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1、Chapter34DNAdamage,repairandmutation,Outline,DNADamagesDNARepairDirectrepairExcisionrepairDouble-strandedbreakrepairDamagebypassDNAmutationTypesandConsequencesofMutationsMutagensReversemutationandSuppressormutationRelationshipofMutagenandCarcinogen,DNAisStable,butfragile,WhatFactorsAlterDNAIntegrity
2、?,EndogenousCellularFactorsEnvironmental-e.g.chemicals,pollution,UVlightDiseasetreatments-e.g.ionizingradiation,chemotherapy,EndogenousCellularFactors,Replicationerrorse.g.imbalanceinthenucleotidepools-resultinmismatchDNAinstabilitydeaminationofbasesdepurination/depyrimidinationofDNAlossofbaseReacti
3、veoxygenspeciesDNA,proteinandlipidoxidation,Deaminationofbases,Depurination/Depyrimidination,DepurinationoccursmorerapidlythandepyrimidinationResultsinadeoxyribosewithoutabase(APsite)E.coli:1depurination/genome/doublingThermusThermophilus:300depurinations/genome/doublingMammaliancell:10,000depurinat
4、ions/genome/doubling,Reactiveoxygenspecies,ProducedbynormalcellularmetabolismMitochondriautilize85%O2incellandareamajorsourceofROSDamageDNA,proteinandlipid.Someformsincellare:Hydrogenperoxide(H2O2)Superoxideradical(O2-)Nitricoxide(NO)Hydroxylradical(HO)Peroxynitrite(O=NOO-)Alkylhydroperoxide(ROOH)Al
5、koxylradical(RO),Environmental(Exogenous)factors,ChemicalAgents(1)NaturalChemicalsAflatoxins-nuts(2)Man-madeChemicalsBenzopyrene-cigarettesmokeChemotherapydrugs-cisplatinPhysicalAgents(1)Sunandtanningboothlamps(UV)(2)IonizingRadiation-raysx-rays,TypesofDNADamage,BasemodificationLossofbases-abasicora
6、purinicsitesReplicationerrors:MismatchStrandbreakageProtein-DNAcross-linksDNA-DNAcross-links,Basemodification,Lossofbases,TheTdimerand6-4photoprouductcausedbyUV,TautomerizationleadstoDNAmismatch,ketoenolcommonbpwithtautomers:AC,AA,GT,AG,DNAisStable,butfragile,FailuretorepaircausesmutationsCaninterfe
7、rewithtranscriptionandreplicationCanleadtoMutageneisCanspeedagingItisessentialthatcellspossessmechanismsforrepairingthisdamageRepairmechanismsareextensiveandefficient100genesparticipateinvariousaspectsofDNArepair,eveninorganismswithverysmallgenomes.Many,perhapsmost,cancersareatleastpartiallyattribut
8、abletodefectsinDNArepair.,Sciencemagazines1994MoleculeoftheYear,DNARepairPathways,Directreversals,ThesedirectcorrectionsareerrorfreesystemsTheyarehighlyspecificprocessesthatrequireonlysinglegeneproductsTheymaybeenergeticallyexpensivebutkineticallyadvantageous(morerapidthanamultistepprocess),Damagest
9、hatcanbedirectlyfixed,PhotoreactivationofDNA,doesnottakeplaceinhumans,photolyase,Repairofalkylationdamage,alkyltransferase,Religationofsinglestrandbreaks,DNAligase,DirectRepairoftheTdimer,Features:Onlyoneenzyme(DNAphotolyase)isenough1)Usesenergyfromlightabsorption2)Containschromophores(lightabsorbin
10、gagents)3)Actionspectrumisblue/nearUVlightrange4)photolyasesarefoundinbacteria,fungi,plantsandmanyvertebrates,butnotinplacentalmammals.Stepsintherepairmechanism:1)Enzymerecognizesandbindstothedamage2)Lightabsorptionbychromophoreconvertsittoanexcitedstate3)Chromophoredonatesanelectrontothecyclobutyld
11、imer4)Dimerisdestabilizedandundergoesaseriesofelectronrearrangementswhichresultinmonomericpyrimidines,RepairbyDirectreversal:photoreactivation,DamageRecognized:Thyminedimers6-4photoproduct,GeneProductsRequired:Photolyase,Relateddisease:Photolyasenotyetfoundinplacentalmammals,Visiblelight,Photoreacti
12、vationoftheTdimer,MechanismofO6-methylguaninemethyltransferase,FourSteps:Recognize,Remove,Resynthesize,ReligateBaseexcisionrepair(BER)-fixesabnormalbases(uracil,hypoxanthine,alkylatedbases)Nucleotideexcisionrepair(NER)-fixeslargestructuralchangesandhelixdistortions(pyrimidinedimers,bulkybaseadducts)
13、Mismatchrepair-fixesmismatches,MechanismsofExcisionRepair,BaseExcisionRepair(BER),ShortPatchRepair-RemovesasingledamagedbaseLongPatchRepair-Removes2-10nucleotidesDNAglycosylasesaretherepairenzymesthatcarryoutbaseexcisionrepairDNAglycosylasesbindtotheDNAandbendit,causingthedamagedbasetoflipout,1.Remo
14、valoftheincorrectbasebyanappropriateDNAN-glycosylasetocreateanAPsite2.NickingofthedamagedDNAstrandbyAPendonucleaseupstreamoftheAPsite,thuscreatinga3-OHterminusadjacenttotheAPsite3.ExcisionoftheAPsite,followedbyextensionofthe3-OHterminusbyaDNApolymerase,StepsinBaseExcisionRepair,BaseExcisionRepair(BE
15、R),DNAGlycosylases,ThecrystalstructureofhumanuracilDNAglycosylaseisshownboundtoaDNAhelix.Notehowtheenzyme,causestheuracilbasetoflipoutoftheDNAhelixandenterthebindingsite.,NucleotideExcisionRepair,NERmachineryrecognizesdamagedregionsinDNAbasedontheirabnormalstructureaswellasontheirabnormalchemistry,t
16、henexcisesandreplacesthem.,DamagerecognitionBindingofaproteincomplexatthedamagedsiteDoubleincisionofthedamagedstrandseveralnucleotidesawayfromthedamagedsite,onboththe5and3sidesRemovalofthedamage-containingfragmentfrombetweenthetwonicksFillinginoftheresultinggapbyaDNApolymeraseLigation,Inallorganisms
17、,NERhasthesamesteps:,TwotypesofNER,GlobalandTranscription-coupledGlobalNERIdentifiesdamagethroughoutthegenomeTranscription-coupledNER(1)Correctsdamagefromthetranscribedstrandofactivegenes(2)ReliesonstallingofRNAPolIIatthesiteofdamage-repairistheninitiated,2UvrAproteinsformacomplexwithoneUvrBproteini
18、nanATP-dependentreactionThecomplexrecognizesUVdamagebythebendinthehelixTheUvrAproteinsdissociatefromthecomplexafterATPhydrolysis.ThisleavesUvrBboundacrossfromthedamage,NucleotideExcisionRepairinE.coli,NowUvrBcanrecruitUvrCproteintothecomplexUvrCactivatesUvrBtonicktheDNA4nts3fromthepyrimidinedimerThe
19、nUvrBactivatesUvrCtonicktheDNA7ntds5fromthepyrimidinedimerThisleavesafragmentofDNAcontainingthedamagethatcannowberemoved,NucleotideExcisionRepairinE.coli,Ahelicase,UvrD,usesATPhydrolysistopowertheunwindingofthedamagedDNAfragment.ThisreomovesUvrCThegapintheDNAisnowfilledinbyDNAPIorII,reomvinguvrBinth
20、eprocessFinally,DNAligasesealsthenick,NucleotideExcisionRepairinE.coli,CriticalforrepairingUV-induceddamage(becausewedontdodirectrepair)Principalisthesameasinbacteria:Buttheproteinsaredifferent(20-30)DefectsinNERproteinscausegeneticdisorders,NERinHumans,DefectsofNERinHumansXerodermapigmentosum(XP),G
21、eneticdisorderwithsymptoms:-extremesensitivitytosunlight(byage2),and1000Xhigherriskofskincancer(byage8)DefectisinrepairofUVdamageGenemappingidentified7repairproteins(calledXPproteins):XP-CandXP-Arecognizepyrimidinedimers;XP-BandXP-Dhavehelicaseactivity;XP-GandXP-Fhavenucleaseactivity,NERinHumans,XP-
22、Crecognizespyrimidinedimers,XP-BandXP-DarehelicasesthatseparatetheDNAstrandsaroundthedamage,XP-GandXP-fareendonucleasesthatcuttheDNAoneithersideofthedamage,XP-Abindstothepyrimidinedimerandhelpstorecruitotherproteinstoacomplex,ThecutfragmentisremovedandthegapisfilledinbyDNAPdore,TCRintheE.coliNERPath
23、way,Twopathways,1.Globalgenomerepair(GGR)2.Transcription-coupledrepair(TCR),TRCF-transcriptionrepaircouplingfactor,MismatchRepair(MMR),Correctsmismatchesandinsertion/deletionsevents(INDELS)afterDNAreplicationIncreasesreplicationfidelityby102-103foldMismatchrepairsystemsscannewly-replicatedDNAduplexe
24、sformismatchedbasesPROBLEM:whichofthe2strandshasthecorrectsequenceandwhichhastheerror?-InE.coli,methylationidentifiestheDNAstrandthatcontainsthecorrectbase-Thestranddiscriminationsignalinmammalsisnotyetknown,MismatchRepair,Sincetheparent(template)strandshouldhavethecorrectsequence,repairmachineryide
25、ntifiestheparentstrandandcopiesittocorrectthemistakeSincemethylationoccurspost-replication,repairproteinsidentifymethylatedstrandasparent,removemismatchedbasesonotherstrandandreplacethem,MismatchRepair,ThemainE.coligenesinvolvedinMMRaremutS,mutHandmutLSixmutShomologs(MSH)geneshavebeenidentifiedineuk
26、aryotes(MSH1-6)MSH2,3,6-formheterodimersthatrecognizemismatchesMutS(MSH2/MSH6)-bindsbase-baseandsinglentINDELSMutS(MSH2/MSH3)-binds2-4ntINDELS,StepsinMismatchRepair,MutSrecognizesmismatchesandbindstothem.BindingofMutLstabilizesthecomplex.TheMutS-MutLcomplexactivatesMutH,whichlocatesanearbymethylgrou
27、pandnicksthenewlysynthesizedstrandoppositethemethylgroup.Ahelicase(UvrD)unwindsfromthenickinthedirectionofthemismatch,andasingle-strandspecificexonucleasecutstheunwoundDNAthegapisfilledinbyDNAPIIIandsealedbyDNAligase.,RepairofMismatchReplicationErrorsinE.coli,1.,2.,1.MutSrecognizesmismatchesandbinds
28、tothem.BindingofMutLstabilizesthecomplex.2.TheMutS-MutLcomplexactivatesMutH,whichlocatesanearbymethylgroupandnicksthenewlysynthesizedstrandoppositethemethylgroup.,Continuedfromthepreviousslide,2.,3.,4.,3.Ahelicase(UvrD)unwindsfromthenickinthedirectionofthemismatch,andasingle-strandspecificexonucleas
29、ecutstheunwoundDNA4.ThegapisfilledinbyDNAPIIIandsealedbyDNAligase.,Double-StrandBreakRepair:NonhomologousEndJoining(NHEJ),Mammalianpathway,Ku:dimerofKu70andKu80,DNA-PKcs:DNA-dependentproteinkinasecatalyticsubunit.MemberofproteinkinasefamilythatincludesATMandATR.,Factorsinvolvedinprocessingofendsnotw
30、ellunderstood.MRNisMre11/Rad50/Nbs1complex.,Xrcc4/DNAligaseIVarerequiredforthefinalligationstep.,Synapsisisachivedthroughmicrohomologies.,Error-prone;smallinsertionsordeletions.MajorpathwayofDSBrepairinmammals,minorpathwayinyeast.,DamageBypass,ManylesionscanstillescapeDNArepairandarepresentatthetime
31、ofDNAreplicationDuringDNAreplication-unrepaireddamagecanblockthemajorreplicativeDNApolymerasePol(,)andhaltreplicationNeedtobypasstheblockormayresultinchromosomaltruncationBERandNERcantbeusedbecausethereplicationforkhasalreadyseparatedtheparentalDNAstrandsTwoSystemsareused-RecombinationalBypassandByp
32、assSynthesis,RecombinationalBypass,UsestemplateswitchingtoavoidtheDNAdamageTheunblockednewlysynthesizedsisterDNAbecomesthesurrogatetemplateforthereplicationthathasstalledThissystemisconsiderederrorfreeIsthoughttouseenzymesinvolvedinhomologousrecombination,ADNAmoleculehasaTdimer,thismoleculeisbeingre
33、plicated.APolIIIwillbeunabletocorrectlycopytheTdimer,DNAPolIIreinitiatesDNAsynthesisdownstreamofTdimers.,Thiscannotberepairedbytheusualrepairsystems.However,theexposedssDNAwiththecorrectlysynthesizeddaughtermolecule,OnedaughtermoleculestillcontainstheTdimerbuttheoppositestrandhasthecorrectsequence.T
34、heotherdaughternowcontainsagapbutthisgapcanberepairedcorrectlybytheusualrepairsystems,RecombinationalBypass,BypassSynthesis,UsesspecializedpolymerasetobypassDNAdamageError-proneprocess-candealwitherrorsatthepriceforsurvivalReplicativepolymeraseisreplacedbybypasspolymeraseandreplicatespastthedamagePo
35、lymerase(,)arethemammalianbypasspolymerasesThesepolymeraseshavelowerfidelityandlowerprocessivitythanthereplicativepolymerases,SOSRepair:Error-pronereplication,TheSOSrepairsystemisinducedinresponsetomajordamagetothebacterialDNAorinresponsetoagentswhichinhibitDNAreplication.Thesystemisacomplexonewitho
36、ver20genesinvolved.Twoofthesearetheimportantregulatorgenes:lexAandrecA.LexAisarepressorthatregulatestheexpressionofalloftheotherSOSrepairgenes,includingrecA.Italsoregulatesitsownsynthesis.LexAisadimer.EachmonomerhasaDNAbindingdomainandadimerizationdomain,however,theproteinwillnotbindtoDNAunlessithas
37、formedadimerfirst.Normally,LexAbindstoitsoperatorstoblockexpressionoftheSOSrepairgenes.,TheRecAproteinisamultifunctionalproteinwithATPaseandssDNAbindingactivities.WhenboundbyssDNA,itisalsoaco-protease.DamageorseverestresstothecellgeneratesssDNAwhichactivatesthisco-proteaseactivity,whichstimulatesthe
38、proteaseactivityoftheLexAprotein.TheautocleavageofLexAseparatestheDNAbindingdomainfromthedimerizationdomain.Asaresult,LexAisnolongerabletorepresstranscription;theSOSrepairgenesaretherebyinducedandexpressed.AmongthegenesthatareinducedareumuCandumuD.UmuDiscleavedbytheRecAcoproteaseactivityandthetrunca
39、tedprotein,UmuD,inassociationwithUmuCformsDNApolymeraseV(D2C)whichreplicatespastgapsintheDNAwithouttheproofreadingfunctionsofPolIII.UmuD2CisarelativelypoorpolymerasethatsynthesizeDNAdistributively.PolVrequirestheandsubunitofPolIIIforoptimalactivity.,whichfunctionsastheslidingclamp,isrequiredforproce
40、ssivityandistheclamploader.,SOSRepair(continued),SOSresponseinE.coli,GenesinducedaspartoftheSOSresponseinE.coli,Error-prone(SOS)bypass,TranslesionBypassDNAPolymerases,PoletainsertsadenosinesoppositeTTdimersingeneralhaslowfidelitylowprocessivitymaybeerror-pronewithotherlesions-PoletaisaproductoftheXP
41、Vgene,PolzetaandRev1Rev1insertsrandombasesoppositedimerPolzetaextendsbypassbyafewbasesBothpolymeraseshavelowfidelityandlowprocessivity,DNAPolymerasesInvolvedinDNARepair,PolymerasesInvolvedinReplicationofNormalandDamagedDNA,IsthereanysimilarSOSresponseinhumanbodies?,DNAdamages,P53P21,Thecellcycleisar
42、restedandstaysatG1,RepairbeforeDNAsreplicate,Tooseveretorepair,Apoptosiswillbeinduced,Damagingagents,DNAmutation,MutationsAreInheritedAlterationsintheDNASequenceMutationsareboththesustaineroflifeandthecauseofgreatsuffering.Ontheonehand,mutationisthesourceofallgeneticvariation,therawmaterialofevoluti
43、on.Ontheotherhand,manymutationshavedetrimentaleffects,andmutationisthesourceofmanyhumandiseasesanddisorders,TypesofDNAMutations,AccordingtomutatedpropertiesPointmutation-Basesubstitutionstransition,apurineisreplacedbyadifferentpurineor,alternatively,apyrimidineisreplacedbyadifferentpyrimidinetransve
44、rsion,apurineisreplacedbyapyrimidineorapyrimidineisreplacedbyapurine.Insertionsanddeletions,Basesubstitutions,AccordingtomutagenMutagenesiscanoccurspontaneouslyorcanbeinduced.Spontaneousmutationsarenaturallyoccurringmutations.Inducedmutationsoccurwhenanorganismisexposedeitherdeliberatelyoraccidental
45、lytoaphysicalorchemicalagent,knownasamutagen,thatinteractswithDNAtocauseamutation.Inducedmutationstypicallyoccuratamuchhigherfrequencythandospontaneousmutationsandhencehavebeenusefulingeneticstudies,TypesofDNAMutations,SpontaneousMutations,SpontaneousmutationscanoccurduringDNAreplication,aswellasdur
46、ingotherstagesofcellgrowthanddivision.SpontaneousDNAReplicationErrorsSpontaneousChemicalChangesDepurinationDeamination,ChangesofDNAbasesasaresultofdeamination,InducedMutations,Mutationscanbeinducedbyexposingorganismstophysicalmutagens,suchasradiation,ortochemicalmutagensChemicalMutagensBaseanalogs-s
47、uchas5-bromouracil(5BU),Base-modifyingagents-suchasnitrousacid,hydroxylamineandmethylmethanesulfonate.Intercalatingagentssuchasproflavin,acridine,andethidiumbromide,Mutageniceffectsofthebaseanalogs,MutationsinducedbyBase-modifyingagents,MutationsinducedbyBase-modifyingagents,MutationsinducedbyInterc
48、alatingagents,AccordingtomutationalphenotypeMissensemutation:AbasesubstitutionthatresultsinadifferentaminoacidintheproteinisreferredtoasamissensemutationNonsensemutation:AnonsensemutationchangesasensecodonintoanonsensecodonSilentmutation:AsilentmutationcreatesadifferentDNAsequencethatspecifiesthesam
49、eaminoacidasthewild-typesequencedoesNeutralmutation:AneutralmutationisamissensemutationthatalterstheaminoacidsequenceoftheproteinbutdoesnotchangeitsfunctionLoss-of-functionmutationsgain-of-functionmutation,TypesofDNAMutations,Suppressormutationthus,anindividualwithasuppressormutationisadoublemutant,possessingboththeoriginalmutationandthesuppressormutationbutexhibitingthephenotypeofanunmutatedwildtype.IntragenicsuppressormutationIntergenicsuppressormutation,Intragenicsuppressormutation,Intergenicsuppressormutation,Recessivemuta