAATGTGCTTGCAAGTG- n a lndit h vector the into cloned was and rti-–ehrs beads protein-G–Sepharose g max d 9 max -ATATCTCGAGACCATGCT- z I.The HI). c temnmmadmaximum and minimum (the c , , D 90 .coli E. Xho 2-18-KEN-Cdc6-Venus m )adtereaction the and M) I/ D Bam 2-18-Cdc6–Venus n uiidby purified and m I.Teviral The HI). expression g 9 -GCTGG- m R gofan 2 values c nM i,D . uhvrp,V . err,C . a,H . i,Y .adYew, and S. Y. Kim, W., H. Nam, R., C. Herrera, N., V. Budhavarapu, H., D. Kim, i,J,Ais .E,Ce,J,Hre,J .adWle,J C. J. Walter, and W. J. Harper, J., Chen, E., E. Arias, T. J., Jin, Hunter, and J. N. Wells, W., Jiang, J. T. K. Kelly, P. and D. Jackson, Tien, M., and Muzi-Falconi, W., J. G. Lukas, Brown, V., P. S., Jallepalli, C. Sørensen, D., J. Reimann, Y., J. Hsu, Aesa isine)fr1 or t4 at hours 16 for Biosciences) (Amersham h uhr elr ocmeiginterests. competing no declare authors The interests Competing the reading critically for Cdc6 Boekhout the Michiel synthesizing and for Voets manuscript. Atmioui Erik el thank Dris We and peptides. Hilkmann Henk Hibbert thank Rick the We and performing Heidebrecht and Tatjana designing Perrakis, Tassos for to grateful are We 1:40; Abcam). Acknowledgements mouse, 1:1000; were (ab49626, indicated antibodies Bu20a, as FITC-conjugated or clone DAKO, 1:1000; or from (M0744, obtained peroxidase- rabbit, Secondary (#A300-948A, BrdU DAKO). (ab70829, DTL/CDT2 Laboratories), Cdt1 Cruz), Abcam), Bethyl Santa 1:1000; 1:1000; Santa (#3092, goat, 1:1000; rabbit, sc-1616, rabbit, A/AIK (I-19, (FL-209, Aurora Actin geminin Cruz), Cruz), Signaling), Cdc6 Santa Cell Transduction), 1:1000; 1:1000; BD rabbit, mouse, indicated 1:1000; the sc-9964, mouse, at (180.2, used (#610455, were CDC27 proteins following dilutions: the against antibodies The Antibodies aes .G,Sonm . urn,E,Cnoe .C,Zu . Kearsey, L., Zou, C., R. Centore, E., Guarino, N., Shobnam, G., C. Havens, C. J. Walter, and G. C. Havens, R. Agami, and A. Duursma, R. Wolthuis, and J. A. Ogink, Castro, L., Clijsters, and T. Lorca, A., Burgess, M. Pagano, and R. Eichner, F., Bassermann, Helin, and F. Marinoni, L., Masiero, R., Zamponi, M., Melixetian, A., Ballabeni, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.145862/-/DC1 online available material Supplementary material Supplementary Program Science for Frontier R.W.]. Organisation Human to Netherlands the RGP0053/2010 the and number R.W.; from [grant to Grant 2007- Vidi (NWO) KWF a Research number R.W.]; Scientific [grant and Society L.C. Cancer Dutch to the 3789 by supported was work This Funding and experiments the designed R.W. manuscript. and the L.C. wrote experiments. the performed L.C. contributions Author ebaebokn n nioyicbto,4 L vnZne al., et Zon (van ELK used. 4% For was incubation, membranes. 2010) antibody nitrocellulose and on blocking blotted 50 membrane and in Zon SDS-PAGE dissolved (van by ELB+ and separated ice-cold 2010) with al., times four et washed were beads incubation, .R. P. ies u4Db-neatn rtisicue d2 hc srqie o S for Cdt1. required factor is replication the which of Cdt2, destruction includes phase proteins Cul4-Ddb1-interacting diverse HsCdc6. of 6193-6198. phosphorylation Cdk by replication phosphorylation. CDK by p65cdc18 Dev. protein by Genes initiator replication entry the of phase Regulation S regulates hEmi1 of APC(Cdh1). inhibiting accumulation E2F-dependent (2002). 11410-11421. usrt eonto yteE bqii iaeCRL4Cdt2. ligase ubiquitin E3 the by recognition C. substrate J. Walter, CRL4Cdt2. and E. ligase S. ubiquitin the for degron Cell a Mol. creates PCNA chromatin-bound S to proliferation. mitosis cellular connecting controls stability in roles distinct play phase. APC/C(Cdh1) and C(Cdc20), cells. mammalian in replication DNA cancer.endogenous of treatment targeted the and Acta control Biophys. cycle cell Biochim. for Implications - system mitosis. in CDT1 stabilizing K. 20) ua eii rmtspeR omto n N elcto by replication DNA and formation pre-RC promotes geminin Human (2004). 21) h R4d2uiutnlgs eitstepoelssof proteolysis the mediates ligase ubiquitin CRL4Cdt2 The (2010). ora fCl cec 21)17 3614 doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal .Cl Biol. Cell J. 35 93-104. , 11 2767-2779. , 201 a.Cl Biol. Cell Nat. 21) ietrl o rlfrtn elnceratgnin antigen nuclear cell proliferating for role Direct (2012). 1013-1026. , 843 150-162. , MOJ. EMBO 20) 5-eedn euaino d6protein Cdc6 of regulation p53-Dependent (2005). nvitro in 20) okn faseilzdPPBxonto Box PIP specialized a of Docking (2009). m 4 o.Cl.Biol. Cell. Mol. 23 apebfe.Tepoen were proteins The buffer. sample l 358-366. , CACc neato experiments. interaction PCNA–Cdc6 3122-3132. , 21) h pnl hcpit APC/ checkpoint, spindle The (2013). 19) utse euaino DNA of regulation Multistep (1999). 21) uniaielv mgn of imaging live Quantitative (2012). 21) h bqii proteasome ubiquitin The (2014). rc al cd c.USA Sci. Acad. Natl. Proc. o.Cell Mol. ˚ ,wietmln.After tumbling. while C, LSONE PLoS 25 6937-6947. , 23 .Bo.Chem. Biol. J. 20) aiyof family A (2006). 709-721. , 7 e45726. , (1997). 287 96 , ,

Journal of Cell Science odvn .L,Padr .adJnsh S. Jentsch, and B. F. Pfander, L., J. G. Diffley, Moldovan, and S. Tanaka, T., Seki, S., Mimura, and Y. Higuchi, Y., Shiomi, H., Komori, T., Ishii, A., Morimoto, M., Michishita, M. Oda, and N. Yoshizawa-Sugata, Z., You, S., Matsumoto, H., Masai, F. J. Diffley, and N. Mailand, Ma,H.T.,Tsang,Y.H.,Marxer,M.andPoon,R.Y. EERHARTICLE RESEARCH ua,C,Srne,C . rmr . atn-ui,E,Lnee,C., Lindeneg, E., Santoni-Rugiu, E., Kramer, S., C. Sørensen, C., X. Lukas, Wu, and Q. Zhao, F., Yan, X., Li, E., A. Liu, Dutta, and U. Narayanan, G., P. Smith, A., M. Milhollen, J., J. Lin, B. Stillman, and C. Liang, eedn idn fmttcccist d6cnrbtst N replication DNA to fork. replication contributes Cdc6 to cyclins mitotic control. of binding dependent CRL4(Cdt2) for important are box, PIP within proteolysis. acids -mediated amino TD with together box, H. Nishitani, how? and when, where, Biochem. replication: DNA chromosome Eukaryotic proteolysis. APC/C-dependent from Cdc6 protecting Cell by cells human in licensing iae2coeae ihtePLK1-SCFbeta-TrCP1-EMI1-anaphase-promoting Biol. Cell. the Mol. genom promote with to axis cooperates complex/cyclosome 2 kinase ees .M,Bre,J n ua,J. Lukas, anaphase-promoting the and complex. of rearrangement J. cyclin-A-dependent and Bartek, E2F requires M., J. Peters, degradation. its induce and 17283-17288. Cdt1 human phosphorylate cancer in senescence and in apoptosis, Cdt1 activation, cells. stabilizing checkpoint by triggering rereplication phase, DNA S elicits MLN4924 drug mutants. NEDD8-targeting cdc6 in cycle cell the during Dev. proteins MCM chromatin-bound PCNA. with association direct Biol. a Cell. through Mol. Xic1 inhibitor kinase cyclin-dependent 122 11 acrRes. Cancer 3375-3386. , Nature 915-926. , Nature 79 89-130. , 21) oiieycagdrsde oae ontemo PIP of downstream located residues charged Positively (2011). 29 30 431 Cell 6500-6514. , 401 4120-4133. , 70 1118-1123. , 815-818. , 129 10310-10320. , ee Cells Genes 665-679. , 19) essetiiito fDArpiainand replication DNA of initiation Persistent (1997). 20) DspooeDArpiainorigin replication DNA promote CDKs (2005). 16 12-22. , eulcto nteasneo mitosis. of absence the in reduplication e 19) cuuaino ylnB1 cyclin of Accumulation (1999). 20) ylndpnetkinases Cyclin-dependent (2004). 20) CA h asr fthe of maestro the PCNA, (2007). 20) ylnA2-cyclin-dependent Cyclin (2009). 20) Phosphorylation- (2004). .Bo.Chem. Biol. J. nu Rev. Annu. (2010). (2010). Genes 279 , iti . Bo M., S., Melixetian, Piatti, R., E. Kramer, F., Marinoni, C., Wagener, O., B. Petersen, eesn .O,Lks . øesn .S,Bre,J n ei,K. Helin, and J. Bartek, S., C. Sørensen, J., Lukas, O., B. Petersen, C., Obuse, M., Saijo, Y., Nakanishi, V., Roukos, N., Sugimoto, H., Nishitani, o,Y,M,J,Wn,W,L,J,Za,W,Lu . u . ag . a,X., Gao, Y., Yang, H., Hu, Z., Liu, W., Zhao, J., Lu, W., Wang, Wolthuis, J., and Mi, H. Y., Riele, Zou, te H., R. Medema, B., Riet, ter J., Ogink, W., Zon, van aslv .T,Tvr . hn . nzvc . ho . u,H,Heimbrook, H., and Sun, L. X., Zhao, C. D., Knezevic, Wang, S., W., Chen, C., K. Tovar, T., Overton, L. Vassilev, C., F. Tsai, D., S. Cappell, L., S. Spencer, J. Pines, J. Pines, ae .adKrcnr .W. M. Kirschner, and M. Rape, fe hc d6snhsscno rmt N elcto nyeast. in replication return’’ DNA no promote of cannot ‘‘point a synthesis defines Cdc6 G1 late which in after CDKs S-phase-promoting of Activation is K. CDC6 Helin, mammalian and of APC-CDH1. M. proteolysis on J. growth-regulated dependent Peters, cell and C., cycle- Matteucci, Cell C., (2000). Gieffers, E., Denchi, Lazzerini hshrlto fmmainCC ycci /D2rgltsissubcellular its regulates A/CDK2 cyclin localization. by CDC6 mammalian of Phosphorylation proteolysis. for Cdt1 human J. target al. EMBO DDB1-Cul4, et and M. SCF-Skp2 Fujita, ligases, K., ubiquitin Nakayama, I., K. Nakayama, T., Tsurimoto, in,B tal. et B. exit. Jiang, mitotic control to recognition box D al. before et . F M. R. 10665. h D2CC cascade. CDK2-CDC6 the ioi ucin fhmnCDK1. human of L. functions Chen, mitotic and C. D. exit. mitotic at activity CDK2 in bifurcation T. Meyer, 588-595. Biol. Cell Trends Dev. npaepooigcmlxculsmtsst -hs entry. S-phase to mitosis couples complex anaphase-promoting Biol. Cell Mol. Rev. 10 ora fCl cec 21)17 3614 doi:10.1242/jcs.145862 1336–1345 127, (2014) Science Cell of Journal 20) ioi:amte fgtigrdo h ih rti ttergttime. right the at protein right the of rid getting of matter a Mitosis: (2006). 21) uimadtecl yl:temn ae fteAPC/C. the of faces many the cycle: cell the and Cubism (2011). 1516-1531. , 25 m . okr .H,Dfly .F n amt,K. Nasmyth, and F. J. Diffley, H., J. Cocker, T., hm, ¨ 21) h rlfrto-uecnedcso scnrle ya by controlled is decision proliferation-quiescence The (2013). 1126-1136. , MOJ. EMBO 21) h P/ eriscci 1d1k nprometaphase in B1Cdk1Cks cyclin recruits APC/C The (2010). 21) U4 rmtsrpiainlcnigb up-regulating by licensing replication promotes CUL4B (2013). 16 55-63. , 12 20) eetv ml-oeueihbtrrvascritical reveals inhibitor small-molecule Selective (2006). 18 427-438. , 396-410. , .Cl Biol. Cell J. ee Dev. Genes rc al cd c.USA Sci. Acad. Natl. Proc. 20) uooosrglto fthe of regulation Autonomous (2004). 200 14 Cell 2330-2343. , 743-756. , 155 .Cl Biol. Cell J. 369-383. , 190 20) w E3 Two (2006). 587-602. , 103 Nature 10660- , (1996). (1999). Genes 1345 432 Nat. ,

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