Beruflich Dokumente
Kultur Dokumente
60120000
Chapter3slide1
SemiconservativeDNAReplication
1.WatsonandCrickDNAmodelimpliesa
mechanismforreplication:
a.UnwindtheDNAmolecule.
b.Separatethetwostrands.
c.Makeacomplementarycopyforeachstrand.
ModelsofDNAReplication
Threemodelshavebeenproposedforit:
Conservativemodel
Semiconservativemodel
Dispersivemodel
a.Conservativemodelproposedbothstrandsofonecopywouldbe
entirelyoldDNA,whiletheothercopywouldhavebothstrands
ofnewDNA.
b.DispersivemodelwasthatdsDNAmightfragment,replicate
dsDNA,andthenreassemble,creatingamosaicofoldandnew
dsDNAregionsineachnewchromosome.
c.SemiconservativemodelisthatDNAstrandsseparate,anda
complementarystrandissynthesizedforeach,sothatsibling
chromatidshaveoneoldandonenewstrand.Thismodelwasthe
winnerintheMeselsonandStahlexperiment.
TheMeselsonStahlExperiment
1. MeselsonandStahl(1958)grewE.coliinaheavy(not
radioactive)isotopeofnitrogen,15Nintheformof
15NH Cl.Becauseitisheavier,DNAcontaining15Nis
4
moredensethanDNAwithnormal14N,andsocanbe
separatedbyCsCldensitygradientcentrifugation
2. OncetheE.coliwerelabeledwithheavy15N,the
researchersshiftedthecellstomediumcontainingnormal
14N,andtooksamplesattimepoints.DNAwasextracted
fromeachsampleandanalyzedinCsCldensitygradients
Fig. 3.2 The Meselson-Stahl experiment, which showed that DNA replicates
semiconservatively
60120000
3.Afteronereplicationcycleinnormal14Nmedium,allDNAhaddensity
intermediatebetweenheavyandnormal.Aftertworeplicationcycles,
thereweretwobandsinthedensitygradient,oneattheintermediate
position,andoneatthepositionforDNAcontainingentirely 14N.
4.Resultscomparedwiththethreeproposedmodels:
a.Doesnotfitconservativemodel,becauseafteronegenerationthereisa
singleintermediateband,ratherthanonewithentirely 15NDNAand
anotherwithentirely14NDNA.
Replication Facts
DNA has to be copied
before a cell divides
DNA is copied during the S
or synthesis phase of
interphase
New cells will need identical
DNA strands
9
copyrightcmassengale
TheE.colichromosome
Onecircular,doublestrandedDNAmoleculeof
about4.6*106bp
Replicationbeginsinonlyoneplace,i.e.asingle
originofreplication(OriCinE.Coli)
Replicationmovesbothdirectionsuntilthetwo
replicationeffortsmeetattheterminationsite
Proteinmachinethataccomplishesreplicationis
thereplisome;
onereplisomeineachdirection
Replicationforksmove1000bp/sec;thusE.coli
canbereplicatedin38min=2280s
Eukaryotic
Biggerchromosomes,moreofthem
Notcircular,usually
Fruitflychromosomes:
Sexchromosome,2longautosomes,
onetinyautosome
1.65*108bp,14000genes
Human
22pairsofautosomes,sexchromosome
3.4*109bp,~22000genes
ReplicationisbidirectionalasinE.coli
Morethanoneoriginsoreplicationiscomparablyfasteventhoughrateislower
EnzymesofProkaryoticDNAReplication
Thedoublehelixisunwoundbytheenzymeshelicase,DNA
topoisomerase,andDNAgyrase
SSBP(singlestrandedbindingprotein)helpskeepstrands
separated
DNApolymeraseIII(polIII)isresponsibleformostofDNA
synthesis
addsnucleotidestothe3endofthedaughterstrandofDNA;
DNAsynthesisisfrom5'to3'
RequiresRNAprimersasaguideforsynthesis
RNAprimersaremadebytheenzymeprimase
12
DNApolymeraseI:involvedinproofreading
andDNArepair
DNAligase:involvedinconnectedendsof
replicatedDNAtogether
RolesofDNAPolymerases
Animation:DNABiosynthesis:HowaNewDNAStrandisMade
1.AllDNApolymeraseslinkdNTPsintoDNAchains(Figure3.4).Main
featuresofthereaction:
a.Anincomingnucleotideisattachedbyits5phosphategrouptothe3
OHofthegrowingDNAchain.EnergycomesfromthedNTP
releasingtwophosphates.TheDNAchainactsasaprimerforthe
reaction.
b.Theincomingnucleotideisselectedbyitsabilitytohydrogenbond
withthecomplementarybaseinthetemplatestrand.Theprocessisfast
andaccurate.
c.DNApolymerasessynthesizeonlyfrom5to3.
2. TheenzymeKornbergisolatedwasbelievedtobetheonlyDNA
polymeraseinE.coli.However,mutationsinthisgene(polA1)were
notlethal,indicatingthatotherDNApolymerasesmustexistinE.coli.
3. Temperaturesensitivemutantsareusedtostudy
essentialgenes.At37CpolAex1strainsare
normal,andtheproteinhasnormalactivityin
vitro.At42C,however,theproteinlacks53
exonucleaseactivity,andbacterialcellswiththis
mutationaredead.
4. AdditionalDNApolymeraseshavebeenisolated,
includingDNApolymeraseII(1970),DNA
polymeraseIII(1971),DNApolymeraseIV,and
DNApolymeraseV.
ThepropertiesofDNApolymerases
5. ThepropertiesofknownE.coliDNApolymerasesare:
a. DNApolymeraseIisasinglepeptideencodedbypolAandusedforDNA
replication.ReplicatesDNAinthe53direction.Has53
exonucleaseactivitytoremovenucleotidesfrom5endofDNAorfroman
RNAprimer.
b. DNApolymeraseIIisasinglepeptideencodedbypolB.UsedforDNA
repair.
c. DNApolymeraseIIIhasthreepolypeptidesubunitsinthecatalyticcoreof
theenzyme:(encodedbythednaEgene),(dnaQ),and(holE).
Holoenzymehasanadditionalsixdifferentpolypeptides.ReplicatesDNA
inthe53direction.
d. DNApolymeraseIVisencodedbythedinBgene,andisusedinDNA
repair.
e. DNApolymeraseVisencodedbyumuDC,andisusedinDNArepair.
6. E.coliDNApolymerasesusedforDNAreplication(DNApolymerase
IandDNApolymeraseIII)have35exonuclease(proofreading)
activity.
InitiationofReplication
1.ReplicationstartswhenDNAattheoriginofreplicationdenaturesto
exposethebases,creatingareplicationfork.Replicationisusually
bidirectionalfromtheorigin.E.colihasoneorigin,oriC,whichhas:
a. Aminimalsequenceofabout245bprequiredforinitiation.
b. Threecopiesofa13bpATrichsequence.
c. Fourcopiesofa9bpsequence.
2. EventsinE.coliinitiatingDNAsynthesis,derivedfrominvitrostudies
(Figure3.5):
a. Initiatorproteinsattach.E.colisinitiatorproteinisDnaA(fromthe
dnaAgene).
b. DNAhelicase(fromdnaB)bindsinitiatorproteinsontheDNA,and
denaturestheATrichregionusingATPasanenergysource.
c. DNAprimase(fromdnaG)bindshelicasetoformaprimosome,which
synthesizesashort(510nt)RNAprimer. .
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.
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.
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.
1.WhenDNAdenaturesattheoriC,replicationforksare
formed.DNAreplicationisusuallybidirectional,butwill
considereventsatjustonereplicationfork(Figure3.6):
a.SinglestrandDNAbindingproteins(SSBs)bindthessDNA
formedbyhelicase,preventingreannealing.
b.Primasesynthesizesaprimeroneachtemplatestrand.
c.DNApolymeraseIIIaddsnucleotidestothe3endoftheprimer,
synthesizinganewstrandcomplementarytothetemplate,and
displacingtheSSBs.DNAismadeinoppositedirectionsonthe
twotemplatestrands.
d.Newstrandmade53insamedirectionasmovementofthe
replicationforkisleadingstrand,whilenewstrandmadein
oppositedirectionislaggingstrand.Leadingstrandneedsonly
oneprimer,whilelaggingneedsaseriesofprimers.
2.HelicasedenaturingDNAcausestighterwindinginother
partsofthecircularchromosome.Gyraserelievesthis
tension.
3.Leadingstrandissynthesizedcontinuously,whilelagging
strandissynthesizeddiscontinuously,intheformof
Okazakifragments.DNAreplicationistherefore
semidiscontinuous.
4.Eachfragmentrequiresaprimertobegin,andisextendedby
DNApolymeraseIII.
5.Okazakidatashowthatthesefragmentsaregraduallyjoined
togethertomakeafulllengthdsDNAchromosome.DNA
polymeraseIusesthe3OHoftheadjacentDNAfragment
asaprimer,andsimultaneouslyremovestheRNAprimer
whileresynthesizingtheprimerregionintheformofDNA.
Thenickremainingbetweenthetwofragmentsissealed
withDNAligase.(Fig.3.7)
Editing&proofreadingDNA
1000bases/second=
lotsoftypos!
DNApolymeraseI
proofreads&correctstypos
repairsmismatchedbases
removesabnormalbases
repairsdamage
throughoutlife
reduceserrorratefrom
1in10,000to
1in100millionbases
6.Keyproteinsareassociatedtoformareplisome.
TemplateDNAprobablybendstoallowsynthesis
ofbothleadingandlaggingstrandsatthe
replicationfork
ReplicationofcircularDNAandthe
supercoilingproblem
1.Somecircularchromosomes(e.g.,E.coli)are
circularthroughoutreplication,creatingatheta
like()shape.Asthestrandsseparateononeside
ofthecircle,positivesupercoilsformelsewhere
inthemolecule.Replicationforkmovesabout
500nt/second,soat10bp/turn,replicationfork
rotatesat3,000rpm.
2.Topoisomerasesrelievethesupercoils,allowing
theDNAstrandstocontinueseparatingasthe
replicationforksadvance.
Fig. 3.11 Diagram showing the unreplicated, supercoiled parent strands and the
portions already replicated
RollingCircleReplication
1.Anothermodelforreplicationisrollingcircle(Figure
3.10),whichisusedbyseveralbacteriophages,including
X174(afteracomplementismadeforthegenomic
ssDNA)and(aftercircularizationbybasepairing
betweenthestickyssDNAcosends)
2.Rollingcirclereplicationbeginswithanick(single
strandedbreak)attheoriginofreplication.The5endis
displacedfromthestrand,andthe3endactsasaprimer
forDNApolymeraseIII,whichsynthesizesacontinuous
strandusingtheintactDNAmoleculeasatemplate.
3.The5endcontinuestobedisplacedasthecirclerolls,
andisprotectedbySSBsuntildiscontinuousDNA
synthesismakesitadsDNAagain.
Fig. 3.10 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.
4.ADNAmoleculemanygenomesinlengthcanbemadeby
rollingcirclereplication.Duringviralassemblyitiscut
intoindividualviralchromosomesandpackagedinto
phagehead.
5.Bacteriophage,regardlessofwhetherenteringthelytic
orlysogenicpathway,circularizesitschromosome
immediatelyafterinfection.
a.Inalysogenicinfection,thecircularDNAintegratedintoa
specificsiteintheE.colichromosomebyacrossoverevent.
b.Inalyticinfection,rollingcirclereplicationproducesalong
concatamerofDNA,andtheaviralendonuclease(productof
thetergene)recognizethecossitesandmakesthestaggeredcuts
thatusedtoassemblenewvirusparticles.
DNAReplicationinEukaryotes
DNAreplicationisverysimilarinbothprokaryotesand
eukaryotes,exceptthateukaryoteshavemorethan
onechromosome.Thelargersizeandcomplex
packagingofeukaryoticchromosomesmeansthey
mustbereplicatedfrommultipleoriginsof
replication.
TheenzymesofeukaryoticDNAreplicationaremore
complexthanthoseofprokaryoticcells.
60120000
Replicons
1. Eukaryoticchromosomesgenerallycontainmuch
moreDNAthanthoseofprokaryotes,andtheir
replicationforksmovemuchmoreslowly.Ifthey
wereliketypicalprokaryotes,withonlyoneorigin
ofreplicationperchromosome,DNAreplication
wouldtakemanydays.
2. Instead,eukaryoticchromosomescontainmultiple
origins,atwhichDNAdenaturesandreplication
thenproceedsbidirectionallyuntilanadjacent
replicationforkisencountered.TheDNA
replicatedfromasingleoriginiscalledareplicon,
orreplicationunit
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
3. Ineukaryotes,repliconsizeissmallerthanitisin
prokaryotes,replicationisslower,andeach
chromosomecontainsmanyreplicons.Numberand
sizeofrepliconsvarywithcelltype.
4. NotalloriginswithinagenomeinitiateDNA
synthesissimultaneously.Cellspecificpatternsof
originactivationareobserved,sothatchromosomal
regionsarereplicatedinapredictableorderineach
cellcycle.
Synthesizingtheendsofthechromosomesis
difficultbecauseofthelackofaprimer.
WitheachroundofDNAreplication,thelinear
eukaryoticchromosomebecomesshorter.
Fig. 3.13 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.
60120000
Chapter3slide36
InitiationofReplication
1. Eukaryoticoriginsaregenerallynotwellcharacterized;thoseofthe
yeastSaccharomycescerevisiaeareamongthebestunderstood.
2. ChromosomalDNAfragments(about100bp)thatareabletoreplicate
autonomouslywhenintroducedintoyeastasextracellular,circular
DNAareknownasARSs(autonomouslyreplicatingsequences).
3. ARSsareyeastreplicators.Thethreesequenceelementstypically
foundinARSsareA,B1,andB2.
4. Initiatorproteininyeastsisthemultiproteinoriginrecognitioncomplex
(ORC),whichbindstoAandB1.Otherreplicationproteinsjoin,
includingonethatunwindsDNAatB2.Theyeastoriginofreplication
isbetweenregionsB1andB2.
5. DNAandhistonesmustbedoubledineachcellcycle.G1preparesthe
cellforDNAreplication,chromosomeduplicationoccursduringS
phase,G2preparesforcelldivision,andsegregationofprogeny
chromosomesoccursduringMphase,allowingthecelltodivide.
6. Cellcyclecontroliscomplex,andonlyoutlinedhere.
Yeasts,inwhichchromosomalreplicationiswellstudied,
serveasaeukaryoticmodelorganism.
7. Initiationofreplicationhastwoseparatesteps,controlled
bycyclindependentkinases(Cdks)thatarepresent
throughoutthecellcycle,exceptduringG1.
a. IntheabsenceofCdskduringG1,replicatorselectionoccurs.
ORCandotherproteinsassembleoneachreplicatortofrompre
replicativecomplexes(preRC).
b. WhencellentersSphase,Cdksarepresent,andactivatepreRCs
toinitiatereplication.
c. CdkactivityinhibitsanotherroundofpreRCformationuntilthe
cellagainentersG1,whenCdksareabsent.
EukaryoticReplicationEnzymes
1.EnzymesofeukaryoticDNAreplicationarentaswell
characterizedastheirprokaryoticcounterparts.The
replicationprocessissimilarinbothgroupsDNA
denatures,replicationissemiconservativeand
semidiscontinuousandprimersarerequired.
2. FifteenDNApolymerasesareknowninmammaliancells:
a. ThreeDNApolymerasesareusedtoreplicatenuclearDNA.Pol
(alpha)extendsthe10ntRNAprimerbyabout30nt.Pol(delta)
andPol(epsilon)extendtheRNA/DNAprimers,oneonthe
leadingstrandandtheotheronthelagging(itisnotclearwhich
synthesizeswhich).
b. OtherDNApolsreplicatemitochondrialorchloroplastDNA,orare
usedinDNArepair.
ReplicatingtheEndsofChromosomes
1.Whentheendsofchromosomesarereplicated
andtheprimersareremovedfromthe5ends,
thereisnoadjacentDNAstrandtoserveasa
primer,andsoasinglestrandedregionisleftat
the5endofthenewstrand.Ifthegapisnot
addressed,chromosomeswouldbecomeshorter
witheachroundofreplication
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
2.Mosteukaryoticchromosomeshaveshort,speciesspecificsequences
tandemlyrepeatedattheirtelomeres.BlackburnandGreiderhave
shownthatchromosomelengthsaremaintainedbytelomerase,which
addstelomererepeatswithoutusingthecellsregularreplication
machinery.
3. IntheciliateTetrahymena,thetelomererepeatsequenceis5
TTGGGG3(Figure3.15).
a. Telomerase,anenzymecontainingbothproteinandRNA,bindstothe
terminaltelomererepeatwhenitissinglestranded,synthesizinga3nt
sequence,TTG.
b. The3endofthetelomeraseRNAcontainsthesequenceAAC,which
bindstheTTGpositioningtelomerasetocompleteitssynthesisofthe
TTGGGGtelomererepeat.
c. Additionalroundsoftelomeraseactivitylengthenthechromosomeby
addingtelomererepeats.
4. Aftertelomeraseaddstelomeresequences,chromosomalreplication
proceedsintheusualway.Anyshorteningofthechromosomeendsis
compensatedbytheadditionofthetelomererepeats.
5. IfthesequenceofthetelomeraseRNAismutated,telomereswill
correspondtothemutantsequence,ratherthantheorganismsnormal
telomeresequence.UsinganRNAtemplatetomakeDNA,telomerase
functionsasareversetranscriptasecalledTERT(telomerasereverse
transcriptase).
6. Telomerelengthmayvary,butorganismsandcelltypeshave
characteristictelomerelengths.Mutantsaffectingtelomerelengthhave
beenidentified,anddataindicatethattelomerelengthisgenetically
controlled.Shorteningoftelomereseventuallyleadstocelldeath,
andthismaybeafactorintheregulationofnormalcelldeath.
44