Molecular Biology 分子生物学课件

MolecularBiology:DNAJunYang,Ph.D.Office:Bldg3,Room601Tel:87217149Officehour:Tuesdays,2-3pmorbyappointmentThestructureofthissectionPartI.ReplicationTherepliconDNAreplicationRecombinationandrepairPartII.MovingelementsTransposonsRetrovirusandretroposonsRearrangementofDNARepliconThelinkbetweenreplicationandcellcycle:TheentiregenomemustbereplicatedpreciselyonceforeverycelldivisionInitiationofDNAreplicationcommitsthecelltoafurtherdivisionIfreplicationproceeds,theconsequentdivisioncannotbepermittedtooccuruntilthereplicationeventcompletedRepliconTheunitofDNAinwhichanindividualactofreplicationoccursEachreplicon“fires”onceandonlyonceineachcellcycleControlelements:origin,terminusRegulation:cis-actingOriginsite,trans-actingregulatorprotein(codedwithinthereplicon,orsomewhereelse)Agenomeinaprokaryoticcellisasinglereplicon:singlecopycontrolPlasmidconstitutesaseparaterepliconmayreplicatewiththebacterialgenomeundersinglecopycontrol;or,undermulticopycontrol(morecopiesofplasmidthanthebacterialchromosome)Quitethecontrary,Eacheukaryoticchromosomecontainsalargenumberofrepliconsmaynotbeactivesimultaneously,butcannotbeactivatedmorethanonceinthecellcycleDefinetheoriginsThe“eye”ReplicationforkThepointatwhichreplicationisoccurring:unidirectionalorbidirectionalq-structureWhenarepliconiscircular,thepresenceofaneyeformstheθ-structureThesuccessivestagesofreplicationofthecircularDNAofpolyomavirusarevisualizedbyelectronmicroscopyThebacterialgenomeisasinglecircularrepliconAbacterialrepliconshouldbeableto:InitiateareplicationcycleControlthefrequencyofinitiationeventsSegregatereplicatedchromosomestodaughtercellsThefirsttwofunctionsarepropertiesoftheoriginSegregationcouldbeanindependentfunction,butinprokaryoticsystemsitisusuallydeterminedbysequencesinthevicinityoftheoriginOriginsineukaryotesdonotfunctioninsegregation,butareconcernedonlywithreplicationCreateanartificialchromosome:thefirststepTheDNAconstitutinganorigincanbeisolatedbyitsabilitytosupportreplicationofanyDNAsequencetowhichitisjoinedDNAfromtheorigincanbeclonedintoamoleculethatlacksorigin,thiswillcreateaplasmidcapableofautonomousreplicationOriginshavebeenidentifiedinbacteria,yeast,chloroplasts,mitochondria,butnotinhighereukaryotesA-TrichE.coliOriginoriC,245bpBidirectionalCircularreplicationTerminationterShortsequence~23bp,functionsinonedirectionRequirestheproductoftusgene,whichrecognizestheconsensussequenceandpreventsthereplicationforkfromproceedingTerminationinE.coliTwoterminationregions,eachcontainsmultipleterminators.Andeachisspecificforonedirectionofforkmovement.“Replicationforktrap”WhathappenswhenareplicationforkencountersanRNApolymeraseengagedintranscription?Areplicationforkmoves>10XfasterthanRNApolymeraseSamedirection:bypass,mechanismunknownOppositedirection:head-oncollisionRarelyhappens:genearrangementonchromosomeMightbelethalEacheukaryoticchromosomecontainsmanyrepliconsTheoriginofyeastrepliconARS(autonomouslyreplicatingsequence)A-Trich14bpcoreregion:Adomain,11bpconsensussequencemutationinthisregionabolishesoriginfunctionB1-B3:anytwoarerequiredforfunctionMutationintheseregionsreducesoriginfunctionORC:originrecognitioncomplex6proteinswithamassof~400kDassociatedwithARSDloopsmaintainmitochondriaoriginsOnlyoneofthetwoparentalstrands(H)isusedastemplateforsynthesisofanewstrandSynthesisproceedsforonlyashortdistance,displacingtheoriginalpartner(L)strand,thusgivingthenameasdisplacementorDloopRNAprimerAnopeningof500–600basesTheshortstrandisunstable,frequentlydegradedandresynthesizedSomemitochondrialDNAspossessseveralDloops,reflectingtheuseofmultipleoriginsChloroplastshavetwoDloopsBasedontheaboveinformation,someconclusionscanbedrawn:AnorigincanbeasequenceofDNAthatservestoinitiateDNAsynthesisusingonestrandastemplateTheopeningoftheduplexdoesnotnecessarilyleadtotheinitiationofreplicationontheotherstrandTheproblemoflinearrepliconsSomepossiblesolutionsConvertingthelinearrepliconintoacircularormultimericmolecule:T4andlambdaphageDNAformsanunusualstructure(e.g.,hairpin)attheterminus,sothereisnofreeend:mitochondrialDNAofParameciumInsteadofbeingpreciselydetermined,theendmaybevariable:telomeresAcovalentlylinkedproteintothe5’terminalbasemakesitpossible:adenovirusDNA,phagef29,poliovirusRNAInseveralvirusesthatusesuchmechanisms,aproteinisfoundcovalentlyattachedtoeach5′end.Inthecaseofadenovirus,aterminalproteinislinkedtothematureviralDNAviaaphosphodiesterbondtoserine,asindicatedinthefigureabove.Theterminalproteinhasadualrole:itcarriesacytidinenucleotidethatprovidestheprimer;anditisassociatedwithDNApolymerase.Thecomplexofpolymeraseandterminalprotein,bearingtheprimingCnucleotide,bindstotheendoftheadenovirusDNA.Thefree3′–OHendoftheCnucleotideisusedtoprimetheelongationreactionbytheDNApolymerase.Thisgeneratesanewstrandwhose5′endiscovalentlylinkedtotheinitiatingCnucleotide.(The5′endofadenovirusDNAisboundtotheterminalproteinthatwasusedinthepreviousreplicationcycle.Theoldterminalproteinisdisplacedbythenewterminalproteinforeachnewreplicationcycle.)RollingcirclesproducemultimersofarepliconAnickopensonestrand,andthenthefree3′OHendgeneratedbythenickisextendedbytheDNApolymerase.Thenewlysynthesizedstranddisplacestheoriginalparentalstrand.Rolling-circleinaction:bacterialconjugationConjugation:aplasmidgenomeorhostchromosomeistransferredfromonebacteriumtoanotherMediatedbyFplasmidAnepisome:existasfreecircularplasmidorintegratedintobacterialchromosomeasalinearsequence~100kbF-positivebacteriaconjugatewithF-negativeonesTransferoftheFfactorCelldivisionandchromosomesegregationSeptumformsinthecenterofabacteriumtodivideitintotwodaughtercellsattheendofadivisioncycleprecededbytheorganizationoftheperiseptalannulusseptumconsistsofthesamecomponentsasthecellenvelopeAdirectlinkbetweenDNAandthemembranecouldaccountforsegregationTruepartitionsystemTrans-actingparAandparBproteins,cis-actingparSsiteReplicationReplisome:themultiproteinstructurethatassemblesatthebacterialreplicatingforktoundertakesynthesisofDNAContainsDNApolymeraseandotherenzymesThreestages:initiation,elongation,andterminationTheidentificationofdnagenesinE.coliDNApolymerasesTwotypesofDNAsynthesisReplicationRepairOnlysomeoftheDNApolparticipateinreplication,sometimestheyarecalledDNAreplicaseEukaryoticDNApolapriming(initiating)replicationbrepairdnuclearreplicaseerepairgmitochodrialreplicationDNApolhasexonucleaseactivityNicktranslation:replacespartofapre-existingstrandofduplexDNAwithnewlysynthesizedmaterialDNApolcontrolsthefidelityofreplicationTheoretically,10-3errorrate,actualrate10-8–10-10DNApolmightimprovethespecificityofcomplementarybaseattwostages:Presyntheticerrorcontrol:scrutinizetheincomingbaseProofreading:scrutinizethebasepairafterthenewbasehasbeenadded,andremovethemostrecentlyaddedbaseifitismismatchedSomeDNApolhaveacommonstructureDNAsynthesisissemicontinuousLeadingstrandLaggingstrandOkazakifragmentsSinglestrandDNAisneededforreplicationHelicase:separatesthestrandsofDNA,usuallyusingthehydrolysisofATPtoprovidethenecessaryenergy.Ahelicaseisgenerallymultimeric,oftenahexamerSinglestrandbindingprotein(SSB):bindsasamonomer,buttypicallyinacooperativemannerinwhichthebindingofadditionalmonomerstotheexistingcomplexisenhancedfX174DNAcanbeseparatedintosinglestrandsbythecombinedeffectsof3functions:nickingwithAprotein,unwindingbyRep,andsingle-strandstabilizationbySSBPrimingisrequiredtostartDNAsynthesisDNApolcannotinitiateDNAsynthesisdenovoAsequenceofRNAissynthesizedonthetemplate,sothatthefree3′OHendoftheRNAchainisextendedbytheDNApolymerase.ThisiscommonlyusedinreplicationofcellularDNA,andbysomevirusesAprimerterminusisgeneratedwithinduplexDNA.Themostcommonmechanismistheintroductionofanick,asusedtoinitiaterollingcirclereplication.ThedifferencefromnicktranslationisthatpolymeraseIsimultaneouslysynthesizesanddegradesDNAfromanick.AproteinprimesthereactiondirectlybypresentinganucleotidetotheDNApolymerase.Thisreactionisusedbycertainviruses.PrimaseAprimaseisrequiredtocatalyzetheactualprimingreactionTheproductofthednaGgeneAsinglepolypeptideof60kD,muchsmallerthanRNApolymeraseUsedonlyunderspecificcircumstances,thatis,tosynthesizeshortstretchesofRNAthatareusedasprimersforDNAsynthesisTwotypesofpriminginE.coliOriCtype,involvedtheassociationofDnaGprimasewiththeproteincomplexatthereplicationforkTheφXsystem,namedforphageφX174,requiresaninitiationcomplexconsistingofadditionalcomponents,calledtheprimosomePrimosomePrimosomeassemblesatauniquesiteonthessDNA,calledtheassemblysite(pas)ThepasistheequivalentofanoriginforsynthesisofthecomplementarystrandofφX174Theprimosomeconsistsofsixproteins:PriA,PriB,PriC,DnaT,DnaB,andDnaCThekeyeventinlocalizingtheprimosomeistheabilityofPriAtodisplaceSSBfromsingle-strandedDNADnaBplaysakeyroleinunwindingandpriminginφXrepliconsPriAistoloadDnaBtoformareplicationforkWhyprimosome?ReplicationishaltedbyadamagedbaseornickinDNAAnE.coliretrievalsystemusesanormalstrandofDNAtoreplacethegapleftinanewlysynthesizedstrandoppositeasiteofunrepaireddamageTheprimosomeisrequiredtorestartastalledreplicationforkaftertheDNAhasbeenrepairedTerminationTus,a36Kdprotein,providesacontra-helicaseactivityandstopsDnaBfromunwindingDNADNApolIIITheholoenzymecontains:catalyticcore,includingtheαsubunitdimerizationcomponent(t)thatlinkstwocoresprocessivitycomponent(β)thatkeepsthepolymeraseontheDNAclamploader(g)thatplacestheprocessivitysubunitsonDNADNApolymeraseIIIholoenzymeassemblesinstages,generatinganenzymecomplexthatsynthesizestheDNAofbothnewstrandsCorepolymeraseandthebclampdissociateatcompletionofOkazakifragmentsynthesisandreassociateatthebeginningCreatethereplicationforkatanoriginPlasmidscarryingtheE.colioriCsequencehavebeenusedtodevelopacell-freesystemforreplicationfromthisoriginInitiationofreplicationatoriCinvitrostartswithformationofacomplexthatrequiressixproteins:DnaA,DnaB,DnaC,HU,Gyrase,andSSBThereactionconvertsacircularsupercoiledtemplateintoanewforminwhichthereisextensiveunwindingoftheduplexThefirststageincomplexformationisbindingtooriCbyDnaAprotein.Thereactioninvolvesactionattwotypesofsequences:9bpand13bprepeats.Togetherthe9bpand13bprepeatsdefinethelimitsofthe245bpminimaloriginTostart:Thefour9bpconsensussequencesontherightsideoforiCprovidetheinitialbindingsitesforDnaA.Itbindscooperativelyuntil20–40monomershaveformedacentralcorearoundwhichoriCDNAiswrappedThentheDnaAproteinactsatthree13bptandemrepeatslocatedintheleftsideoforiC.TheDNAstrandsaremeltedateachofthesesitestoformanopencomplex.Allthree13bprepeatsmustbeopenedforthereactiontoproceedtothenextstageTheDnaBandDnaCproteinsformacomplexinwhich6DnaCmonomersbindeachhexamerofDnaB.Thisgeneratesaproteinaggregateof480kD,correspondingtoasphereofradius6nmTheDnaB·DnaCcomplextransfersonehexamerofDnaBtoformthereplicationforkDnaBprovidesthehelicasethatunwindstheDNA,itdisplacesDnaAfromthe13bprepeatsandcommencesunwindingDnaBalsohasthesameabilityheretoactivatetheDnaGprimasethatithasfortheOkazakifragmentsTwofurtherproteinsarerequiredtosupporttheunwindingreactionGyraseprovidesaswivelthatallowsonestrandtorotatearoundtheotherSSBstabilizesthesingle-strandedDNAasitisformedThelengthofduplexDNAthatusuallyisunwoundtoinitiatereplicationisprobably<60bpproteinHUisageneralDNA-bindingproteininE.coliItspresenceisnotabsolutelyrequiredtoinitiatereplicationinvitro,butitstimulatesthereactionHUhasthecapacitytobendDNA,andislikelytobeinvolvedinsomegeneralstructuralcapacityTheconnectionbetweentranscriptionandreplicationRNApolymerasecouldberequiredtoreadintotheoriginfromadjacenttranscriptionunits;byterminatingatsitesintheorigin,itcouldprovidethe3′-OHendsthatprimeDNApolymeraseIIIAlternatively,theactoftranscriptioncouldbeassociatedwithastructuralchangethatassistsinitiationMethylationandreplicationinitiationOnlyfullymethylatedoriginscaninitiatereplication;hemimethylateddaughteroriginscannotbeusedagainuntiltheyhavebeenrestoredtothefullymethylatedstateLicensingfactortheory:controlsofre-replicationOneroundofreplicationeitherinactivatesordestroysthefactor,andanotherroundcannotoccuruntilfurtherfactorisprovidedRecombinationandRepairHomologousrecombination:requireshomologySite-specificrecombination:requireshomology,responsibleforphagegenomeintegratingintobacterialgenomeTransposition:homologynotrequiredCopychoice:RNAviruses,inwhichthepolymeraseswitchesfromonetemplatetoanotherwhileitissynthesizingRNAPatchrecombinantsSplicerecombinantsInreality,recombinationisinitiatedbydoublestrandbreaks(DSBs),followedbyforming3’-singlestrandendsInyeast,the5′endsofthedouble-strandbreaksareconnectedtotheproteinSpo11,thebreakisconvertedintoapermanentstructurebyaninteractionwithanotherproteinthatdissociatestheSpo11complex.ThenremovalofSpo11isfollowedbynucleaseaction.Atleast9otherproteinsarerequiredtoprocessthedouble-strandbreaks.Onegroupofproteinsisrequiredtoconvertthedouble-strandbreaksintoprotruding3′OHsingle-strandedends.Anothergroupthenenablesthesingle-strandedendstoinvadehomologousduplexDNAchisequenceandRecBCDchi:HotspotsActivatedbyDSBsseveralkbsawaysMovinginonedirectionRecBCDNucleaseHelicaseATPaseProvidesss-endswith3’freeendRecAcatalyzessingle-strandassimilationRecAcantakethesinglestrandwiththe3′endthatisreleasedwhenRecBCDcutsatchi,andcanuseittoreactwithahomologousduplexsequence,thuscreatingajointmoleculeRecAAslowpresynapticphaseinwhichRecApolymerizesonsingle-strandedDNA;Afastpairingreactionbetweenthesingle-strandedDNAanditscomplementintheduplextoproduceaheteroduplexjoint;AslowdisplacementofonestrandfromtheduplextoproducealongregionofheteroduplexDNARuvsystemresolvesHollidayjunctionsRuvA,B,andCTopologicalmanipulationofDNASeparationofthestrandsofaDNAdoublehelixcouldbeachievedbyseveralmeansSupercoils:positiveandnegativeIntroducingatransientnickDNAtopoisomerasesSomecanrelax(remove)onlynegativesupercoilsfromDNA;otherscanrelaxbothnegativeandpositivesupercoils.SomecanintroducenegativesupercoilsTypeIenzymesactbymakingatransientbreakinonestrandofDNA.TypeIIenzymesactbyintroducingatransientdouble-strandbreakSpecificsitesforphageintegrationInthelyticlifestyle,lambdaDNAexistsasanindependent,circularmoleculeintheinfectedbacterium.Inthelysogenicstate,thephageDNAisanintegralpartofthebacterialchromosome(calledprophage)Toenterthelysogeniccondition,freelambdaDNAmustbeintegratedintothehostDNA.Tobereleasedfromlysogenyintothelyticcycle,prophageDNAmustbeexcisedfromthechromosomeAttachment(att)sitesthebacterialattachmentsite(attλ)iscalledattB,consistingofthesequencecomponentsBOB′.Theattachmentsiteonthephage,attP,consistsofthecomponentsPOP′Integrationgeneratestwonewsites,attLandattRDifferentproteinfactorsarerequiredforintegrationandexcisionIntegration(attB⊙attP)requirestheproductofthephagegeneintandabacterialproteincalledintegrationhostfactor(IHF).Excision(attL⊙attR)requirestheproductofphagegenexis,inadditiontoIntandIHFRepairsystemsanddamageDamageSinglebasechangesStructuraldistortionRepairDirectrepairExcisionrepairMismatchrepairTolerancesystems:error-prone,highereukaryotesRetrievalsystems:bacteriaSinglebaseStructuraldistortionStepsinexcisionrepairUvrsystemisresponsibleforExcisionRepairinE.coliEukaryoteshaveasimilarsystemThepresenceofVeryShortPatchrepair(VSP),shortpatchrepair,andlongpatchrepairError-pronerepairandmutationtherepairofdamagedλphageDNAisaccompaniedbytheinductionofmutationsifthephageisintroducedintocellsthathadpreviouslybeenirradiatedwithUV.ThemutagenicresponsealsooperatesonthebacterialhostDNAADNApolymerasethatinsertsincorrectbases,whichrepresentmutations,whenitpassesanysiteatwhichitcannotinsertcomplementarybasepairsinthedaughterstrandumuDandumuC:DNApolVModelofTLS(translesionbypasssynthesis)TrendsCellBiol2000;10;201Low-fidelitycopyingofundamagedDNAbyspecializedhumanDNApolymerasesSci2002;296:1627DetectionofNon-targetedMutagenesisinMammalianCellsMismatch:howdoyouknowtoremovethewrongone?Someprecautionsthedeaminationof5-methyl-cytosinetothyminegeneratesaG:Tpair,VSPsystemhasabiastocorrectthemtoG:Cpairs(ratherthantoA:Tpairs)mutL,SalsoremovesTfromG:TandC:TThemethylationstatusofnewlysynthesizedDNAstrand:damsystemdamgenecodesforamethylasewhosetargetistheadenineinthesequenceGATCCTAGConsistsofseveralproteinsRetrievalsysteminE.colithedamageisconfinedtotheoriginaldistortion(althoughthesamerecombination-repaireventsmustberepeatedaftereveryreplicationcycleunlessanduntilthedamageisremovedbyanexcisionrepairsystem)RecAtriggersSOSresponseSOSresponseinE.colidescribesthecoordinateinductionofmanyenzymes,includingrepairactivities,inresponsetoirradiationorotherdamagetoDNA;resultsfromactivationofproteaseactivitybyRecAtocleaveLexArepressorSOSresponseincreasedcapacity(long-patchexcisionrepairsystemandtheRecrecombination-repairpathways)torepairdamagedDNAcelldivisionisinhibitedLysogenicprophagesmaybeinducedRecAactivationasinitialstepssDNAmightbethesignalInducesthecleavageofLexALexALexAisasmall(22kD)proteinthatisrelativelystableinuntreatedcells,whereitfunctionsasarepressoratmanyoperonsLexAhasalatentproteaseactivitythatisactivatedbyRecA,autocatalyticcleavageinactivatestheLexArepressorfunction,andcoordinatelyinducesalltheoperonstowhichitwasboundLexArepressesitstargetgenesbybindingtoa20bpstretchofDNAcalledanSOSbox,Autogenousrepression:therearetwoadjacentSOSboxesatthelexAlocusRepairsystemsineukaryotesLessclearRadfamily(fromthestudiesofradiation)TLSXerodermapigmentosum(XP),arecessivediseaseresultinginhypersensitivitytosunlight,inparticulartoultraviolet.Thedeficiencyresultsinskindisorders:defectofERNon-homologousendjoining(NHEJ),therecombinationofimmunoglobulingenes:DNA-PK