老師課件給學(xué)生05 sec f

SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭SectionF

DNADamage,RepairandbinationF1Mutagenesis(誘變)F2DNADamage(損傷)F3DNARepair(修復(fù))F4bination(重組)2014-03-06SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭F1Mutagenesis(誘變)Mutation(突變)ReplicationFidelity(復(fù)制忠誠(chéng)性)Chemicalmutagens(化學(xué)誘變劑)Directmutagenesis(直接誘變)Indirectmutagenesis(間接誘變)SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Mutation-I(突變)Definition:Mutationsarepermanent(永久的),heritable(可遺傳的)alterationinthebasesequenceofDNA.Classification:(1)Pointmutation;(2)Frame-shiftmutation1.Pointmutation:Asinglebasechange,includes:

transition(轉(zhuǎn)換):onepurine(orpyrimidine)isreplacedbytheother

purine(orpyrimidine);

transversion(顛換):apurinereplacesapyrimidine(T→A).T→A蘇→絲ASectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Mutation-I(突變)Classification:1.2Frame-shift

(移碼)mutation:Insertionsordeletionsinvolvetheaddition(增加)orloss(減少)ofoneormorebases，andthetranslatedproteinsequencetotheC-terminalsideofthemutationiscompletelychanged.TASectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Mutation-II(突變)Phenotypic(表型)effectsofmutation:Silent(沉默)mutation:Ifthemutationis(1)inanon-codingornonregulatoryDNA;(2)inthethirdpositionofacodon.Missense

(錯(cuò)意)mutation:Ifthemutationresultsinanalteredaminoacidinageneproduct,thenitisamissensemutationwhoseeffectcanvaryfromnothingtodeath,dependingonthepositionoftheaminoacidaffected.Consequencesofmutation:Tumorigenesis(癌變):Mutationsthataffecttheprocessesofcellgrowthandcelldeathcanresultintumorigenesis;Geneticpolymorphisms(遺傳多態(tài)性):Theaccumulationofmanysilentandothernon-deathmutationsinpopulationsproducesgeneticpolymorphisms.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭ReplicationFidelity(復(fù)制忠誠(chéng)性)ThehighaccuracyofDNAreplication(oneerrorper1010basesincorporated)dependsonacombinationofproperbasepairingoftemplatestrandandingnucleotideintheactivesiteoftheDNApolymerase,proofreadingoftheincorporatedbaseby3’5’exonucleaseandmismatchrepair.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Chemicalmutagens(化學(xué)誘變劑)Mostchemicalmutagensarecarcinogens(致癌劑)Baseanalogs(類似物):arethenormalbaseswithalteredbasepairingpropertiesandcancausedirectmutagenesis.Nitrousacid(亞硝酸):deaminatescytosinetoproduceuracil,whichbase-pairswithadenineandcausesG?C

A?T.Alkylatingagents(烷化劑):producealkylated(methylated)bases.Intercalators(嵌入劑):generateinsertionanddeletionmutation.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Directmutagenesis(直接誘變)Mechanism:IfabaseanalogisnotremovedbyaDNArepairmechanismbeforepassageofreplicationfork,thenanincorrectbasewillbeincorporated.ThesecondreplicationfixesthemutationpermanentlyintheDNA.Forexample:thymineanalog5-bromo-uracil(5-溴尿嘧啶B)base-pairswithadenine腺(A)andalsowithguanine鳥(G).Afterreplication,thustheBcausesA?T

G?C.AGCTTCCTA

TCGAAGGATAGCTBCCTA

TCGAAGGATT

BAGCTBCCTA

TCGAGGGATAGCTBCCTA

TCGAAGGATAGCTBCCTA

TCGAAGGATAGCTCCCTA

TCGAGGGATB

GG

CB:AB

:A脫氨突變被固定SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Indirectmutagenesis(間接誘變)Definition:Indirectmutagenesisresultsfrom:1.Theactivationoftrans-lesion(轉(zhuǎn)移損傷)DNAsynthesis(SOS)2.Thesuppressionofproofreadingsystem,oneormoreincorrectbasesareincorporatedinDNA.Mechanism:1.Mostlesions(損傷)inDNAarerepairedbyerror-free(無錯(cuò))directreversal(恢復(fù))orexcisionrepair(切除修復(fù))mechanismsbeforepassageofareplicationfork.2.Ifthisisnotpossible,trans-lesionDNAsynthesis(轉(zhuǎn)移損傷DNA合成)maytakeplaceandoneormoreincorrectbaseseincorporatedoppositethelesion,soasto(以便)maintaintheintegrity(完整性)ofthechromosome.3.Proofreadingmaybesuppressed(抑制)duringthisprocess.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭F2DNADamage(損傷)DNAlesions(損傷)Spontaneouslesions(自發(fā)性損傷)Oxidativedamage(氧化性損傷)Alkylation(烷基化)Bulkyadducts(聚化加合物)SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭DNAlesions(DNA損傷)TTACG…–A:T–GCTGCHOHATAACGT:AA:T…...G:C…...DPCDefinition:AlesionisanalterationofthenormalchemicalorphysicalstructureoftheDNA.Mechanism:Someofthenitrogenandcarbonatomsintheheterocyclicring(雜環(huán))systemsarechemicallyquitereactive.Manyexogenousagents,suchaschemicalsandradiation,cancausestructurechangestothesepositionsandresultinDNAlesions.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭DNAlesions(DNA損傷)Consequences:1.DNAmutation:IfsuchalesionwasallowedtoremainintheDNA,amutationcouldefixedintheDNAbydirectorindirectmutagenesis;2.Celldeath:Ifthechemicalchangemayproduceaphysicaldistortion(變形)intheDNAwhichblocksreplicationand/ortranscription,thencausingcelldeath.TTACG…–A:T–GCTGCHOHATAACGT:AA:T…...G:C…...DPCSectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Spontaneouslesions(自發(fā)性損傷)Mechanism:1.Deamination:Cytosine(腺)deaminatestoproduceuracil(尿).AnyuracilfoundinDNAisremovedbyanenzymecalleduracilDNAglycosylase(糖化酶)andisreplacedbycytosine.2.Depurination:ItisanotherspontaneoushydrolyticreactionthatinvolvescleavageoftheN-glycosylicbond(糖苷鍵)betweenN-9ofthepurinebasesAandGandC-1'ofthedeoxy-ribosesugarandhencelossofpurinebasesfromtheDNAbackbone.3.Depyrimidination:itcanalsooccurwithverylowfrequency.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Oxidativedamage(氧化性損傷)Definition:Reactiveoxygenspecies(ROS)suchassuperoxide(O2-?),hydrogenperoxide(H2O2)andhydroxylradicals(?OH)produceavarietyoflesions,suchdamageoccursspontaneouslybutisincreasedbysomeexogenousagentsincluding

-rays.Examples:=OH8-羥基鳥嘌呤8-OxoguanineH2N8=ONH2HO2-羥基腺嘌呤2-OxoadenineO=O-CH2OH5-羥甲基尿嘧啶5-hydroxymethyluracilSectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Alkylation(烷基化)Examplesforalkylatingagents(烷基化試劑):O=CH3–S–O–CH3O=H2N–C–NCH2CH3N=OO=甲基磺酸甲酯乙基亞硝基尿

Examplesformethylatedbases(甲基化堿基):H2N=OCH3CH37-甲基鳥嘌呤3-甲基腺嘌呤H2N=OCH3O6-甲基鳥嘌呤NH2SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Bulkyadducts(聚化加合物)Definition:Theyarebulkycovalentadductswhichareformedbyphysicalorchemicalagents.Theyresultinlossofbasepairingabilitywiththeoppositestrand.Examples:1.Physicalagent:UVlight.相鄰胸腺嘧啶環(huán)化形成二聚體；不能進(jìn)行堿基配對(duì)。S-NC–NC=OC=CS-NC–NC=OC=CP環(huán)丁烷嘧啶二聚體2.Chemicalagent:Aromaticarylatingagents(芳香族烷化劑)苯并芘鳥嘌呤加合物P450G–NHHOHOOHSectionF:DNADamageRepair華中師范大學(xué)生科院楊旭F3DNARepair(修復(fù))Photo-reactivation(光復(fù)活)Alkyltransferase(烷基轉(zhuǎn)移酶)Excisionrepair(切除修復(fù))Mismatchrepair(錯(cuò)配修復(fù))SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Photo-reactivation(光復(fù)活)Definition:Inthepresenceofvisiblelight,theDNAphotolyases(光解酶,photo-reactivatingenzymes,光復(fù)活酶)canresolvecyclo-butanepyrimidinedimers(環(huán)丁烷嘧啶二聚體)intomonomeragain,thisprocessisknownasphoto-reactivation.Mechanism:Theseenzymeshaveprostheticgroups(輔基-色素)whichabsorbbluelightandtransfertheenergytothecyclo-butaneringwhichisthencleaved.Photoreactivationisspecificforpyrimidinedimers.Itisadirectreversalofalesionandiserror-free.Examples:E.coliphotolyasehastwochromophores(色素分子):1.N5N10-亞甲基四氫葉酸;2.還原性黃素腺嘌呤二核苷酸.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Photo-reactivation(光復(fù)活)ReactionmechanismofDNAphotolyaseSectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Alkyltransferase(烷基轉(zhuǎn)移酶)Mechanism:Mutagenicprotectionisafforded(被提供)byanalkyltransferase,whichremovesthealkylgroupfromthedirectlymutagenicO6-alkylguanine(O6-甲基鳥嘌呤).Thealkylgroupistransferred(被轉(zhuǎn)移)totheproteinitselfandinactivatesit.Thus,eachalkyltransferasecanonlybeusedonce.Photo-reactivationandalkyl-transferaseeffectareallbelongtotheerror-freedirectreversalforms(無差錯(cuò)直接修復(fù)).H2N=OCH3H2N=OCys-SHCys-SHCH3SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Nucleotideexcisionrepair(NER)對(duì)于DNA雙鏈之間不能形成氫鍵配對(duì)的損傷進(jìn)行修復(fù)Nucleotideexcisionrepair(NER):

anendonucleasemakesnicks(切口)oneithersideofthelesion,

thenthelesionisremovedtoleaveagap.

ThisgapisthenfilledbyaDNApolymeraseI,

andDNAligasemakesfinalphosphodiesterbond.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Baseexcisionrepair(BER)Baseexcisionrepair(BER):

thelesionisremovedbyaspecificDNAglycosylase(DNA糖基化酶),whichcleavetheN-glycosylicbondbetweenthealteredbaseandthesugar.

ThisresultsAPsite(脫嘌呤/脫嘧啶位點(diǎn)),thenitiscleavedandexpandedtoagapbyanAPendonuclease(AP內(nèi)切核酸酶)andexonuclease(外切核酸酶).

ThisgapisthenfilledbyaDNApolymeraseI,

andDNAligasemakesfinalphosphodi-esterbond.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Mismatchrepair(錯(cuò)配修復(fù))Mechanism:(保存母鏈，修正子鏈)1.Mismatch:Inareplicationalmispair,forexampleG?TorC?A,thewrongbaseisonlyinthedaughterstrand.Formismatchrepair,

wrongpairand

wrongstrandmustbeknown.2.Distinguish:

thewrongpair(mismatch)isidentifiedandattachedbyMutS/MutLproteincomplex,

andthewrong(daughter)strandisidentifiedbyMutH

endonucleaseaccordingto

thehemi-methylated(半甲基化的)sequenceGATC.3.Excisionrepair:MutH

endonucleasethenmakesnicks(切口)atGATCsiteoneithersideofthelesioninthenewlysynthesizedstrand,anddoexcisionrepair.SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭F4bination(重組)Homologousbination(同源重組)Site-specificbination(位點(diǎn)特異性重組)Transposition(轉(zhuǎn)座作用)SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Homologousbination-I(同源重組)Definition:ThisprocessinvolvestheexchangeofhomologousregionsbetweentwoDNAmolecules,itisalsoknownaspost-replicationrepair.Theintegrity(完整性)ofDNAcontainingun-repairedlesionscanbefixedduringreplicationbyhomologousbination.Mechanism:1.Ineukaryotes,thiscommonlyoccursduringmeiosis(減數(shù)分裂),whenthehomologousduplicatedchromosomeslineupinparallelinmetaphaseI(中期I)andthenon-sisterchromatidsexchangeequivalentsectionsbycrossingover.來自父親的染色單體來自母親的染色單體SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭Hollidaymodel2.Inprokaryotes:ThismodelwasproposedbyRobinHollidayin1964.Mechanism:TwohomologousDNAalign(排列成行)witheachother;Nuclease/RecBCDproteincomplexmakesnicks(切口)nearthechisequence(GCTGGTGG);ThessDNAescoatedinRecAproteintoformRecA-ssDNAfilaments.Theycrossoverandsearchtheoppositesequence;Thenicksaresealed(封閉)andafour-branchedHollidaystructureisformed,thenbranchmigrates;TheHollidayintermediate(中間體)canberesolvedintotwoDNAduplexesinoneoftwoways:SectionF:DNADamageRepair華中師范大學(xué)生科院楊旭HollidaymodelABabABabABabSectionF:DNADamageRepair華中師范大學(xué)生科院楊旭attPofλpOp’attBofE.coli

BOB’BOP’POB’Int(Integrase)Xis(Excisionase)Site-specificbinationDefinition:ismediatedbyproteinsthatrecognizespecificDNAsequences.ItdoesnotrequireRecAorssDNA.

Examples:Prokaryotes:Bacteriophage

DNA/E.coliDNASectionF:DNADamageRepair華中師范大學(xué)生科院楊旭--CGTAAGCTCGCCGGCGGCAGGATTGA----GCATTCGACGCCGCGCCGTCCTAACT----CGTAAGCTGCGGCAGGATTGA----GCATTCGACGCCGTCCTAACT----CGTAAGCTCGCCG

GCGGCAGGATTGA----GCATTCGACGCCGCGCCGTCCTAACT--transposase--CGTAAGCTGCGGCAGGATTGA----GCATTCGACGCCGTCCTAACT--Transposition(轉(zhuǎn)座作用)Definition:Transposons(轉(zhuǎn)座子)aresmallDNAsequencesthatcanmovetoalmostanypositioninacell’sgenome.Transpositionhasalsobeencalledillegitimate(異常)binationbecauseitrequiresnohomologybetweensequencesnorisitsite-specific.Examples:ISinE.coliInsertionSequence(插入序列):Theycompriseatransposase(轉(zhuǎn)座酶)geneflankedby(側(cè)連)ITR.ThetransposasemakesastaggeredcutinthechromosomalDNAandtheninsertsacopyofISelements.ThegapssealedbyDNApolymeraseIandDNA