生物化學(xué)課件DNA的生物合成和損傷修復(fù)DNA

Chapter15DNABiosynthesisandDNADamageRepairSectionOneTheGeneralFeaturesofReplicationofChromosomalDNADNAreplicatessemiconservatively.⑴.WatsonandCrickpredictedthatDNAmightsemiconservativelyreplicate.

thehypothesis___________p___________d___________p___________p____________d____________p⑵.In1958MeselsonandStahldemons-tratedthesemiconservativenatureofDNAreplicationinEcoli.Theexperiment:CsClequilibriumgradientdensityultracentri-

fugationof15NlabeledEcoliDNA.

ThedensityofDNAwasincreasedbylabelingitwith15N,aheavyisotopeofnitrogen.ThiswasdonebygrowingEcoli15ge-nerationsinamediumthatcontained15NH4Clasitsonlynitrogensource.

15NDNAwasextractedandsubjectedtoCsClequilibriumgradientdensityultracen-trifugation.TheDNAbandpositionwasrecorded.Therewasonebandof15NDNA.Thebacteriaweretransferredtoan14NH4Clmediumandgrownforonegeneration.TheDNAdensitywasdeterminedagain.ThepositionoftheDNAbandwascomparedwiththatofthe15NDNA.Whatwastheresult?TherewasoneDNAband.Ithadalowerdensitythan15NDNAbecauseitspositionwasaboveonthatof15NDNA.Itwas15N/14NhybridDNA.Afteranothergenerationgrowinginthe

14NH4ClmediumthebacterialDNAdensitywasdetermined.ThereweretwoDNAbands.OnehalfoftheDNAwas14NDNA,andanotherhalfwashybridDNA.Insucceedinggenerationstheratioof14NDNAtohybridDNAincreasedgradually.ThehybridDNAbecamelessandless..

summaryDNAreplicatesinasemiconservativeman-ner.Whenthetwoparentalstrandssepa-rate,eachservesasthetemplateformakinganew,complementarystrand.2.ThepointatwhichseparationofthestrandsandsynthesisofnewDNAtakesplaceisknownasthereplicationfork.ThereplicationforkisY-shaped.Twoarms(V)areseparatedstrandswhichactasthetemplateandDNAsynthesisisactivelytakingplace.Thebody(I)istheparentalDNA.

3.DNAreplicationisusuallybidirectional.

⑴.Replicon:Anypiecewhichreplicatesasasingleunitiscalledareplicon.AllbacterialchromosomesandmanyphageandvirusDNAmoleculesarecircularandcomprisesinglereplicons.Incontrasteukaryoticchromosomesconsistofmultiplereplicons.⑵.Origin:Theinitiationwithinarepliconalwaysoccursatafixedpointknownastheorigin.⑶.Terminus:Inacircularrepliconthereisasingleterminationsiteroughly180°oppositetheuniqueorigin.

SummaryInacircularrepliconreplicationbeginsfromthefixedoriginandformstworepli-cationforks.Thetworeplicationforksproceedbidirec-tionallyawayfromtheoriginandthestrandsarecopiedastheyseparateuntiltheterminusisreached.4.DNAreplicationissemidiscontinuous.⑴.ThemechanismofDNAreplicationallowsonlyforsynthesisina5’3’direction.⑵.ThetwostrandsofDNAareantiparallel.QuestionHowistheparentalstrandthatruns5’3’pastthereplicationforkcopied?

Theanswerissemidiscontinuousreplica-tion.

Ateachreplicationforkonestrand(thelead-ingstrand),whosetemplateruns3’5’pastthereplicationfork,issynthesizedasonecon-tinuouspiece,whiletheotherstrand(thelag-gingstrand),whosetemplateruns5’3’pastthereplicationforkismadediscontinuouslyasshortfragmentsinthereversedirection.TheseshortfragmentsarecalledOkazakifragments.TheyarejoinedbyDNAligaseandformthelaggingstrand.5.OriginscontainshortAT-richrepeatse-quences.⑴.Prokaryoticandeukaryoticoriginshavecommonfeatures:a.Theyconsistofmultipleuniqueshortrepeatsequences.b.Thesesequencesarerecognitionandbindingsitesofmulti-subunitinitiationfactors.c.ThesesequencesareusuallyAT-rich.⑵.Ecoli‘soriginiscalledoriC.Itis254bplongandcontainsthree13-bpdirectrepeatsandfour9-bpinvertedre-peats.6.DNAreplicationneedspriming.⑴.DNApolymerasescannotinitiateDNAreplicationbystartinganewDNAchain.Theycanonlyaddnucleotidestothe3’endofanexistentpieceofRNAorDNAunderthedirectionofthetemplate.TheexistentpieceofRNAorDNAarecalledprimer.⑵.TheleadingstrandandallOkazakifrag-mentsareprimedbysynthesisofashortpieceofRNA(anRNAprimer),whichisthenelongatedwithDNAbyDNApoly-merase.⑶.TherearealsoDNAprimingornucleotidepriming.7.Multi-enzymesandproteinsparticipateinDNAreplication.⑴.TopoisomerasesregulatethetypeandlevelofsupercoilingofdsDNA.⑵.HelicasesunwindthedsDNA.⑶.SSBsbindandstabilizethesingleDNAstrand.⑷.PrimasesynthesizestheRNAprimer.⑸.DNApolymeraseselongateDNAchains.⑹.DNAligasejoinsOkazakifragments.8.DNAreplicationisofhighfidelity.⑴.Therearetwotypesofreplicationerrors.a.base(nucleotide)substitution.b.nucleotideinsertionordeletion.⑵.Tofreadingcontrol⑶.mismatchrepair.

SectionTwoFeaturesofDNAPolymerasesThesubstratesofDNApolymerasesare2.TheactivecenterofDNApolscatalyzesDNAsynthesis.⑴.TheactivecentercandifferentiatedNTPfromNTP.⑵.DNApolscanchoosetherightnucleotideforbase-pairingwiththetemplatenucleo-tide.3.Thesemi-closedright-handedstructureoftheDNApol.iscomposedofthreedomains.⑴.thumbdomain,fingersdomainandpalmdomain⑵.twoactivecenters:polymeraseactivecenterand3’-5’exonucleaseactivecenter.Theyarelocatedinthepalmdomain.⑶.Thepalmdomainhasthreefunctions:ofreadingcontroltoremovethe‘mis-paired‘nucleotide.⑷.Thefingersdomainbindsthetemplatestrandandinteractswiththenucleotidethatentersthepolymeraseactivecenter.⑸.Thethumbdomainkeepstheprimer-templatejunctioninpositionintheactivecenterandmakesthepolymerasebindthesubstratestightly.4.Theproteinof‘slidingclamp’,thatencirclestheDNAandinteractswiththeDNApol,isresponsiblefortheprocessivityoftheDNApolymerase.ProcessivityoftheDNApolmeansDNApolgoingonsynthesisofDNArapidlywithoutstopping.SectionThreeDNAReplicationinEcoli1.EcoliDNAreplicationinitiatesatoriCinaprocessmediatedbyamulti-proteincom-plex.⑴.ProteinfactorsparticipateininitiationatoriCinclude:DnaA,DnaB,DnaC,HU,to-poisomeraseII(gyrase)andSSB.

⑵.DnaAproteinformsacomplexof20-40molecules,eachboundtoanATPmole-cule,aroundwhichtheoriCDNAwithfour9-bprepeatsbecomeswrapped.⑶.Thisfacilitatesmeltingofthree13-bprepeatswhichopentoallowbindingofDnaBprotein.⑷.WiththehelpofDnaC,DnaBbindstheopenedDNA.DnaBisahelicaseandcanunwinddsDNAbyusingtheenergyofATPhydrolysis.⑸.SSBbindsthesingleDNAstrand.⑹.GyraseintroducesnegativesupercoilsintothedsDNAaheadofthereplicationfork.⑺.Thepreprimingcomplexisformed.2.PrimasesynthesizesRNAprimer.⑴.DnaGisaprimase.ItbindsthetemplateandisactivatedbyDnaB.⑵.TheactivatedprimasesynthesizesRNAprimer.3.DNApolIIIelongatesDNAandDNApolIremovestheprimer.⑴.DNApolIIIistheprincipalenzymeinelongationofDNA.⑵.Thestructureoftheholoenzymeiscomposedof10differentsubunitsintotalnumberof16.(αεθ)2ζ2γ2δδ’χψβ2Twocoreenzymes(αεθ)2areheldtogetherbyaγcomplex.

αsubunit:DNAsynthesis

εsubunit:proofreading

βsubunit:the‘slidingclamp’AsingleholoenzymeisresponsibleforthesynthesisofbothleadingstrandandOkazakifragmentsoflaggingstrand.TheholoenzymeofDNApolIIIhastwocoreenzymes.Oneisresponsibleforthesynthesisofleadingstrand.TheotherforthesynthesisofOkazakifragments.Becausethetemplateofthelaggingstrandisloopedoutbothleadingandlaggingstrandsynthesismoveinthesamedirection.WhenthelaggingstrandcoreenzymecompletesanOkazakifragment,itre-leasesthestrand.Thentheprimosome(DnaB-DnaGcom-plex)synthesizesanotherprimerandthecoreenzymeelongatesandcompletesanotherOkazakifragment.⑶.DNApolIhasonlyonepolypeptide.Ithasthreeenzymeactivities:a.thepolymeraseactivityb.the3’5’exonucleaseactivityc.the5’3’exonucleaseactivity.Subtilicincancutitintotwofragments.Thelargefragmentiscalledklenowfragment.Ithasthepolymeraseactivityandthe3’5’exonucleaseactivity.

The5’3’exonucleaseremovestheprimer.

ThepolymerasefunctionsimultaneouslyfillsthegapwithDNAbyelongatingthe3’-endoftheadjacentOkazakifragment.ThefinalphosphodiesterbondbetweenthefragmentsismadebyDNAligase.4.TusproteinrecognizesandbindstotheTERsite.Thatpreventsthereplicationforkadvancing.DNAreplicationterminates.

5.Onlyatthefull-methylatedoriCcaninitiatereplication.Thereare11copiesofsequenceGATCinoriC.ThedammethylasethatrecognizesthesequenceGATCandplacesamethylgroupontheA.GATCisapalindrome.TheoppositestrandalsoreadsGATCinthe5’3’direction.5’GATC3’3’CTAG5’Onlyatthefull-methylatedoriCcaninitiatereplication.DuringDNAreplicationoriCisalsore-plicated.Theparentalstrandismethylated,butthenewlysynthesizeddaughterstrandisn’t.AlthoughGATCinthedaughterstrandisalsodestinedtobecomemethylated,about10minuteselapsebeforethiscanhappen.6.TwotypesoftopoisomerasesarerequiredinDNAreplication.DNAunwindingatthereplicationforkcangeneratepositivesupercoilingofthedsDNAaheadofthereplicationfork.DNAgyraseusestheenergyofATPhydrolysistointroducenegativesu-percoilingintoDNAhenceremovingsupercoiling..Ecolichromosomeiscircular.WhenDNAreplicationiscompleted,therearetwodaughtercircularDNAlinkedtogether(catenane).EcolitopoisomeraseIVcanunlinkthecatenane.SectionFourDNAReplicationinEukaryotesTherearefivetypesofcommoneukaryoticDNApolymerases:α,β,γ,δ,ε.2.EukaryoticandprokaryoticenzymesandproteinfactorsthatparticipateinDNAreplicationatthereplicationforkarecomparable.3.Afterthepolymeraseα/

primasecomplexinitiatesreplicationpolymeraseδstartselongation.4.Therearetwomechanismsofremovingprimers.

a.

RNaseHIandFEN1-dependentmechanism.b.helicaseDna2andEFN1-dependentmechanism.

5.Theeukaryoticchromosomerepli-catesonlyonceinacellcycle.a.pre-RCfoprmsintheG1-phaseandisactivatedintheS-phase.b.Cdkcontrolstheformationandactivationofpre-RC.6.Telomeraseparticipatesinthereplica-tionoftelomereDNA.a.theproblemofreplicatingtheendsoflinearchromosomes:

Theendsoflinearchromosomescan’tbefullyreplicatedbysemidiscontinuousreplicationasthereisnoDNAtoelon-gatetoreplacetheRNAremovedfromthe5’-endofthelaggingstrand.ThusgeneticinformationcouldbelostfromtheDNA.2.Telomereandtelomerasesolvetheproblem.Toovercomethis,theendofeukaryoticchromosomes(telomeres)consistsofhundredsofcopiesofasimplenon-informationalrepeatsequence(TTAGGG)withthe3’-endoverhangingthe5’-end.TheenzymetelomerasecontainsashortRNAmolecule,partofwhosesequenceiscomplimentarytothisrepeat.ThisRNAactsasatemplateforthead-ditionoftheserepeatstothe3’-over-hangbyrepeatedcyclesofelongation.Thecomplimentarystrandisthensynthe-sizedbynormallaggingstrandsynthesisleavinga3’-overhang.

SectionFiveDNAReplicationinMitochondriaandPhagesmtDNAreplicatesinD-loops.2.Phage’scircularDNAreplicatesinrollingcircle.SectionSixTheRepairofDNADamage1.PhysicalorchemicalagentsmaycauseDNAdamage.Therearereplicationerrors.ThereareseveralDNArepairsystemsinbothprokaryoticandeukaryoticcells.2.Mismatchrepairsystemrepairsthereplicationerrors.Replicationerrorsthatescapeproof-readinghaveamismatchinthedaug-hterstrand.Hemi-methylationoftheDNAafterrepli-cationallowsthedaughterstrandtobedistinguishedfromtheparentalstrand.ThemismatchedbaseisrecognizedandboundbyMutS.MutSandDNAcomplexrecruitsMutL.MutH,anendonuclease,joinsthemandmakesanickinthenewlysynthesizedDNAstrand.HelicaseUvrDandanexonucleaseremoveapieceofssDNAthatcontainstheerror.DNApolymeraseIIIfillsthegapandDNAligasesealsthenick.

3.DNADamageRepairSystems

a.DirectRepairSystem

ThemostcommonDNAdamageisformationofthyminedimerbyUVradiation.IntheTTdimerthereisacyclobutaneringbetweenthetwoneighbouringTresiduesinthesameDNAstrand.Thephotoreactivationrepairsystemisadirectrepairsystem.DirectrepaircanrepairDNAdamagewithoutremovingabaseornucleotide.Inthephotoreactivationrepairthephotolyaseisactivatedbyvisiblelightandmakesuseofthelightenergytobreakthecyclobutanering.

TheTTdimerarerestoredtotheoriginalstructure.b.BaseExcisionRepairSystemSinglebasedamagessuchasdeaminationofC,depurination,anddepyrimidinationarealsoverycommon.Glycosidasescanrecognizethedamagedbaseandremoveit.ThatresultsinanAPsite(apurinicorapyrimidinicsite).APendonucleasehydrolyzesthephos-phordiesterbondatthe5’-endoftheAPsite.APexonucleasecleavesthephosphor-diesterbondatthe3’-endoftheAPsite.andthedeoxyribose-phosphateisre-moved.Thegapleftisfilledwithanucleotidecomplementarytothetemplate.DNAligasemakesthefinalphosphor-diesterbond.c.NucleotideExcisionRepairSystemNucleotideexcisionrepairsystemrecog-nizesthedistortionoftheDNAdoublehelix.ThedistortionmaybecausedbyTT,CTorCCdimer.InEcolitheNERcomponentsconsistofUvrA,UvrB,UvrC,andUvrDproteins.UvrArecognizesthedistortionofDNAandcombineswithUvrBandATPtoseparatethedsDNA.UvrBrecruitsUvrC,anendonuclease.UvrCmakesnicksatbothsidesofthedamageontheDNAstrand.

UvrD,ahelicase,removesthedamagecontainedfragment.

DNApolymeraseIfillsthegap.DNAligaselinksthe3’-OHand5’-Pbyphosphordiesterbond.XerodermaPigmentosum(XP)HumanNERsystemconsistsofanumberofXPproteinsincludingXPA,XPB,XPC,XPD,XPF,XPGandERCC1.ThecomplexofXPproteinsscanstheDNA,recognizesthelesionandremovesapieceofdamagedDNAabout25nuc-leotidesinlength.ThegapisrepairedbyDNApolymeraseandDNAligase.PatientshavingrecessivemutationsinXPproteingenescannotrepairUV-inducedDNAdamagesintheskinbe-bauseoflackingofXPproteinactivities.Thatcausescancerandisknownasxerodermapigmentosum.d.Recombinationalrepair(RR)systemisresponsibleforrepairingDNAdouble-strandbreaks.TheexchangeofhomologousregionsbetweentwoDNAmoleculesiscalledhomologousrecombination(HR).Homologousrecombination(HR)playsanimportantpartinorganisms.OneoftheHRfunctionsistherepairofDNAdouble-strandbreaks.ThehypotheticalmechanismofRRisasfollows:brokenDNA5’-----------------------------------------------3’3’-----------------------------------------------5,5’--------------------------------------------------3’3’--------------------------------------------------5’intacthomologousDNAThebrokenregionundergoes5’-exonucleasedigestion.5’-------------------3’5’-----------------3’3’------------5’3’-------------------------5’5’-----------------------------------------------3’3’-----------------------------------------------5’unwindingandstrandinvasion

5’----------------3’5’--------------------3’3’------------5’------------------------5’

5’--------------3’---------------------3’3’-----------------------------------------------5’DNAsynthesis5’------------------------------------------3’3’-------------5’-------------------------5’3’---------------------------------5’5’----------------------------------------------3’resolutionofHolidayjunctions5’-----------------------------------------3’3’--------------------------------------------5’3’-----------------------------------------5’5’--------------------------------------------3’InEcoliRecBCDproteincomplexhasbothnucleaseandhelicaseactivities.RecAproteinparticipatesintheformationoftheHollidayjunction.DNApolymerasesynthesizesDNA.RuvA,RuvB,andRuvCproteinsresolutetheHollidayjunction.e.TranslesionalDNApolymerasescansynthesizeDNAtranslesionally.TranslesionalDNApolsrequiretemplateforDNAsynthesis,buttheydonotstrictlyobeythebase-pairingrulesinincorporationofnucleotides.HencetranslesionDNAsynthesis(TLS)iserror-prone.Ahighmutationratewilloccur.

InEcoliTLSisapartofacellularstressresponsetoextensiveDNAdamageknownasSOSresponse.TheSOSsysteminvolvesalotofgeneswhicharerelatedtoDNAreplicationandrepair.SOSsystemisregulatedbyLexAandRecAproteins.LexAbindstothecontrolregionofSOSgenesencodedforrepairenzymesandshutsoffthegenes.SevereDNAdamagesactivatetheRecAmediatedcleavageanddestructionofLexA.ThatresultsinefficientexpressionofSOSresponsegenes.DNArepairoccurswitherror-proneproperties.SectionSeven

ReverseTranscriptionThediscoveryofreversetranscriptasedevelopsthecentraldogmaputforthbyCrick.thecentraldogmareplicationreplicat