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Russell
CHAPTER 3
DNA Replication
editedbyYueWenWangPh.D.
Dept.ofAgronomy,NTU
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Chapter3slide1
SemiconservativeDNAReplication
1.WatsonandCrickDNAmodelimpliesa
mechanismforreplication:
a.UnwindtheDNAmolecule.
b.Separatethetwostrands.
c.Makeacomplementarycopyforeachstrand.
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Chapter3slide2
2.ThreepossiblemodelswereproposedforDNA
replication:
a.Conservativemodelproposedbothstrandsofonecopywould
beentirelyoldDNA,whiletheothercopywouldhaveboth
strandsofnewDNA.
b.DispersivemodelwasthatdsDNAmightfragment,replicate
dsDNA,andthenreassemble,creatingamosaicofoldandnew
dsDNAregionsineachnewchromosome.
c.SemiconservativemodelisthatDNAstrandsseparate,anda
complementarystrandissynthesizedforeach,sothatsibling
chromatidshaveoneoldandonenewstrand.Thismodelwasthe
winnerintheMeselsonandStahlexperiment.
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Chapter3slide3
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide4
TheMeselsonStahlExperiment
Animation:TheMeselsonStahlExperiment
1. MeselsonandStahl(1958)grewE.coliinaheavy
(notradioactive)isotopeofnitrogen,15Nintheformof
15NH Cl.Becauseitisheavier,DNAcontaining15Nis
4
moredensethanDNAwithnormal14N,andsocanbe
separatedbyCsCldensitygradientcentrifugation(Box
3.1).
2. OncetheE.coliwerelabeledwithheavy15N,the
researchersshiftedthecellstomediumcontainingnormal
14N,andtooksamplesattimepoints.DNAwasextracted
fromeachsampleandanalyzedinCsCldensitygradients
(Figure3.2).
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Chapter3slide5
Fig. 3.2 The Meselson-Stahl experiment, which showed that DNA replicates
semiconservatively
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide6
Box Fig. 3.1 Equilibrium centrifugation of DNA of different densities in a cesium chloride
density gradient
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide7
3.Afteronereplicationcycleinnormal14Nmedium,allDNAhad
densityintermediatebetweenheavyandnormal.Aftertworeplication
cycles,thereweretwobandsinthedensitygradient,oneatthe
intermediateposition,andoneatthepositionforDNAcontaining
entirely14N.
4.Resultscomparedwiththethreeproposedmodels:
a.Doesnotfitconservativemodel,becauseafteronegenerationthere
isasingleintermediateband,ratherthanonewithentirely 15NDNA
andanotherwithentirely14NDNA.
b.ThedispersivemodelpredictedthatasinglebandofDNAof
intermediatedensitywouldbepresentineachgeneration,gradually
becominglessdenseasincreasingamountsof 14Nwereincorporated
witheachroundofreplication.Instead,MeselsonandStahlobserved
twobandsofDNA,withtheintermediateformdecreasingovertime.
c.Thesemiconservativemodelfitsthedataverywell.
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Chapter3slide8
SemiconservativeDNAReplicationin
Eukaryotes
1.TovisualizeDNAof
eukaryoticchromosomes
replicating,CHO
(Chinesehamsterovary)
cellsaregrownin5
bromodeoxyuridine
(BUdR),abaseanalogfor
thymine.Aftertworounds
ofreplication,mitotic
chromosomesarestained
withfluorescentdyeand
Giemsastain(Figure3.3).
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Chapter3slide9
2.DNAcontainingTstainsdarkly,whileDNA
containingtwoBUdRstrandsstainslightly.Observed
thatafteronegeneration,bothchromatidsstainthesame,
eachwithoneBUdRstrandandoneTstrand.Aftertwo
generations,theystaindifferentlyandarecalledharlequin
chromosomes,onelight(bothstrandshaveBUdR)and
onedark(onestrandhasBUdRandotherstrandhasT).
3. Showedthateukaryotesalsousesemiconservative
DNAreplication.
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Chapter3slide10
EnzymesInvolvedinDNASynthesis
1.FirstisolationofanenzymeinvolvedinDNA
replicationwasin1955.ArthurKornbergwonthe
1959NobelPrizeinPhysiologyorMedicinefor
thiswork.
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Chapter3slide11
DNAPolymeraseI
1.AccomplishedinvitrosynthesisofE.coliDNA.His
reactionmixtureincluded:
a.DNAfragments(template).
b.RadioactivelylabeleddNTPs(dATP,dGTP,dTTPand
dCTP).
c.E.colilysate.
2.EnzymeoriginallycalledtheKornbergenzyme,now
knownasDNAPolymeraseI.Onceisolated,could
characterizeitsactivity,showingthattheabove
componentsarerequired,alongwithMg21ionsfor
maximumactivity.
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Chapter3slide12
RolesofDNAPolymerases
1.AllDNApolymeraseslinkdNTPsintoDNAchains(Figure3.4).
Mainfeaturesofthereaction:
a.Anincomingnucleotideisattachedbyits5phosphategrouptothe
3OHofthegrowingDNAchain.EnergycomesfromthedNTP
releasingtwophosphates.TheDNAchainactsasaprimerforthe
reaction.
b.Theincomingnucleotideisselectedbyitsabilitytohydrogenbond
withthecomplementarybaseinthetemplatestrand.Theprocessisfast
andaccurate.
c.DNApolymerasessynthesizeonlyfrom5to3.
2.TwoadditionalDNApolymeraseswerelaterisolated,DNAPolIIin
1970andDNAPolIIIin1971.
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Chapter3slide13
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide14
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide15
ThepropertiesofDNApolymerases
3.Thepropertiesoftheseenzymesare(Table3.1):
a.DNApolymeraseIisasinglepeptideencodedbypolA.Thereare
about400moleculesinanE.colicell.ReplicatesDNAinthe53
direction.Has53exonucleaseactivitytoremovenucleotidesfrom
5endofDNAorfromRNAprimer.
b.DNApolymeraseIIisasinglepeptideencodedbypolB.Numberof
moleculesperE.colicellisntknownforcertain,butprobablyaround
1020.Roleinthecellisunknown.
c.DNApolymeraseIIIhas10polypeptidesubunitsencodedby10
differentgenes.Thecatalyticcoreoftheenzymeiscomposedofthree
subunits,(encodedbythednaEgene),(dnaQ)and(holE).There
are1020moleculesofthisenzymeinanE.colicell.ReplicatesDNA
inthe53direction.
4.AllthreeE.coliDNApolymeraseshave35exonuclease
(proofreading)activity.
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Chapter3slide16
MolecularModelofDNAReplication
1. Table3.2showskeygenesandDNA
sequencesinvolvedinreplication.
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Chapter3slide17
InitiationoMolecularModelofDNA
Replication
1.Replicationstartsatoriginofreplication,withdenaturationto
exposethebasesandcreateabidirectionalreplicationbubble.E.coli
hasoneorigin,oriC,whichhasaminimalsequenceofabout245bp
requiredforinitiation.
2.EventsininitiatingDNAsynthesis,derivedfrominvitrostudies
(Figure3.5):
a.Gyrase(atypeoftopoisomerase)relaxessupercoilsintheregion.
b.Initiatorproteinsattach.
c.DNAhelicase(fromdnaB)bindsinitiatorproteinsontheDNA,and
denaturestheregionusingATPasanenergysource.
d.DNAprimase(fromdnaG)bindshelicasetoformaprimosome,
whichsynthesizesashort(1161nt)RNAprimer.Primersbeginwith
twopurines,typicallyAG.
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Chapter3slide18
Fig. 3.5 Model for the formation of a replication bubble at a replication origin in
E. coli and the initiation of the new DNA strand
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide19
SemidiscontinuousDNAReplication
Animation:MolecularModelofDNAReplication
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Chapter3slide20
Fig. 3.6a, b Model for the events occurring around a single replication fork of the
E. coli chromosome
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide21
Fig. 3.6c-e Model for the events occurring around a single replication fork of the
E. coli chromosome
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide22
1.WhenDNAdenaturesattheoriC,replicationforksare
formed.DNAreplicationisusuallybidirectional,butwill
considereventsatjustonereplicationfork(Figure3.6):
a.SinglestrandDNAbindingproteins(SSBs)bindthessDNA
formedbyhelicase,preventingreannealing.
b.Primasesynthesizesaprimeroneachtemplatestrand.
c.DNApolymeraseIIIaddsnucleotidestothe3endofthe
primer,synthesizinganewstrandcomplementarytothe
template,anddisplacingtheSSBs.DNAismadeinopposite
directionsonthetwotemplatestrands.
d.Newstrandmade53insamedirectionasmovementof
thereplicationforkisleadingstrand,whilenewstrandmadein
oppositedirectionislaggingstrand.Leadingstrandneedsonly
oneprimer,whilelaggingneedsaseriesofprimers.
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Chapter3slide23
2.HelicasedenaturingDNAcausestighterwindinginother
partsofthecircularchromosome.Gyraserelievesthis
tension.
3.Leadingstrandissynthesizedcontinuously,whilelagging
strandissynthesizeddiscontinuously,intheformof
Okazakifragments.DNAreplicationistherefore
semidiscontinuous.
4.Eachfragmentrequiresaprimertobegin,andisextended
byDNApolymeraseIII.
5.Okazakidatashowthatthesefragmentsaregradually
joinedtogethertomakeafulllengthdsDNAchromosome.
DNApolymeraseIusesthe3OHoftheadjacentDNA
fragmentasaprimer,andsimultaneouslyremovestheRNA
primerwhileresynthesizingtheprimerregionintheformof
DNA.Thenickremainingbetweenthetwofragmentsis
sealedwithDNAligase.(Fig.3.7)
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Chapter3slide24
Fig. 3.7 Action of DNA ligase in sealing the gap between adjacent DNA fragments to
form a longer, covalently continuous chain
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide25
6.Keyproteinsareassociatedtoformareplisome.
TemplateDNAprobablybendstoallowsynthesis
ofbothleadingandlaggingstrandsatthe
replicationfork(Fig.3.8)
7.Earlystagesofbidirectionalreplicationare
summarizedinFigure3.9.
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Chapter3slide26
Fig. 3.8 Model for the replication machine, or replisome, the complex of key
replication proteins, with the DNA at the replication fork
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide27
iActivity:UnravelingDNAReplication
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Chapter3slide28
Fig. 3.9 Diagram of the formation at a replication origin sequence of two replication
forks that move in opposite directions
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide29
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide30
ReplicationofcircularDNAandthe
supercoilingproblem
1.Somecircularchromosomes(e.g.,E.coli)are
circularthroughoutreplication,creatingatheta
like()shape(Fig.3.10).Asthestrandsseparate
ononesideofthecircle,positivesupercoilsform
elsewhereinthemolecule.Replicationfork
movesabout500nt/second,soat10bp/turn,
replicationforkrotatesat3,000rpm.
2.Topoisomerasesrelievethesupercoils,
allowingtheDNAstrandstocontinueseparating
asthereplicationforksadvance.(Fig.3.11)
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Chapter3slide31
Fig. 3.11 Diagram showing the unreplicated, supercoiled parent strands and the
portions already replicated
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide32
RollingCircleReplication
1.Anothermodelforreplicationisrollingcircle,whichis
usedbyseveralbacteriophages,includingX174(aftera
complementismadeforthegenomicssDNA)and
(aftercircularizationbybasepairingbetweenthesticky
ssDNAcosends)
2.Rollingcirclereplicationbeginswithanick(single
strandedbreak)attheoriginofreplication.The5endis
displacedfromthestrand,andthe3endactsasaprimer
forDNApolymeraseIII,whichsynthesizesacontinuous
strandusingtheintactDNAmoleculeasatemplate.
3.The5endcontinuestobedisplacedasthecircle
rolls,andisprotectedbySSBsuntildiscontinuous
DNAsynthesismakesitadsDNAagain.
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Chapter3slide33
Fig. 3.12 The replication process of double-stranded circular DNA molecules through
the rolling circle mechanism
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide34
4.ADNAmoleculemanygenomesinlengthcanbemade
byrollingcirclereplication.Duringviralassemblyitis
cutintoindividualviralchromosomesandpackagedinto
phagehead.
5.Bacteriophage,regardlessofwhetherenteringthe
lyticorlysogenicpathway,circularizesitschromosome
immediatelyafterinfection.
a.Inalysogenicinfection,thecircularDNAintegratedintoa
specificsiteintheE.colichromosomebyacrossoverevent.
b.Inalyticinfection,rollingcirclereplicationproducesalong
concatamerofDNA,andtheaviralendonuclease(productof
thetergene)recognizethecossitesandmakesthestaggeredcuts
thatusedtoassemblenewvirusparticles.
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Chapter3slide35
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide36
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide37
DNAReplicationinEukaryotes
1. DNAreplicationisverysimilarinboth
prokaryotesandeukaryotes,exceptthat
eukaryoteshavemorethanonechromosome.
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Chapter3slide38
DNAReplicationandtheCellCycle
1.Eacheukaryoticchromosomemustbecopied,sobothDNAand
histonesmustbedoubledineachcellcycle.Chromosomeduplication
occursduringtheSphaseofthecellcycle,andsegregationofprogeny
chromosomesduringtheMphase.
2.Detailsofcellcyclecontrolarebeyondthescopeofthisdiscussion.
Yeasts,inwhichchromosomalreplicationiswellstudied,serveasa
eukaryoticmodelorganism.
3.Manygenesareinvolvedinthesystemofchecksandbalancesthat
controlsthecellcycle(Figure3.15).Somemajorcheckpointsinthe
system:
a.TomovefromG1intoSphase,cellsmustbelargeenough,andthe
environmentfavorable.ThischeckpointiscalledSTARTinyeastsand
G1checkpointinmammaliancells.
b.TheG2checkpointcontrolsentryintoMphase,assessingwhether
DNAhasbeenduplicated,thecellislargeenoughandtheenvironment
isfavorable.
c.AttachmentofchromosomestothemitoticspindleduringMphase
functionsasanothercheckpoint,triggeringseparationofthe
chromatids.
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Chapter3slide39
4.Checkpointsusecyclinproteinsandcyclindependentkinases
(Cdks).YeastsuseasingleCdkforboththeG1andG2checkpoints,
whilemammalsuseatleasttwoCdksforeachcheckpoint.Intheyeast
system,forexample:
a.AttheSTART(G1)checkpoint,oneormoreG1cyclinsbindand
activatetheCdk.TheactivatedCdkphosphorylatesproteinsneededto
initiateSphase.Cyclinlevelsthendecreasebyproteolysis.
b.AttheG2checkpoint,mitoticcyclin(s)bindtoCdk,formingtheM
phasepromotingfactor(MPF).DephosphorylationofMPFbyother
enzymesactivatesit,anditthencatalyzesphosphorylationofproteins
thatmovethecellintoMphase.Mitoticcyclinisdegradedafter
metaphase,causingMPFinactivationsothatmitosiscanbecompleted.
5.Cellcyclecontrolisverycomplex,andwillbediscussedmorefully
withthegeneticsofcancer.
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Chapter3slide40
Fig. 3.15 Some of the molecular events that control progression through the cell cycle
in yeasts
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide41
EukaryoticReplicationEnzymes
1.EnzymesofeukaryoticDNAreplicationarentaswellcharacterized
astheirprokaryoticcounterparts.Thereplicationprocessissimilarin
bothgroupsDNAdenatures,replicationissemiconservativeand
semidiscontinuousandprimersarerequired.
2.FiveDNApolymerasesareknowninmammaliancells:
a.(alpha)isnuclear,usesRNAprimers,isinvolvedinnuclearDNA
replicationandhasnotbeenshowntoproofread.
b.(beta)isnuclear,servesinDNArepairanddoesnotshow
proofreadingactivity.
c.(delta)isnuclear,usesRNAprimers,isinvolvedinnuclearDNA
replicationandiscapableofproofreading.
d.(gamma)ismitochondrial,replicatingthemitochondrialDNA
usingRNAprimersandproofreading.
e.(epsilon)isnuclear,hasproofreadingactivityandmaybeusedfor
DNArepair.
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Chapter3slide42
Replicons
1.Eukaryoticchromosomesgenerallycontainmuchmore
DNAthanthoseofprokaryotes,andtheirreplication
forksmovemuchmoreslowly.Iftheywereliketypical
prokaryotes,withonlyoneoriginofreplicationper
chromosome,DNAreplicationwouldtakeaverylong
time.
2.Instead,eukaryoticchromosomescontainmultiple
origins,atwhichDNAdenaturesandreplicationthen
proceedsbidirectionallyuntilanadjacentreplicationfork
isencountered.TheDNAreplicatedfromasingleorigin
iscalledareplicon,orreplicationunit(Figure3.16).
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Chapter3slide43
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide44
3.Repliconsizeissmallerineukaryotesthan
prokaryotes,andeachchromosomecontains
manyreplicons.Numberandsizeofreplicons
mayvarywithcelltype.
4.NotalloriginswithinagenomeinitiateDNA
synthesissimultaneously.Cellspecificpatternsof
originactivationareobserved,sothat
chromosomalregionsarereplicatedina
predictableorderineachcellcycle.
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Chapter3slide45
Fig. 3.17 Temporal ordering of DNA replication initiation events in replication units
of eukaryotic chromosomes
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide46
OriginsofReplication
1.Eukaryoticoriginsaregenerallynotwellcharacterized;thoseofthe
yeastSaccharomycescerevisiaeareamongthebestunderstood.
2.ChromosomalDNAfragmentsthatareabletoreplicate
autonomouslywhenintroducedintoyeastasextracellular,circular
DNAareknownasARSs(autonomouslyreplicatingsequences).
3.SomeARSsappeartobeyeastoriginsofreplication,butothers
probablyarenot.
4.FoursequenceelementshavebeenidentifiedinARSs.TheA
elementisessentialtotheARS,andcombineswithB1and/orB2
elementstoformthecoreoftheARStowhichthemultiproteinorigin
recognitioncomplex(ORC)binds.TheB3elementbindsaprotein
calledAbf1(ARSbindingfactor)toenhancereplication.
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Chapter3slide47
ReplicatingtheEndsofChromosomes
1.Whentheendsofchromosomesarereplicated
andtheprimersareremovedfromthe5ends,
thereisnoadjacentDNAstrandtoserveasa
primer,andsoasinglestrandedregionisleftat
the5endofthenewstrand.Ifthegapisnot
addressed,chromosomeswouldbecomeshorter
witheachroundofreplication(Figure3.18).
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Chapter3slide48
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide49
2.Mosteukaryoticchromosomeshaveshort,speciesspecific
sequencestandemlyrepeatedattheirtelomeres.BlackburnandGreider
haveshownthatchromosomelengthsaremaintainedbytelomerase,
whichaddstelomererepeatswithoutusingthecellsregularreplication
machinery.
3.IntheciliateTetrahymena,thetelomererepeatsequenceis5
TTGGGG3.Telomerase,anenzymecontainingbothproteinand
RNA,bindstotheterminaltelomererepeatwhenitissinglestranded,
synthesizinga3ntsequence,TTG.The3endofthetelomeraseRNA
containsthesequenceAAC,whichbindstheTTGpositioning
telomerasetocompleteitssynthesisoftheTTGGGGtelomererepeat.
Additionalroundsoftelomeraseactivitylengthenthechromosomeby
addingtelomererepeats.
4.Aftertelomeraseaddstelomeresequences,chromosomalreplication
proceedsintheusualway.Anyshorteningofthechromosomeendsis
compensatedbytheadditionofthetelomererepeats.
5.Telomerelengthmayvary,butorganismsandcelltypeshave
characteristictelomerelengths.Mutantsaffectingtelomerelengthhave
beenidentified,anddataindicatethattelomerelengthisgenetically
controlled.Shorteningoftelomereseventuallyleadstocelldeath,and
thismaybeafactorintheregulationofnormalcelldeath.
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Chapter3slide50
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter3slide51
AssemblingNewDNAintoNucleosomes
1.WheneukaryoticDNAisreplicated,itcomplexeswithhistones.
Thisrequiressynthesisofhistoneproteinsandassemblyofnew
nucleosomes.
2.TranscriptionofhistonegenesisinitiatedneartheendofG1phase,
andtranslationofhistoneproteinsoccursthroughoutSphase.
3.NewlyreplicatedDNAisorganizedintonucleosomesveryquickly.
DNAsynthesisrequiresnucleosomestodisassemblesotheDNAcan
bedenatured,butonly200300bpwillbenucleosomefreearoundthe
replicationforks.
4.Datastronglysuggestthatnewnucleosomesarecomposedof
entirelynewhistones,andexistingnucleosomesareconserved.Oldand
newnucleosomesbindrandomlytothesiblingchromatidsafter
replication.ITISNOTCONCLUSIVEYET!
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Chapter3slide52
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Chapter3slide53
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Chapter3slide54