Major Molecular Events of DNA Replication - Nature

DNA is always synthesized in the 5'-to-3' direction, meaning that nucleotides are added only to the 3' end of the growing strand. As shown in Figure 2, ... Thispagehasbeenarchivedandisnolongerupdated   MajorMolecularEventsofDNAReplication By: LeslieA.Pray,Ph.D. © 2008 NatureEducation  Citation: Pray, L. (2008) MajormoleculareventsofDNAreplication. NatureEducation 1(1):99 ArthurKornbergcomparedDNAtoataperecordingofinstructionsthatcanbecopiedoverandover.Howdocellsmakethesenear-perfectcopies,anddoestheprocessevervary? Aa Aa Aa   Scientistshavedevoteddecadesofefforttounderstanding howdeoxyribonucleicacid(DNA)replicatesitself.Insimpleterms,replication involvesuseofanexistingstrandofDNAasatemplateforthesynthesisofa new,identicalstrand.AmericanenzymologistandNobelPrizewinnerArthur Kornbergcomparedthisprocesstoataperecordingofinstructionsforperforming atask:"[E]xactcopiescanbemadefromit,asfromataperecording,sothat thisinformationcanbeusedagainandelsewhereintimeandspace"(Kornberg, 1960). Inreality,theprocessofreplicationisfarmorecomplex thansuggestedbyKornberg'sanalogy.Researcherstypicallyutilizesimplebacterial cellsintheirexperiments,buttheystilldonothavealltheanswers, particularlywhenitcomestoeukaryoticreplication.Nonetheless,scientists arefamiliarwiththebasicstepsinthereplicationprocess,andtheycontinue torelyonthisinformationasthebasisforcontinuedresearchand experimentation. TheMolecularMachineryofBacterialDNAReplication Atypicalbacterialcellhasanywherefromabout1million to4millionbasepairsofDNA,comparedtothe3billionbasepairsinthe genomeofthecommonhousemouse(Mus musculus).Still,eveninbacteria,withtheirsmallergenomes,DNA replicationinvolvesanincrediblysophisticated,highlycoordinatedseriesof molecularevents.Theseeventsaredividedintofourmajorstages:initiation, unwinding,primersynthesis,andelongation. InitiationandUnwinding Duringinitiation,so-calledinitiatorproteinsbindtothe replicationorigin,abase-pairsequenceofnucleotidesknownasoriC.This bindingtriggerseventsthatunwindtheDNAdoublehelixintotwo single-strandedDNAmolecules.Severalgroupsofproteinsareinvolvedinthisunwinding (Figure1).Forexample,theDNAhelicasesare responsibleforbreakingthehydrogenbondsthatjointhecomplementary nucleotidebasestoeachother;thesehydrogenbondsareanessentialfeature ofJames WatsonandFrancisCrick'sthree-dimensionalDNAmodel.Becausethenewlyunwoundsinglestrands haveatendencytorejoin,anothergroupofproteins,thesingle-strand-binding proteins,keepthesinglestrandsstableuntilelongationbegins.Athird familyofproteins,thetopoisomerases,reducesomeofthetorsionalstrain causedbytheunwindingofthedoublehelix. Figure1: FacilitationofDNAunwinding.DuringDNAreplication,severalproteinsfacilitatetheunwindingoftheDNAdoublehelixintotwosinglestrands.Topoisomerases(red)reducetorsionalstraincausedbytheunwindingoftheDNAdoublehelix;DNAhelicase(yellow)breakshydrogenbondsbetweencomplementarybase-pairs;single-strandbindingproteins(SSBs)stabilizetheseparatedstrandsandpreventthemfromrejoining.©2014NatureEducationAdaptedfromPierce,Benjamin.Genetics:AConceptualApproach,2nded.Allrightsreserved. Aspreviouslymentioned,thelocationatwhicha DNAstrandbeginstounwindintotwoseparatesinglestrandsisknownasthe originofreplication.AsshowninFigure1, whenthedoublehelixunwinds,replicationproceedsalongthetwosingle strandsatthesametimebutinoppositedirections(i.e.,lefttorightonone strand,andrighttoleftontheother).Thisformstworeplicationforksthat movealongtheDNA,replicatingastheygo. PrimerSynthesis Primersynthesismarksthebeginningoftheactualsynthesis ofthenewDNAmolecule.Primersareshortstretchesofnucleotides(about10 to12basesinlength)synthesizedbyanRNApolymeraseenzymecalledprimase. PrimersarerequiredbecauseDNApolymerases,theenzymesresponsibleforthe actualadditionofnucleotidestothenewDNAstrand,canonlyadd deoxyribonucleotidestothe3'-OHgroupofanexistingchainandcannotbegin synthesisdenovo.Primase,onthe otherhand,canaddribonucleotidesde novo.Later,afterelongationiscomplete,theprimerisremovedand replacedwithDNAnucleotides. Elongation Finally,elongation--theadditionofnucleotidestothenew DNAstrand--beginsaftertheprimerhasbeenadded.Synthesisofthegrowing strandinvolvesaddingnucleotides,onebyone,intheexactorderspecifiedby theoriginal(template)strand.Recallthatoneofthekeyfeaturesofthe Watson-CrickDNAmodelisthatadenineisalwayspairedwiththymineand cytosineisalwayspairedwithguanine.So,forexample,iftheoriginalstrand readsA-G-C-T,thenewstrandwillreadT-C-G-A. DNAisalwayssynthesizedinthe5'-to-3'direction,meaning thatnucleotidesareaddedonlytothe3'endofthegrowingstrand.Asshown inFigure2,the5'-phosphategroupofthenew nucleotidebindstothe3'-OHgroupofthelastnucleotideofthegrowing strand.Scientistshaveyettoidentifyapolymerasethatcanaddbasestothe 5'endsofDNAstrands.Figure2: NewDNAissynthesizedfromdeoxyribonucleosidetriphosphates(dNTPs).(A)Adeoxyribonucleosidetriphosphate(dNTP).(B)DuringDNAreplication,the3'-OHgroupofthelastnucleotideonthenewstrandattacksthe5'-phosphategroupoftheincomingdNTP.Twophosphatesarecleavedoff.(C)Aphosphodiesterbondformsbetweenthetwonucleotides,andphosphateionsarereleased.©2014NatureEducationAdaptedfromPierce,Benjamin.Genetics:AConceptualApproach,2nded.Allrightsreserved. TheDiscoveryofDNAPolymerase WhilestudyingE.coli bacteria,enzymologistArthurKornbergdiscoveredthatDNApolymerasescatalyze DNAsynthesis.Kornberg'sexperimentinvolvedmixingallofthebasic "ingredients"necessaryforE.coli DNAsynthesisinatesttube,includingnucleotides,E.coliextract,andATP,andthenpurifyingandtestingthe enzymesinvolved.Usingthismethod,KornbergnotonlydiscoveredDNA polymerases,buthealsoperformedsomeoftheinitialworkdemonstratinghow enzymesaddnewnucleotidestogrowingDNAchains(Kornberg,1959). Scientistshavesinceidentifiedatotaloffivedifferent DNApolymerasesinE.coli,eachwith aspecializedrole.Forexample,DNApolymeraseIIIdoesmostoftheelongation work,addingnucleotidesonebyonetothe3'endofthenewandgrowingsingle strand.Otherenzymes,includingDNApolymeraseIandRNaseH,areresponsible forremovingtheRNAprimerafterDNApolymeraseIIIhasbegunitswork, replacingitwithDNAnucleotides(Ogawa&Okazaki,1984).Whenthese enzymesfinish,theyleaveanickbetweenthesectionofDNAthatwasformerly theprimerandtheelongatedsectionofDNA.AnotherenzymecalledDNAligasethen actstosealthebondbetweenthetwoadjacentnucleotides. DNAPolymeraseOnlyMovesinOneDirection AfteraprimerissynthesizedonastrandofDNAandtheDNA strandsunwind,synthesisandelongationcanproceedinonlyonedirection.As previouslymentioned,DNApolymerasecanonlyaddtothe3'end,sothe5'end oftheprimerremainsunaltered.Consequently,synthesisproceedsimmediately onlyalongtheso-calledleadingstrand.Thisimmediatereplicationisknownas continuousreplication.Theotherstrand(inthe5'directionfromtheprimer) iscalledthelaggingstrand,andreplicationalongitiscalleddiscontinuous replication.Thedoublehelixhastounwindabitbeforethesynthesisof anotherprimercanbeinitiatedfurtheruponthelaggingstrand.Synthesiscan thenoccurfromthe3'endofthatnewprimer.Next,thedoublehelixunwindsa bitmore,andanotherspurtofreplicationproceeds.Asaresult,replication alongthelaggingstrandcanonlyproceedinshort,discontinuousspurts (Figure3).Figure3: ReplicationoftheleadingDNAstrandiscontinuous,whilereplicationalongthelaggingstrandisdiscontinuous.AfterashortlengthoftheDNAhasbeenunwound,synthesismustproceedinthe5'to3'direction;thatis,inthedirectionoppositethatoftheunwinding.©2014NatureEducationAdaptedfromPierce,Benjamin.Genetics:AConceptualApproach,2nded.Allrightsreserved. FigureDetail ThefragmentsofnewlysynthesizedDNAalongthelagging strandarecalledOkazakifragments,namedinhonoroftheirdiscoverer, JapanesemolecularbiologistReijiOkazaki.Okazakiandhiscolleaguesmade theirdiscoverybyconductingwhatisknownasapulse-chaseexperiment,which involvedexposingreplicatingDNAtoashort"pulse"ofisotope-labelednucleotides andthenvaryingthelengthoftimethatthecellswouldbeexposedto nonlabelednucleotides.Thislaterperiodiscalledthe"chase"(Okazakietal.,1968).Thelabelednucleotides wereincorporatedintogrowingDNAmoleculesonlyduringtheinitialfew secondsofthepulse;thereafter,onlynonlabelednucleotideswereincorporated duringthechase.ThescientiststhencentrifugedthenewlysynthesizedDNAand observedthattheshorterchasesresultedinmostoftheradioactivity appearingin"slow"DNA.Thesedimentationratewasdeterminedbysize:smaller fragmentsprecipitatedmoreslowlythanlargerfragmentsbecauseoftheir lighterweight.Astheinvestigatorsincreasedthelengthofthechases, radioactivityinthe"fast"DNAincreasedwithlittleornoincreaseof radioactivityintheslowDNA.Theresearcherscorrectlyinterpretedthese observationstomeanthat,withshortchases,onlyverysmallfragmentsofDNA werebeingsynthesizedalongthelaggingstrand.Asthechasesincreasedin length,givingDNAmoretimetoreplicate,thelaggingstrandfragmentsstarted integratingintolonger,heavier,morerapidlysedimentingDNAstrands.Today, scientistsknowthattheOkazakifragmentsofbacterialDNAaretypicallybetween 1,000and2,000nucleotideslong,whereasineukaryoticcells,theyareonly about100to200nucleotideslong. TheChallengesofEukaryoticReplication Bacterialandeukaryoticcellssharemanyofthesamebasic featuresofreplication;forinstance,initiationrequiresaprimer,elongation isalwaysinthe5'-to-3'direction,andreplicationisalwayscontinuousalong theleadingstrandanddiscontinuousalongthelaggingstrand.Butthereare alsoimportantdifferencesbetweenbacterialandeukaryoticreplication,some ofwhichbiologistsarestillactivelyresearchinginanefforttobetter understandthemoleculardetails.Onedifferenceisthateukaryoticreplication ischaracterizedbymanyreplicationorigins(oftenthousands),notjustone, andthesequencesofthereplicationoriginsvarywidelyamongspecies.Onthe otherhand,whilethereplicationoriginsforbacteria,oriC,varyinlength (fromabout200to1,000basepairs)andsequence,exceptamongcloselyrelated organisms,allbacterianonethelesshavejustasinglereplicationorigin (Mackiewiczetal.,2004). EukaryoticreplicationalsoutilizesadifferentsetofDNA polymeraseenzymes(e.g.,DNApolymeraseδandDNApolymeraseε insteadofDNApolymeraseIII).Scientistsarestillstudyingtherolesofthe 13eukaryoticpolymerasesdiscoveredtodate.Inaddition,ineukaryotes, theDNA templateiscompactedbythewayitwindsaroundproteinscalledhistones. ThisDNA-histonecomplex,calledanucleosome,posesauniquechallengeboth forthecellandforscientistsinvestigatingthemoleculardetailsof eukaryoticreplication.WhathappenstonucleosomesduringDNAreplication? Scientistsknowfromelectronmicrographstudiesthatnucleosomereassembly happensveryquicklyafterreplication(thereassemblednucleosomesarevisible intheelectronmicrographimages),buttheystilldonotknowhowthishappens (Annunziato,2005). Also,whereasbacterialchromosomesarecircular,eukaryotic chromosomesarelinear.DuringcircularDNAreplication,theexcisedprimeris readilyreplacedbynucleotides,leavingnogapinthenewlysynthesizedDNA.In contrast,inlinearDNAreplication,thereisalwaysasmallgapleftatthe veryendofthechromosomebecauseofthelackofa3'-OHgroupforreplacement nucleotidestobind.(Asmentioned,DNAsynthesiscanproceedonlyinthe 5'-to-3'direction.)Iftherewerenowaytofillthisgap,theDNAmolecule wouldgetshorterandshorterwitheverygeneration.However,theendsoflinear chromosomes—thetelomeres—haveseveralpropertiesthatpreventthis. DNAreplicationoccursduringtheSphaseofcelldivision.InE.coli, thismeansthattheentiregenomeisreplicatedinjust40minutes,atapace ofapproximately1,000nucleotidespersecond.Ineukaryotes,thepaceismuch slower:about40nucleotidespersecond.Thecoordinationoftheprotein complexesrequiredforthestepsofreplicationandthespeedatwhich replicationmustoccurinorderforcellstodivideareimpressive,especially consideringthatenzymesarealsoproofreading,whichleavesveryfewerrorsbehind. Summary ThestudyofDNAreplicationstartedalmostassoonasthestructureof DNAwaselucidated,anditcontinuestothisday.Currently,thestagesof initiation,unwinding,primersynthesis,andelongationareunderstoodinthe mostbasicsense,butmanyquestionsremainunanswered,particularlywhenit comestoreplicationoftheeukaryoticgenome.Scientistshavedevoteddecades tothestudyofreplication,andresearcherssuchasKornbergandOkazakihave madeanumberofimportantbreakthroughs.Nonetheless,muchremainstobe learnedaboutreplication,includinghowerrorsinthisprocesscontributeto humandisease. ReferencesandRecommendedReading Annunziato,A.T.Splitdecision:WhathappenstonucleosomesduringDNAreplication?JournalofBiologicalChemistry280,12065–12068(2005) Bessman,M.J.,etal.Enzymaticsynthesisofdeoxyribonucleicacid.II.Generalpropertiesofthereaction.JournalofBiologicalChemistry233,171–177(1958) Kornberg,A.Thebiologicalsynthesisofdeoxyribonucleicacid.NobelLecture,December11,1959.(linktotranscript) ———.Biologicalsynthesisofdeoxyribonucleicacid.Science131,1503–1508(1960) Lehman,I.R.,etal.Enzymaticsynthesisofdeoxyribonucleicacid.I.PreparationofsubstratesandpartialpurificationofanenzymefromEscherichiacoli.JournalofBiologicalChemistry233,163–170(1958) Losick,R.,&Shapiro,L.DNAreplication:BringingthemountaintoMohammed.Science282,1430–1431(1998) Mackiewicz,P.,etal.Wheredoesbacterialreplicationstart?RulesforpredictingtheoriCregion.NucleicAcidsResearch32,3781–3791(2004) Ogawa,T.,&Okazaki,T.FunctionofRNaseHinDNAreplicationrevealedbyRNaseHdefectivemutantsofEscherichiacoli.MolecularandGeneralGenetics193,231–237(1984) Okazaki,R.,etal.MechanismofDNAchaingrowth.I.Possiblediscontinuityandunusualsecondarystructureofnewlysynthesizedchains.ProceedingsoftheNationalAcademyofSciences59,598–605(1968) Outline | Keywords | AddContenttoGroup ArticleHistory Close Share | Cancel Revoke | Cancel Keywords KeywordsforthisArticle AddkeywordstoyourContent Save | Cancel FlagInappropriate TheContentis: Objectionable Explicit Offensive Inaccurate Comments FlagContent | Cancel Close share Close Digg MySpace Google+ StumbleUpon EmailyourFriend YourFirstName * YourLastName * YourEmailAddress * YourFriend'sEmailaddress * YourMessage* Submit | Cancel * Required Close Thiscontentiscurrentlyunderconstruction. 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