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- 3. OVERVIEWOFTHECELLCYCLE 1055 cytokinesis mitosis metaphase-to-anaphasetransition INTERPHASE DNAreplic chromosomes are packaged into separate nuclei at telophase.Cltokinesis then cleaves the cell in two, so that each daughter cell inherits one of the two nuclei (Figure l7-3). <TACT><TCAA> Most cells require much more time to grow and double their mass of pro- teins and organelles than they require to duplicate their chromosomes and divide. Partly to allow more time for growth, most cell cycles have exrra gap phases-a G1phase between M phase and S phase and a Gz phase between S phase and mitosis. Thus, the eucaryotic cell cycle is traditionally divided into four sequential phases:Gr, S,G2,and M. Gr, S,and G2together are called inter- phase (Figure l7-4, and seeFigure 17-3).In a typical human cell proliferating in culture, interphase might occupy 23 hours of a 24-hour cycle,with I hour for M phase.Cell growth occurs throughout the cell cycle,exceptduring mitosis. The two gap phases are more than simple time delays to allow cell growth. They also provide time for the cell to monitor the internal and external environ- ment to ensure that conditions are suitable and preparations are complete before the cell commits itself to the major upheavalsof Sphaseand mitosis.The G1 phase is especially important in this respect. Its length can vary greatly depending on external conditions and extracellular signalsfrom other cells. If extracellular conditions are unfavorable, for example, cells delay progress through G1and may even enter a specializedresting stateknown as Go(Gzero), in which they can remain for days,weeks,or even yearsbefore resuming prolif- eration. Indeed, many cellsremain permanently in Geuntil they or the organism dies. If extracellular conditions are favorable and signals to grow and divide are present, cells in early G1or G0progressthrough a commitment point near the end of G1 knorvn as Start (in yeasts) or the restriction point (in mammalian cells).We will use the term Start for both yeast and animal cells.After passing this point, cells are committed to DNA replication, even if the extracellular sig- nals that stimulate cell growth and division are removed. M PHASE mitosis (nuclear division) G2PHASE 5 PHASE Figure17-3 Theeventsofeucaryotic celldivisionasseenundera microscope,Theeasilyvisibleprocesses of nucleardivision(mitosis)andcell division(cytokinesis),collectivelycalled M phase,typicallyoccupyonlya small fractionof the cellcycle.Theother,much longer,part of the cycleisknownas interphase,whichincludesSphaseand the gap phases(discussedin text).The fivestagesof mitosisareshown:an abruptchangein the biochemicalstate of the celloccursat the transitionfrom metaphaseto anaphase.A cellcanpause in metaphasebeforethistransitionpoint, but onceit passesthispoint,the cell carrieson to the endof mitosisand throughcytokinesisinto interphase. Figure17-4 Thefour phasesof the cell cycle.In mostcells,gap phasesseparate the majoreventsof 5 phaseand M phase' Gristhe gapbetweenM Phaseand 5 phase,while G2isthe gaPbetween SphaseandM phase. cytokinesis (cytoplasmic (DNAreplication) G1PHASE
- 4. 1056 Chapter17:TheCellCycle Cell-CycleControllsSimilarinAllEucaryotes Some features of the cell cycle, including the time required to complete certain events, vary greatly from one cell type to another, even in the same organism. The basic organization of the cycle, however, is essentially the same in all eucaryotic cells, and all eucaryotes appear to use similar machinery and control mechanisms to drive and regulate cell-cycle events. The proteins of the cell- cycle control system, for example, first appeared over a billion years ago. Remarkably, they have been so well conserved over the course of evolution that many of them function perfectly when transferred from a human cell to a yeast cell. we can therefore study the cell cycle and its regulation in a variety of organ- isms and use the findings from all of them to assemblea unified picture of how eucaryotic cells divide. In the rest of this section, we briefly review the three eucaryotic systemsmost commonly used to study cell-cycle organization and control: yeasts,animal embryos, and cultured mammalian cells. Cell-CycleControlCanBeDissectedGeneticallybyAnalysisof YeastMutants Yeastsare tiny, single-celled fungi, with a cell-cycle control system remarkably similar to our own. TWospeciesare generally used in studies of the cell cycle.The fission yeast schizosaccharomycespombe is named after the African beer it is used to produce. It is a rod-shaped cell that growsby elongation at its ends.Divi- sion occurs when a septum, or cell plate, forms midway along the rod (Figure r7-5A). The budding yeast Saccharomycescereuisiaeis used by both brewers and bakers. It is an oval cell that divides by forming a bud, which first appears during G1and grows steadily until it separatesfrom the mother cell aftei mito- sis (Figure l7-58). gene,becausewe avoid the complication of having a second copy of the genein the cell. Many important discoveriesabout cell-cycle control have come from sys- tematic searchesfor mutations in yeaststhat inactivate genesencoding essen- tial components of the cell-cycle control system.The genesaffectedby iome of YEAST(Schhosaccharomycespom be) Gr ISTART YEAST(5accharomycescerevisiae) Figure17-5 A comparisonof the cell cyclesof fissionyeastsand budding yeasts.(A)Thefissionyeasthasa typical eucaryoticcellcyclewith Gr,5,G2,and M phases.Thenuclearenvelopeof the yeastcell,unlikethatof a higher eucaryoticcell,doesnot breakdown duringM phase.Themicrotubulesof the mitoticspindle(lightgreen)forminside the nucleusandareattachedto spindle polebodies(darkgreen)at its periphery. Thecelldividesbyforminga partition (knownasthecellplate)andsplittingin two.(B)ThebuddingyeasthasnormalG1 andSphasesbut doesnot havea normal G2phase.Instead,a microtubule-based spindlebeginsto form latein Sphase;as in fissionyeasts,the nuclearenvelope remainsintactduringmitosis,andthe spindleformswithinthe nucleus.ln contrastwith a fissionyeastcell,the cell dividesby budding. G r @
- 9. THECELL-CYCLECONTROLSYSTEM lsall DNAreplicated? lsenvironmentfavorable? G2lMCHECKPOINT Are all chromosomes attachedto the spindle? METAPHASE-TO-ANAPHASE TRANSITION 1061 Figure'17-14Thecontrolof the cell cycle.A cell-cyclecontrolsystemtriggers the essentialprocessesof thecellcycle- suchasDNAreplication,mitosis,and cytokinesis.Thecontrolsystemis representedhereasa centralarm-the controller-that rotatesclockwise, triggeringessentialprocesseswhenit reachesspecificcheckpointson theouter dial.Informationaboutthecompletionof cell-cycleevents,aswellassignalsfrom theenvironment,cancausethe control systemto arrestthe cycleat these checkpoints.ThemostProminent checkDointsoccurat locationsmarked with vellowboxes. STARTCHECKPOINT lsenvironmentfavorable? in these cells is independent of the eventsit controls, so that its timing mecha- nisms continue to operate even if those eventsfail. In most cells,however,the control systemdoesrespond to information receivedback from the processesit controls. Sensors,for example, detect the completion of DNA synthesis,and if some malfunction prevents the successfulcompletion of this process,signals are sent to the control systemto delayprogressionto M phase.Suchdelayspro- vide time for the machinery to be repaired and also prevent the disaster that might result if the cycle progressedprematurely to the next stage-and segre- gated incompletely replicated chromosomes,for example. The cell-cyclecontrol systemis based on a connected seriesof biochemical switches, each of which initiates a specific cell-cycle event. This system of switchespossessesmany important engineeringfeaturesthat increasethe accu- racy and reliability of cell-cycle progression. First, the switches are generally binary (on/off) and launch eventsin a complete, irreversible fashion. It would clearly be disastrous,for example, if events like chromosome condensation or nuclear envelope breakdor.trnwere only partially initiated or started but not completed. Second,the cell-cycle control system is remarkably robust and reli- able,partly becausebackup mechanisms and other featuresallow the systemto operate effectivelyunder a variety of conditions and even if some components fail. Finally, the control system is highly adaptable and can be modified to suit specific cell types or to respond to specific intracellular or extracellularsignals. In most eucaryotic cells,the cell-cyclecontrol systemtriggerscell-cyclepro- gression at three major regulatory transitions, or checkpoints (see Figure I7-I4).The first checkpoint is Start (or the restriction point) in late Gr,where the cell commits to cell-cycle entry and chromosome duplication, as mentioned earlier.The second is the GzlM checkpoint, where the control system triggers the early mitotic events that lead to chromosome alignment on the spindle in metaphase.The third is the metaphase-to-anaphase transition, where the con- trol systemstimulates sister-chromatid separation,leading to the completion of mitosis and cytokinesis.The control systemblocks progressionthrough each of these checkpoints if it detectsproblems inside or outside the cell. If the control system sensesproblems in the completion of DNA replication, for example, it will hold the cell at the G2iM checkpoint until those problems are solved.Simi- larly, if extracellular conditions are not appropriate for cell proliferation, the control system blocks progression through Start, thereby preventing cell divi- sion until conditions become favorable. TRIGGERANAPHASEAND PROCEEDTO CYTOKINESIS ENTERCELLCYCLEAND PROCEEDTO S PHASE
- 10. 1062 Chapter17:TheCellCycle TheCell-CycleControlSystemDependson CyclicallyActivated Cyclin-DependentProteinKinases(Cdks) Central components of the cell-cyclecontrol systemare members of a family of protein kinases knor,rmas cyclin-dependent kinases (Cdks). The activities of these kinases rise and fall as the cell progressesthrough the cycle, leading to cyclical changesin the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle. An increase in Cdk activity at the G2lM checkpoint, for example, increasesthe phosphorylation of proteins that control chromosome condensation, nuclear envelope breakdown, spindle assembly,and other eventsthat occur at the onset of mitosis. Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other proteins that regulatethese kinases.The most important of theseCdk regulatorsareproteins known ascyclins. Cdks,astheir name implies, are dependent on cyclins for their activity: unless they are tightly bound to a cyclin, they have no protein kinase activity (Figure f7-f 5). Cyclins were origi- nally named becausethey undergo a cycle of syrthesis and degradation in each cell cycle.The levelsof the cdk proteins, by contrast, are constant, at leastin the simplest cell cycles.Cyclical changesin cyclin protein levelsresult in the cyclic assembly and activation of the cyclin-cdk complexes; this activation in turn triggerscell-cycle events. There are four classesofcyclins, each defined by the stageofthe cell cycleat which they bind cdks and function. All eucaryotic cells require three of these classes(Figure l7-f 6): l. G1/S-cyclinsactivate Cdks in late Gr and thereby help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels fall in Sphase. 2. S-cyclins bind Cdks soon after progressionthrough Start and help stimu- late chromosome duplication. S-cyclin levels remain elevateduntil mito- sis, and these cyclins also contribute to the control of some early mitotic events. 3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2lM checkpoint. Mechanisms that we discusslater destroy M-cyclins in mid- mitosis. In most cells,a fourth classof cyclins,the Gl-cyclins, helps govern the activities of the Gr/S cyclins,which control progressionthrough Start in late G1. In yeastcells,a singlecdk protein binds all classesof cyclinsand triggersdif- ferent cell-cycle events by changing cyclin partners at different stages of the cycle.In vertebrate cells,by contrast, there are four cdks. TWointeract with Gr- cyclins,one with G1/S-and S-cyclins,and onewith M-cyclins. In this chapter,we simply refer to the different cyclin-Cdk complexesasG1-Cdk,Gr/S-Cdk, S-Cdk, and M-Cdk. Thble l7-l lists the names of the individual Cdks and cyclins. How do different cyclin-cdk complexes trigger different cell-cycle events? The answer,at least in part, seemsto be that the cyclin protein does not simply activateits cdk partner but also directs it to specific target proteins. As a result, ' , , G r t M i_-metaphase-anaphase i M G 1 APC/C cyclin-dependent kinase(Cdk) Figure17-15Twokeycomponentsof the cell-cyclecontrolsystem.When cyclinformsa complexwith Cdk,the proteinkinaseisactivatedto trigger specificcell-cycleevents.Withoutcyclin, Cdkisinactive. Figure17-16 Cyclin-Cdkcomplexesof the cell-cyclecontrol system.The concentrationsof the threemajorcyclin typesoscillateduringthecellcycle,while the concentrationsof Cdks(not shown) do not changeandexceedtheamounts of cyclins.In lateG1,risingG1lS-cyclin levelsleadto the formationof G1lS-Cdk complexesthattriggerprogression throughthe Startcheckpoint.S-Cdk complexesform at the startof Sphase andtriggerDNAreplication,aswellas someearlymitoticevents.M-Cdk complexesformduringG2but areheldin an inactivestateby mechanismswe describelater.Thesecomplexesare activatedat theendof G2andtriggerthe earlyeventsof mitosis.A separate regulatoryprotein,the APC/C,whichwe discusslater,initiatesthe metaphase-to- anaphasetransition. G,iS-cyclin Gr/s-Cdk S-Cdk
- 11. THECELL.CYCLECONTROLSYSTEM Table17-1TheMajorCyclinsandCdksofVertebratesandBuddingYeast G1-Cdk GrlS-Cdk 5-Cdk M-Cdk cyclinD* Cdk4Cdk6 cyclinE Cdkz cyclinA Cdk2,Cdkl** cyclinB Cdkl Cln3 Cln1,2 clbs,6 clb1,2,3,4 cdkl"* cdkl cdkl cdkl " TherearethreeD cyclinsin mammals(cyclinsD1,D2,andD3) **Theoriginalnameof CdklwasCdc2in bothvertebratesandflssionyeast,andCdc2Bin buddingyeast each cyclin-Cdk complex phosphorylates a different set of substrate proteins. The same cyclin-Cdk complex can also induce different effects at different times in the cycle,probably becausethe accessibilityof some Cdk substrateschanges during the cell cycle.Certain proteins that function in mitosis, for example,may become availablefor phosphorylation only in G2. Studiesof the three-dimensional structuresof Cdk and cyclin proteins have revealed that, in the absence of cyclin, the active site in the Cdk protein is partly obscured by a slab of protein, like a stone blocking the entrance to a cave (Figure l7-L7A). Cyclin binding causesthe slab to move away from the active site,result- ing in partial activation of the Cdk enz)ryne(Figure l7-l7B). Full activation of the cyclin-Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active site. This causesa small conformational change that further increasesthe activ- ity of the Cdk, allowing the kinase to phosphorylate its target proteins effectively and thereby induce specific cell-cycle events (Figure I7-I7C). <TAGA> InhibitoryPhosphorylationandCdkInhibitoryProteins(CKls)Can SuppressCdkActivity The rise and fall of cyclin levels is the primary determinant of Cdk activity dur- ing the cell cycle. Severaladditional mechanisms, however, fine-tune Cdk activ- ity at specific stagesofthe cycle. Phosphorylation at a pair of amino acidsin the roof of the kinase active site inhibits the activity of a cyclin-Cdk complex. Phosphorylation of thesesitesby a protein kinase knor,vnasWeel inhibits Cdk activity, while dephosphorylation of these sites by a phosphatase knor,rmas Cdc25 increases Cdk activity (Figure 17-18). We will seelater that this regulatory mechanism is particularly impor- tant in the control of M-Cdk activity at the onset of mitosis. Binding of Cdk inhibitor proteins (CKIs) also regulates cyclin-Cdk com- plexes.The three-dimensional structure of a cyclin-Cdk-CKl complex reveals activatingphosphate (c) Cdk-activatingkinase(CAK)cyclin 1063 Figure17-17Thestructuralbasisof Cdk activation.Thesedrawingsarebasedon three-dimensionalstructuresof human Cdk2,asdeterminedby x-ray crystallography.Thelocationof the bound ATPisindicated.Theenzymeisshownin threestates.(A)Inthe inactivestate,without cyclinbound,the activesiteisblockedby a regionof the proteincalledtheT-loop(red). (B)Thebindingof cyclincausestheT-loopto moveout of the activesite,resultingin partialactivationof the Cdk2.(C) Phosphorylationof Cdk2(byCAK)at a threonineresiduein theT-loopfurther activatesthe enzymebychangingtheshape of theT-loop,improvingthe abilityof the enzymeto bind itsproteinsubstrates. activesite (A) TNACTTVE (B) PARTLYACTIVE
- 12. 1064 Chapter17:TheCellCycle that CKI binding stimulates a large rearrangement in the structure of the Cdk active site,rendering it inactive (Figure f 7-fg). Cellsuse CKIs primarily to help govern the activities of Gr/S- and S-Cdksearly in the cell cycle. TheCell-CycleControlSystemDependson CyclicalProteolysis fhereas activation of specificcyclin-Cdk complexesdrives progressionthrough the Start and G2lM checkpoints (see Figure 17-16), progression through the metaphase-to-anaphasetransition is triggered not by protein phosphorylation but by protein destruction, leading to the final stagesof cell division. The key regulator of the metaphase-to-anaphasetransition is the anaphase- promoting complex, or cyclosome (APC/C),a member of the ubiquitin ligase family of enzymes.As discussedin Chapter 3, many of these enzyrnesare used in numerous cell processesto stimulate the proteolytic destruction of specific regulatory proteins.They transfer multiple copiesof the small protein ubiquitin to specific target proteins, resulting in their proteolytic destruction by the pro- teasomes. Other ubiquitin ligases mark proteins for purposes other than destruction. The APC/C catalyzesthe ubiquitylation and destruction of two major pro- teins.The first is securln,which normally protects the protein linkagesthat hold sister chromatid pairs together in early mitosis. Destruction of securin at the metaphase-to-anaphasetransition activatesa proteasethat separatesthe sisters and unleashesanaphase.The S- and M-cyclins are the second major targetsof the APCic. Destroying thesecyclins inactivatesmost cdks in the cell (seeFigure 17-16).As a result, the many proteins phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by various phosphatasesthat are present in the anaphase cell. This dephosphorylation of Cdk targets is required for the completion of M phase,including the final steps in mitosis and the processof cytokinesis.Following its activation in mid-mitosis, the APC/c remains activein G1,thereby providing a stableperiod of Cdk inactivity.MhenG1/S-Cdksare acti- vated in late Gr, the APC/G is turned off, thereby allowing cyclin accumulation to start the next cell cycle. The cell-cycle control system also uses another ubiquitin ligase called SCF (after the names of its three subunits). It ubiquitylates certain cKI proteins in late G1and thereby helps control the activation of S-cdks and DNA replication. The APC/C and SCF are both large, multisubunit complexes with some related components, but they are regulated differently. APC/C activity changes during the cell cycle,primarily as a result of changesin its associationwith an activating subunit-either cdc20 during anaphase or cdhl from late mitosis through early G1.These subunits help the APC/G recognize its target proteins (Figure l7-2oL). SCFactivity also depends on subunits called F-box proteins, which help the complex recognize its target proteins. unlike Apc/c activity, however, scF activity is constant during the cell cycle. ubiquitylation by scF is controlled instead by changesin the phosphorylation stateof its target proteins, as F-box subunits recognize only specifically phosphorylated proteins (Figure I7-20H. actrve cyclin-Cdk complex inactive p27-cyclin-Cdk comprex Cdk activating phosphate INACTIVE Figure17-18Theregulationof Cdk activity by inhibitory phosphorylation. Theactivecyclin-Cdkcomplexisturned off when the kinaseWeel phosphorylates two closelyspacedsitesabovethe active site.Removalof thesephosphatesby the phosphataseCdc25activatesthe cyclin-Cdkcomplex.Forsimplicity,only oneinhibitoryphosphateisshown.CAK addstheactivatingphosphate,asshown in Figure17-17. Figure17-19Theinhibitionof a cyclin-Cdkcomplexby a CKl.This drawingisbasedon thethree- dimensionalstructureof the human cyclinA-Cdk2complexboundto the CKIp27,asdeterminedby x-ray crystallography.Thep27bindsto both thecyclinandCdkin the complex, distortingthe activesiteof the Cdk.lt alsoinsertsintotheATP-bindingsite, furtherinhibitingtheenzymeactivity.
- 15. 5 PHASE Summary Thecell-cyclecontrol systemtriggersthe euentsof the cell cycleand ensuresthat these euentsare properly timed and occur in the correctorder Thecontrol systemresponds to uarious intracellular and extracellular signalsand arreststhe cyclewhen the cell eitherfails to completean essentialcell-cycleprocessor encountersunfauorableenui- ronmentalor intracellularconditions. Central componentsof the cell-cyclecontrol systemare cyclin-dependentprotein kinases(Cdks),which dependon cyclin subunitsfor their actiuity. Oscillationsin the actiuitiesof uariouscyclin-Cdk complexescontrol uariouscell-cycleeuents.Thus,acti- uation of S-phasecyclin-Cdk complexes(S-Cdk)initiates Sphase,while actiuation of M-phase cyclin-Cdk complexes(M-Cdk) triggersmitosis. Themechanismsthat con- trol the actiuities of cyclin-Cdk complexesinclude phosphorylation of the Cdk sub- unit, binding of Cdk inhibitor proteins(CIQs),proteolysisof cyclins,and changesin the transcription of genesencoding Cdk regulators. The cell-cyclecontrol systemalso dependscrucially on two additional enzymecomplexes,theAPC|Cand SCFubiquitin ligases,which catalyzethe ubiquitylation and consequentdestructionof specificregu- latory proteins that control critical euentsin thecVcle. SPHASE The linear chromosomes of eucaryotic cells arevast and dyramic assembliesof DNA and protein, and their duplication is a complex process that takes up a major fraction of the cell cycle. Not only must the long DNA molecule of each chromosome be duplicated accurately-a remarkablefeat in itself-but the pro- tein packaging surrounding each region of that DNA must also be reproduced, ensuring that the daughter cellsinherit all featuresof chromosome structure. The central event of chromosome duplication is replication of the DNA. A cell must solvetwo problems when initiating and completing DNA replication. First, replication must occur with extreme accuracy to minimize the risk of mutations in the next cell generation. Second,every nucleotide in the genome must be copied once, and only once, to prevent the damaging effects of gene amplification. In Chapter 5,we discussthe sophisticatedprotein machinery that performs DNA replication with astonishing speedand accuracy.In this section, we consider the elegantmechanisms by which the cell-cyclecontrol systemini- tiates the replication processand, at the sametime, prevents it from happening more than once per cycle. S-CdkInitiatesDNAReplicationOncePerCycle DNA replication begins at origins of replication, which are scattered at numer- ous locations in everychromosome. During Sphase,the initiation of DNA repli- cation occurs at these origins when specializedprotein machines (sometimes called initiator proteins) unwind the double helix at the origin and load DNA replication enzymes onto the two single-stranded templates. This leads to the elongationphase of replication, when the replication machinery movesoutward from the origin at tuvoreplicationforks (discussedin Chapter 5). To ensure that chromosome duplication occurs only once per cell cycle, the initiation phase of DNA replication is divided into two distinct steps that occur at different times in the cell cycle. The first step occurs in late mitosis and early Gr, when a large complex of initiator proteins, called the prereplica- tive complex, or pre-RC, assemblesat origins of replication. This step is some- times called licensingof replication origins because initiation of DNA synthe- sis is permitted only at origins containing a pre-RC.The second step occurs at the onset of S phase,when components of the pre-RC nucleate the formation of a larger protein complex called the preinitiation complex. This complex then unwinds the DNA helix and loads DNA polymerasesand other replication enzymes onto the DNA strands,thereby initiating DNA synthesis,asdescribed in Chapter 5. Once the replication origin has been activated in this way, the 1067