ThemutabilityandrepairofDNA第九單元學(xué)習(xí)

ThemutabilityandrepairofDNA第九單元學(xué)習(xí)WelcometoMyMolecularBiologyClassMolecularBiologyoftheGene,5/E

Watsonetal.(2004)PartI:ChemistryandGeneticsPartII:MaintenanceoftheGenomePartIII:ExpressionoftheGenomePartIV:RegulationPartV:Methods3/22/05PartII:MaintenanceoftheGenomeDedicatedtothestructureofDNAandtheprocessesthatpropagate,maintainandalteritfromonecellgenerationtothenextCh6:ThestructuresofDNAandRNACh7:Chromosomes,chromatinsandthenucleosomeCh8:ThereplicationofDNACh9:ThemutabilityandrepairofDNACh10:HomologousrecombinationatthemolecularlevelCh11:Site-specificrecombinationandtranspositionofDNA3/22/05TheconsequenceofhighratesofmutationMutationingermline(生殖細(xì)胞)woulddestroythespeciesMutationinsoma(體細(xì)胞)woulddestroytheindividual.MaintenanceofthecorrectnessoftheDNAsequenceisdefinitelycrucialforlivingorganisms.Keepingtheerrorrateaslowas10-10(page191)isveryexpensive.Buildupaseriousattitudetoscience!!!IabsolutelydonotagreewithWastonetal.atthepointsdescribed3rd&4thparagraphsonpage235Whatarethemorereasonableexplanationsforthe10-10mutationfrequencyinlivingorganisms?Whataretheevidencesthatsuchalowmutationratecandrivetheevolvementofanewspeciesifthecellchangesareknownharmful?ListeningtonatureMutation(突變)isnaturallyavoidedbyrepairoftheerrorsanddamages,suggestingitisgenerallynotwelcomeinlivingorganisms.Recombination(重組)isgoodandnaturallypromoted;itisresponsiblefordiversityinsideofspecies.Transposition(轉(zhuǎn)座)isdifferentfrommutationandrecombinationbecause(1)producingmechanismisdifferent;(2)nomechanismtocorrectit;(3)existinginnatureinawell-controlledmanner(10-5).Notrepairedbutcontrolled.CHAPTER9:ThemutabilityandrepairofDNAMolecularBiologyCourseReplicationerrorsandtheirrepairDNAdamageRepairofDNAdamage3teachinghoursTwoimportantsourcesformutation(unavoidable)InaccuracyinDNAreplication(10-7isnotaccurateenough)Errors(錯(cuò)誤)Chemicaldamagetothegeneticmaterial(environment)Lesions(損害,傷害arosefromspontaneousdamage)Damage(損害,傷害causedbychemicalagentsandradiationTorepairanerrorordamageFirst,DetecttheerrorsSecond,Mend/repairtheerrorsorlesionsinawaytorestoretheoriginalDNAsequence.QuestionstobeaddressedHowistheDNAmendedrapidlyenoughtopreventerrorsfrombecomingsetinthegeneticmaterialasmutationHowdoesthecelldistinguishtheparentalstrandfromthedaughterstrandinrepairingreplicationerrorsHowdoesthecellrestoretheproperDNAsequencewhentheoriginalsequencecannolongerberead?Howdoesthecelldealwithlesionsthatblockreplication?Topic1:ReplicationerrorsandtheirrepairCHAPTER9ThemutabilityandrepairofDNAHowthereplicationerrorsareresulted?Thenatureofthereplicationerrorsismismatch.2.Howmismatchesarerecognizedandcorrectlyrepair?ThenatureofmutationsPointmutations:Transitions(pyrimidinetopyrimidine,purinetopurine)Transversions(pyrimidinetopurine,purinetopyrimidine)ReplicationerrorsandreplicationInsertionsDeletionsGrossrearrangementofchromosome.Thesemutationsmightbecausedbyinsertionbytransposonorbyaberrantactionofcellularrecombinationprocesses.Rateofspontaneousmutationatanygivensiteonchromosomalrangesfrom10-6to10-11perroundofDNAreplication,withsomesitesbeing“hotspot”.Mutation-pronesequenceinhumangenomearerepeatsofsimpledi-,tri-ortetranucleotidesequences,knownasDNAmicrosatellites(微衛(wèi)星DNA).Thesesequences(1)areimportantinhumangeneticsanddisease,(2)hardtobecopiedaccuratelyandhighlypolymorphicinthepopulation.Howthereplicationerrorsareresulted?Eachbaseshasitspreferredtautomericform(Ch6)Thestrictnessoftherulesfor“Waston-Crick”pairingderivesfromthecomplementaritybothofshapeandofhydrogenbondingpropertiesbetweenadenineandthymineandbetweenguanineandcytosine.SomereplicationerrorsescapeproofreadingThe3’-5’exonucleaseactivityofreplisomeonlyimprovesthefidelityofDNAreplicationbyafactorof100-fold.Themisincorporatednucleotideneedstobedetectedandreplaced,otherwiseitwillcausemutation(Fig.9-2).ReplicationerrorsandreplicationFigure9-2GenerationofMutation2.Howthereplicationerrorsarerepaired?MismatchrepairremoveserrorsthatescapeproofreadingIncreasetheaccuracyofDNAsynthesisfor2-3ordersofmagnitudes.Twochallenges:(1)rapidlyfindthemismatches/mispairs,(2)AccuratelycorrectthemismatchReplicationerrorsandreplicationTalkingaboutthestoryofE.colirepairsystemMutSscanstheDNA,recognizingthemismatchfromthedistortiontheycauseintheDNAbackboneMutSembracesthemismatch-containingDNA,inducingapronouncedkinkintheDNAandaconformationalchangeinMutSitselfFigure9-4CrystalstructureofMutSMutSisadimer.Onemonomerinteractswiththemismatchspecifically,andtheothernonspecifically.DNAiskinkedMutS-mismatch-containingDNAcomplexrecruitsMutL,MutLactivatesMutH,anenzymecausinganincisionornickononestrandnearthesiteofthemismatch.Nickingisfollowedbythespecifichelicase(why?)(UrvD)andoneofthreeexonucleases(why?).DNApolymeraseIIIHelicaseExonuclease,Detail1:HowdoestheE.colimismatchrepairsystemknowwhichofthetwomismatchednucleotidetoreplace?ThenewlysynthesizedstrandisnotmethylatedbyDammethylaseinafewminutesafterthesynthesis.Figure9-5Detail2:DifferentexonucleasesareusedtoremovessDNAbetweenthenickcreatedbyMutHandthemismatch.Figure9-6EukaryoticcellsalsorepairmismatchesanddosousinghomologstoMutS(MSH)andMutL(MLH).Theunderlyingmechanismsarenotthesameandnotwellunderstood.Topic2:DNAdamageCHAPTER9ThemutabilityandrepairofDNA3/22/05DNAundergoesdamagespontaneously(自發(fā)的)fromhydrolysis(水解)anddeamination(轉(zhuǎn)氨)ResultedfromtheactionofwaterDNAdamageFigure9-7:MutationduetohydrolyticdamageDeaminationCUHydrolysiscreatesapurinicdeoxyriboseDeamination5-mCT

ThepresenceofUandapurinicdeoxyriboseinDNAresultedfromhydrolyticreactionsisregardedasunnatural,thusiseasilyberecognizedandrepaired.Can5-mCTlesionberepaired?VertebrateDNAfrequentlycontains5-methylcytosineinplaceofcytosineasaresultoftheactionofmethyltransferase.Thismodifiedbaseplaysaroleinthetranscriptionalsilencing(Ch17).DNAisdamagedbyalkylation(烷基化),oxidation(氧化)andradiation(輻射)DNAdamageFigure9-8GmodificationAlkylatingchemical:Nitrosamines(亞硝胺)Reactiveoxygenspecies(O2-,H2O2,OH?)“O2-” hyperoxide“H2O2” Peroxide“OH?” hydroxylFigure9-9Thyminedimer.UVinducesacyclobutane(環(huán)丁烷)ringbetweenadjacentT.Radiationdamage1GammaradiationandX-rays(ionizingradiation)causedouble-strandbreaksandareparticularlyhazardous(hardtoberepaired).Radiationdamage2Mutationsarealsocausedbybaseanalogs(堿基類似物)andintercalatingagents(嵌入劑)DNAdamageBaseanalogs:similarenoughtothenormalbasestobeprocessedbycellsandincorporatedintoDNAduringreplication.Buttheybasepairdifferently,leadingtomispairingduringreplication.Themostmutagenicbaseanalogis5-bromoUracil(5-BrU)(溴尿嘧啶).Figure9-10aBaseanaloguesFigure3-33G-Upair烯醇異構(gòu)體酮異構(gòu)體IntercalatingagentsareflatmoleculescontainingseveralpolycyclicringsthatinteractwiththenormalbasesinDNAthroughhydrogenbondsandbasestacking.Figure9-10bIntercalatingagents溴乙非啶二氨基吖啶/原黃素吖啶,氮蒽Topic3:RepairofDNAdamageCHAPTER9ThemutabilityandrepairofDNA3/22/05TwoconsequenceofDNAdamageSomedamages,suchasthyminedimer,nickorbreaksintheDNAbackbone,createimpedimentstoreplicationortranscriptionSomedamagescreatesalteredbasesthathasnoeffectonreplicationbutcausemispairing,whichinturncanbeconvertedtomutation.RepairofDNAdamageDirectreversalofDNAdamagebyphotoreactivation(光活化作用)andalkyltransferase(烷基轉(zhuǎn)移酶)Baseexcisionrepair(切割修復(fù))NucleotideexcisionrepairRecombination(DSB)repairsTranslesionDNAsynthesisMechanismstorepairadamageSeeTable9-1forsummaryRepairofDNAdamageDirectreversalofDNAdamageError-freerepairRepairofDNAdamagePhotoreactivationFigure9-11MonomerizationofthyminedimersbyDNAphotolyasesinthepresenceofvisiblelight.MethyltransferaseRemovesthemethylgroupfromthemethylatedO6-methylguanine.Themethylgroupistransferredtotheproteinitself,inactivatingtheprotein.Figure9-12BaseExcisionrepairenzymeremovedamagedbasesbyabase-flippingmechanismRepairofDNAdamageGlycosylaseRecognizesthedamagedbaseRemovesthedamagedbaseAPendonulease&exonulcease3.Cleavestheabasicsugar-phosphatebackboneExonulcease/DNApolymerase/ligase4.Workssequentiallytocompletetherepairevent.Figure9-14:base-flippingrecognitionbyglycosylaseFigure9-13:removesthedamagedbaseandrepairoxoG:Arepair.

AglycosylaserecognizesthemispairandremovesA.Afail-safeglycosylasealsoremovesTfromT:Gmispairs,asifitknowshowTisproduced.Fail-safesystems(最后保險(xiǎn)系統(tǒng))Figure9-15:NucleotideExcisionrepairenzymescleavedamagedDNAoneithersideofthelesionRepairofDNAdamageRecognizedistortionstotheshapeoftheDNAdoublehelixRemoveashortsingle-strandedsegmentthatincludesthelesion.DNApolymerase/ligasefillinthegap.Figure9-16**Figure9-17.Transcription-couplerepair:nucleotideexcisionrepair(NER)systemiscapableofrescuingRNApolymerasethathasbeenarrestedbythepresenceoflesionsintheDNAtemplate

TFIIHTFIIHisatranscriptionfactorincludingXPAandXPD(UvrB)RecombinationrepairsDNAbreaksbyretrievingsequenceinformationfromundamagedDNARepairofDNAdamageDouble-strandbreak(DSB)repairpathwayDetailsareinchapter10Figure10-4.DamageintheDNAtemplatecanleadtoDSBformationduringreplicationFIGURE10-3DSBrepairmodelforhomologousrecombinationTranslesionDNAsynthesisenablesreplicationtoproceedacrossDNAdamageRepairofDNAdamageError-pronerepair***OccurswhentheaboverepairsarenotefficientenoughsothatareplicatingpolymeraseencountersalesionTranslesionsynthesisisalsocalledafail-safeorlastresortmechanism.TranslesionsynthesisiscatalyzedbyaspecializedclassofDNApolymerasesthatsynthesizeDNAdirectlyacrossthedamagesite.Transl