8The Replication of DNA复制.docx
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8The Replication of DNA复制.docx
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8TheReplicationofDNA复制
TheReplicationofDNA
Chapter8
A.TheChemistryofDNASynthesis
B.DNAPolymerase
C.Proofreading
D.ReplicationFork
E.Okazakifragments
F.RNAPrimer
G.RNaseH
H.DNAHelicase
I.Single-StrandedBindingProteins
J.Topoisomerase
K.SpecializedDNAPolymerases
L.SlidingClamps
M.DNASynthesisattheReplicationFork
N.InitiationofReplication
O.EukaryoteReplication
P.FinishingReplication
A.TheChemistryofDNASynthesis
DNAsynthesisrequires4components:
1.AtemplateDNA(aparentalstrandthatiscopied)
2.aprimerthatprovidesa3’hydroxyltowhichnewbasepairscanbeadded
3.thefourdeoxynucleotidetriphosphates(dATP,dTTP,dGTP,anddCTP)
4.DNApolymerase
DNApolymeraserequiresa3’hydroxylterminustowhichitcanaddnewbasepairs.
AprimerisashortpieceofRNAthatprovidesa3’OHforDNAsynthesis.
TheprimerisboundtothetemplateDNAformingaprimer:
templatejunctionwhereDNAsynthesiscanoccur.
Theprimermusthavea3’OHtowhichthenewbasepaircanbeadded.
Thetemplatedetermineswhichnucleotidewillbeaddedtotheprimer.
InDNAsynthesisnucleotidesareaddedtothe3’OHoftheprimer:
templatejunction.
Nucleotidetriphosphatesmustbeavailabletobeaddedtotheprimer.
Nucleotidetriphosphateshave3phosphategroups.Thesearelabeledα(alpha),β(beta),andγ(gamma).
DNAsynthesisrequires4dNTPs.Thesearenucleotidetriphosphates.
ThephosphatesarelabeledwithGreekletters:
α(alpha),β(beta),andγ(gamma).
DNAandRNAarebothsynthesizedbyextendingthe3’end.Thehydroxyl(OH)atthe3’endattachestotheα-phosphorylgroupofthenewnucleotidetriphosphate.Apyrophosphate,whichistheβandγphosphatesisreleasedbythereaction.
Thereactionis:
The∆Gofthisreactionisonly-3.5kcal/mol.
Additionalenergyisprovidedbybreakingthepyrophosphateintotwophosphategroups.
Thischangesthereactionto:
Thisreactionhasalargenegative∆Gof-7kcal/mole.Thislarge∆(delta)Gpushesthisreaction.
B.DNAPolymerase
DNApolymerasecatalyzestheformationofthecovalentbondbetweenthe3’OHandtheαphosphate.
DNApolymerasehasasingleactivesitethatanyofthefourdNTPscanfitin.
DNApolymerasedetectstheabilityofthenewnucleotidetobasepairtothetemplateDNA.
Onlywhenacorrectbasepairispresentarethe3’OHandtheα-phosphateofthenewnucleotideintheoptimumpositionforDNApolymerasetocatalyzethereaction.
DNApolymerasedistinguishesbetweendNTPs(deoxyribonucleotidetriphosphosphates)andrNPTs(ribonucleotidetriphosphosphates).
InDNApolymerasetheactivesitewherethenucleotidebindshasnoroomforthe
2’OHthatispresentinrNTPs.
Inthesideoftheactivesitethereare2aminoacidsthatmakevanderWaalsinteractions(noncovalentbonds)withthesugarring.Thereisnoroomforthe2’OH.
ThemolecularstructuresofdifferentDNApolymeraseshavebeendetermined.
Whatmethodscanbeusedtodeterminethestructureofaprotein?
x-raycrystallographyandNMR
ThestructuresofdifferentDNApolymeraseshavebeendeterminedusingx-raycrystallography.
FromthesestructuresitisclearthattheDNAfitsinacleftintheDNApolymerasemolecule.Thiscleftresemblesthepalmofapartiallyclosedhand.Thereare3domainsinDNApolymeraseandtheyarecalledthefingers,thumbandpalm.
Thefingersandthumbaremadeofalphahelices.
Palm
Thepalmofthehandisaβsheetthathastheactivesite.
Thereare2divalentmetalions(usuallyMg++orZn++boundintheactivesite.
Onemetalionpullsthehydrogenawayfromthe3’OHtoproducea3’O-thatcanbindtotheα-phosphate.
OnemetalionpullstheHawayfromthe3’OHtoproducea3’O-(oxygenion).
Theothermetalionattractsthenegativechargesofthephosphatesofthenewnucleotide.Thishelpsstabilizethepyrophosphate.
Asecondmetalionstabilizesthephosphates.
TheaminoacidsinthepalmoftheDNApolymerasehydrogenbondtotheDNA.Thistestswhetherornotthecorrectbasepairhasbeenadded.Thehydrogenbondsdonotformiftheincorrectbasepairisadded.
Ifthewrongbaseisadded,catalysisslowsandtheDNApolymerasehaslessaffinityfortheDNA.
Whenthewrongbaseispresent,thereducedaffinityandslowedcatalysiscanallowthenewlysynthesizedDNAtobereleasedfromtheactivesite.ThenitcanbeboundinaseparateactivesitethatproofreadstheDNA.
Fingers
ThefingersoftheDNApolymerasebindtothenewnucleotide.Ifitisthecorrectnucleotide,thefingerdomainmovestopushthenucleotideclosertothemetalionsandthetemplate.
Thumb
ThethumbformsnoncovalentbondswiththeDNAthathasjustbeenaddedtothenewstrand.
Thisholdstheprimerinplaceinrelationshiptotheactivesite.ItalsohelpsholdtheDNApolymeraseonthegrowingDNAstrand.
Thethumbbindstotherecentlyaddednucleotides.ThishelpsholdtheDNApolymeraseontheDNA.
StepsinDNASynthesis
1.ThenewnucleotidebasepairswiththeDNAtemplate.
2.ThefingersoftheDNApolymerasemovethenucleotideintotheactivesite.
3.Aphosphodiesterbondisformed.
4.Thefingersmovebackandtheprimer:
templatejunctionmovesbyonebasepair.
DNApolymeraseisveryfast.Itisabletoadd1000nucleotidespersecond.
ThespeedofDNApolymeraseismostlyduetoitsprocessivity.
Processivity
Processivity:
Theabilityofanenzymetorepeatedlycontinueitscatalyticfunctionwithoutdissociatingfromitssubstrate.
ADNApolymerasecanbindtotheprimer:
templatejunctionandaddafewnucleotidesorasmanyas50,000.
TheinitialbindingoftheDNApolymerasetotheprimer:
templatejunctionisslowandratelimiting.Ittakesabout1second.OncetheDNApolymeraseisbound,itcanveryquicklyaddnucleotides.
AnonprocessiveDNApolymerasecouldadd1nucleotidepersecond.Aprocessiveenzymecanaddasmanyas1000nucleotidesinasecond.
ProcessiveenzymeslikeDNApolymerasecatalyzemanyreactionsbeforetheyreleasethesubstrate.
TheprocessivityofDNApolymeraseispartlyduetoit’sabilitytoslidealongtheDNAtemplate.Afteranewnucleotideisadded,DNApolymerasepartlyreleasestheDNA,buttheelectrostaticnoncovalentbondsbetweenthethumbandtheDNAaremaintained.TheDNApolymerasethenquicklyrebindstotheDNAinpositiontoaddanothernucleotide.
Thus,theinteractionsbetweentheDNAandthethumbpartoftheDNApolymerasecontributetotheprocessivityoftheenzyme.
Processivityistrulyamazing.ItallowsDNApolymerasetoreplicateDNAatanincrediblerate,sothecellcyclecanproceedandcellscanreplicate.
C.Proofreading
Specializedenzymescanalsobreakthephosphodiesterbondsinnucleicacids.
WhatisthenameforenzymesthatcutDNA?
DNases(deoxyribonucleases)
ThosethatspecificallycutRNAarecalledRNases(ribonucleases).
Enzymesthatnibblefromtheendofanucleicacidandremoveonebasepairatatimearecalledexonucleases.
Thosethatcutwithinastrandarecalledendonucleases.
DNApolymerasewillattachanincorrectbaseatafrequencyofabout1in105-106basepairs.
Thisispartlyduetobasepairstautomerizing.Thiscancausethewrongbasetobeadded.
Whatisatautomer?
DifferentTautomericForms:
Review
Eachofthebasescanexistin2alternativetautomericstates.Thesestatesareinequilibrium.
Tautomer:
analteredchemicalstructure
Thenormalbasepairisomerizes(changesform).
Mostofthetimethebaseisinthenormalstate.Onlyrarelyarethealteredformspresent.
Thealteredformscanformdifferenthydrogenbonds,sotautomerizationcancausemutationsintheDNA.
Thenitrogenatomsboundtotheadenineandcytosineareusuallyintheaminoform,butcanchangetotheiminoform.
Theoxygenatomsboundtoguanineandthymineareusuallyintheketoform,butcanchangetotheenolform.
Mostoftimetheincorrectbaseisimmediatelyremovedbythe3’to5’exonucleasethatispartofDNApolymerase.Theincorrectbasepairisremovedsothatthecorrectbasepaircanbeadded.
Thisisalsocalledtheproofreadingexonuclease.
Proofreadingisre-readingsomethingyouhavewrittentofindtheerrors.Itisalsocalledediting.
TheDNApolymeraseproofreadsthenewlymadeDNAtocheckformistakes.
Whenanincorrectbasehasbeenadded,thenoncovalentinteractionswiththepalmregionoftheDNApolymerasearechanged.Thisslowscatalysissoanotherbaseisnotadded.Therateofsynthesisisslowedbecausethe3’OHoftheincorrectbaseisnotinthecorrectlocationforthenextbasepairtobeadded.
Duetothechangeinnoncovalentbonds,theprimer:
templatejunctionmovesawayfromthepolymerasesiteinthepalmanddowntotheexonucleasesite.Theincorrectbaseisremovedbytheproofreadingexonucleaseandthentheprimer:
templatejunctionmovesbacktotheactivesitefortheDNApolymerase.
AmispairedbasepaircausesDNApolymerasetoslowanddisruptsnoncovalentbondsinthepalm.
Theprimer:
templatejunctionmovestotheexonucleaseactivesiteandthemispairedbaseisremoved.
Thentheprimer:
templatejunctionmovesbacktothepolymeraseactivesiteandDNAsynthesiscontinues.
D.ReplicationFork
IncellsbothstrandsofDNAarecopiedatthesametime.The2strandsmustseparatesotheycanbothbeusedastemplates.
ThejunctionbetweenthenewlyseparatedstrandsandtheunreplicatedDNAduplexiscalledthereplicationfork.
Thisiscalledareplicationfork.
Itremindedscientistsofaforktoeatwith.
replicationfork
5’to3’direction
AllDNAandRNApolymerasesworkinthe5’to3’directionandcannotwork3’to5’.Newbasepairscanonlybeaddedtothe3’OH.ThiscomplicatesDNAreplica
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