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The cell-cycle control system alsodependscrucially on two additional enzymecomplexes,the APC|Cand SCFubiquitinligases,which catalyzethe ubiquitylation and consequentdestruction of specificregulatory proteins that control critical euentsin the cVcle.S PHASEThe linear chromosomes of eucaryotic cells are vast and dyramic assembliesofDNA and protein, and their duplication is a complex process that takes up amajor fraction of the cell cycle. Not only must the long DNA molecule of eachchromosome be duplicated accurately-a remarkable feat in itself-but the protein packaging surrounding each region of that DNA must also be reproduced,ensuring that the daughter cells inherit all features of chromosome structure.The central event of chromosome duplication is replication of the DNA. Acell must solve two problems when initiating and completing DNA replication.First, replication must occur with extreme accuracy to minimize the risk ofmutations in the next cell generation.
Second, every nucleotide in the genomemust be copied once, and only once, to prevent the damaging effects of geneamplification. In Chapter 5, we discussthe sophisticated protein machinery thatperforms DNA replication with astonishing speed and accuracy.In this section,we consider the elegant mechanisms by which the cell-cycle control system initiates the replication process and, at the same time, prevents it from happeningmore than once per cycle.S-CdkInitiatesDNAReplicationOncePerCycleDNA replication begins at origins of replication, which are scattered at numerous locations in every chromosome.
During S phase, the initiation of DNA replication occurs at these origins when specialized protein machines (sometimescalled initiator proteins) unwind the double helix at the origin and load DNAreplication enzymes onto the two single-stranded templates.
This leads to theelongationphase of replication, when the replication machinery moves outwardfrom 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 thatoccur at different times in the cell cycle. The first step occurs in late mitosisand early Gr, when a large complex of initiator proteins, called the prereplicative complex, or pre-RC, assemblesat origins of replication. This step is sometimes called licensing of replication origins because initiation of DNA synthesis is permitted only at origins containing a pre-RC.
The second step occurs atthe onset of S phase, when components of the pre-RC nucleate the formationof a larger protein complex called the preinitiation complex. This complexthen unwinds the DNA helix and loads DNA polymerases and other replicationenzymes onto the DNA strands, thereby initiating DNA synthesis, as describedin Chapter 5. Once the replication origin has been activated in this way, the10671068Chapter17:TheCellCyclep r e r e p l i c a t i vceo m p l e x e sa t r e p l i c a t i o no r i g i n sIreplicationelongation forksM-Cdk activation|.hrotoro."segregation,.4^Glpre-Rc is dismantled and cannot be reassembledat that origin until the following G1.As a result, origins can be activated only once per cell cycle.The cell-cycle control system governs both assembly of the pre-RC andassembly of the pre-initiation complex (Figure lZ-22).
Assembly of the pre-RCis inhibited by Cdk activiry and, in most cells, is stimulated by the ApC/C. preRC assembly therefore occurs only in late mitosis and early G1,when cdk activity is low and APC/C activity is high. At the onset of S phase, activation of S-Cdkthen triggers the formation of a preinitiation complex, which initiates DNA synthesis. In addition, the pre-RC is partly dismantled. BecauseS-Cdk and M-Cdkactivities remain high (and APC/C activity remains low) until late mitosis, newpre-RCs cannot be assembled at fired origins until the cell cycle is complete.Figure 17-23 illustrates some of the proteins involved in the initiation ofDNA replication.
A key player is a large, multiprotein complex called the originrecognition complex (oRc), which binds to replication origins throughout thecell cycle. In late mitosis and early Gr, the proteins cdc6 and cdtl bind to theORC at origins and help load a group of six related proteins called the Mcm proteins. The resulting large complex is the pre-RC, and the origin is now licensedfor replication.The six Mcm proteins of the pre-RC form a ring around the DNA that isthought to serve as the major DNA helicase that unwinds the origin DNA whenDNA synthesis begins and as the replication forks move out from the origin.Thus, the central purpose of the pre-RC is to load the helicase that will play acentral part in the subsequent DNA replication process.Once the pre-RC has assembled in Gr, the replication origin is ready to fire.The activation of s-cdk in late G1triggers the assembly of severaladditional protein complexes at the origin, leading to the formation of a giant preinitiationcomplex that unwinds the helix and begins DNA synthesis.At the same time as it initiates DNA replication, S-Cdk triggers the disassembly of some pre-RC components at the origin.
cdks phosphorylate both theoRC and cdc6, resulting in their inhibition by various mechanisms. Furthermore, inactivation of the APC/G in late Gr also helps turn off pre-RC assembly.In late mitosis and early G1, the APC/C triggers the destruction of a protein,Figure 17-22 Control of chromosomeduplication.Preparationsfor DNAreplicationbeginin Gr with the assemblyof prereplicativecomplexes(pre-RCs)atreplicationorigins.S-Cdkactivationleadsto the formationof multiproteinpreinitiationcomplexesthat unwindtheDNAat originsand beginthe processofDNA replication.Two replicationforksmoveout from eachoriginuntiltheentirechromosomeis duplicated.Duplicatedchromosomesare thensegregatedin M phase.The activationofreplicationoriginsin S phasealsocausesdisassemblyof the prereplicativecomplex,which does not reformat theoriginuntilthe followingG1-therebyensuringthat eachoriginis activatedonly oncein eachcellcycle.1MgS PHASEO R C( o r i g i nr e c o g n i t i o nc o m p l e x )u1p r e r e p l i c a t i vceo m p l e x( p r e - R C )COMPLETIONOFDNA REPLICATIONt61Mgeminin, that binds and inhibits the pre-RC component Cdtl.
Thus, when theAPC/C is turned off in late G1,geminin accumulates and inhibits Cdtl.In thesevarious ways, S- and M-Cdk activities, combined with lowAPC/C activity, blockpre-RC formation during S phase and thereafter.How then, is the cell-cycle control system reset to allow replication to occur in the next cell cycle? The answeris simple. At the end of mitosis, APC/C activation leads to the inactivation ofCdks and the destruction of geminin. Pre-RC components are dephosphorylated and Cdtl is activated, allowing pre-RC assembly to prepare the cell for thenext S phase.of ChromatinDuplicationRequiresDuplicationChromosomeStructureThe DNA of the chromosomes is extensively packaged in a variety of proteincomponents, including histones and various regulatory proteins involved inthe control ofgene expression (discussedin Chapter 4). Thus, duplication ofaFigure17-23 Control of the initiation ofDNA replication.The ORCremainsassociatedwith a replicationoriginthroughoutthe cellcycle.In earlyG1,Cdc6and Cdtl associatewith the ORC.The resultingproteincomplexthenon theMcm ring complexesassemblesadjacentDNA,resultingin the formationof the prereplicativecomplex(pre-RC).from anotherS-Cdk(with assistanceprotein kinase,not shown)thenof severalthe assemblystimulatesadditionalproteinsat the originto formcomplex.DNAthe preinitiationpolymeraseand other replicationproteinsare recruitedto the origin,theMcm protein ringsare activatedas DNAhelicases,and DNAunwindingallowsto begin.S-CdkalsoDNAreplicationblocksrereplicationby triggeringthedestructionof Cdc6and the inactivationofthe ORC.Cdtl is inactivatedby theproteingeminin.Gemininis an APC/Ctarget and its levelsthereforeincreasein5 and M phases,when APC/Cis inactive.Thus,the componentsofthe pre-RC(Cdc6,Cdt1,Mcm) cannotform a newpre-RCat the originsuntil M-Cdkisinactivatedand the APC/Cis activatedatthe end of mitosis(seetext).1070Chapter17:TheCell Cyclechromosome is not simply a matter of duplicating the DNA at its core but alsorequires the duplication of these chromatin proteins and their proper assemblyon the DNA.The production of chromatin proteins increasesduring S phase to providethe raw materials needed to package the newly synthesized DNA.
Most importantly, S-Cdksstimulate a large increase in the synthesis of the four histone subunits that form the histone octamers at the core of each nucleosome. These subunits are assembledinto nucleosomes on the DNA by nucleosome assemblyfactors, which typically associatewith the replication fork and distribute nucleosomes on both strands of the DNA as they emerge from the DNA synthesismachinery.Chromatin packaging helps to control gene expression.In some parts of thechromosome, the chromatin is highly condensed and is called heterochromatin,whereas in other regions it has a more open structure and is called euchromatin.These differences in chromatin structure depend on a variety of mechanisms,including modification of histone tails and the presenceof non-histone proteins(discussedin Chapter 4). Becausethese differences are important in gene regulation, it is crucial that chromatin structure, like the DNA within, is reproducedaccurately during S phase.
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