上海師范大學(xué) 分子生物學(xué)5 DNA損傷修復(fù)及基因操作概述-kaigy課件

SECTIONF

DNA損傷和修復(fù)DNAdamage,repairlinkF1 Mutagenesis(誘變）Mutation(突變)Replicationfidelity（復(fù)制忠實性）Mutagens:chemical&physicalMutagenesis:direct&indirectF1-1Mutation突變突變：DNA堿基水平上的永久性的，可以遺傳的改變Permanent,heritable

alterationsinthebasesequenceofDNA產(chǎn)生突變的原因ReasonsDNA復(fù)制或減數(shù)分裂重組過程中的自發(fā)性錯誤SpontaneouserrorsinDNAreplicationormeioticrecombination由于理化試劑損傷DNA所致

ThedamagingeffectsofphysicalorchemicalmutagensonDNA

DNA非編碼區(qū)NoncodingDNA非調(diào)節(jié)區(qū)NonregulatoryDNA密碼子的第三個堿基

3rdpositionofacodon沉默突變Silentmutation

DNA編碼區(qū)改變了氨基酸CodingDNAalteredAA錯義突變Missensemutation表型效應(yīng)PhenotypiceffectsNoDNA編碼區(qū)產(chǎn)生新終止密碼子產(chǎn)生截短的蛋白CodingDNAstopcodontruncatedprotein無義突變Nonsensemutation點突變的表型效應(yīng)EffectsofapointmutationYesorNoYes插入或缺失Insertionsordeletions移碼突變Frameshiftmutations由于編碼蛋白的基因被改變，結(jié)果導(dǎo)致所翻譯出的蛋白從突變點到C末端都完全被改變了TheORFofaproteinencodedgeneischangedsothattheC-terminalsideofthemutationiscompletelychanged.插入或缺失涉及一個或多個堿基的增加或丟失TheadditionorlossofoneormorebasesinaDNAregionExamplesofdeletionmutations

F1-2復(fù)制忠實性Replicationfidelity

MutationrelevantE.coli在復(fù)制中的自發(fā)突變的概率為1/1010

SpontaneouserrorsinDNAreplicationisveryrare,oneerrorper1010baseinE.coli.DNA復(fù)制保持忠實性的意義：利于確保上下代間遺傳信息的準(zhǔn)確傳遞ImportantforpreservethegeneticinformationfromonegenerationtothenextbyE.colipolymeraseProofreadingF1Mutaagenesis物理誘變劑Physicalmutagens高能離子化輻射：如X射線等可引起斷鏈及堿基戊糖損傷High-energyionizingradiation:X-raysandg-raysstrandbreaksandbase/sugardestruction非離子化輻射如紫外線可引起嘧啶二聚體Nonionizingradiation:UVlightpyrimidinedimers化學(xué)誘變劑Chemicalmutagens 堿基類似物Baseanalogs:directmutagenesis 亞硝酸Nitrousacid:deaminatesCtoproduceU 烷化劑Alkylatingagents 芳基化劑ArylatingagentsF1-3&4:誘變Mutagenesis理化誘變劑所產(chǎn)生的絕大多數(shù)損傷，可在與復(fù)制叉相遇之前就被一種或多種修復(fù)機制所修復(fù)。thegreatmajorityoflesionsintroducedbychemicalandphysicalmutagensarerepairedbyoneormoreoftheerror-freeDNArepairmechanismsbeforethelesionsisencounterbyareplicationfork直接誘變DirectmutagenesisDNA中穩(wěn)定的，具有改變了配對特性的未修復(fù)的堿基，在DNA復(fù)制中使得這種損傷（非永久性的）固定下來成為突變（永久可遺傳的）。

Thestable,unrepairedbasewithalteredbasepairingpropertiesintheDNAisfixedtoamutationduringDNAreplication.5-BrU:G:A烯醇式enolformBrOHHOBr酮基異構(gòu)體KetoformHOAGCTTCCTATCGAAGGATAGCTBCCTATCGAAGGATBaseanalogincorporationAGCTBCCTATCGAGGGATAGCTTCCTATCGAAGGAT1stroundofreplicationAGCTBCCTATCGAAGGATAGCTCCCTATCGAGGGAT2ndroundofreplicationA·TG·Ctransition5－溴尿嘧啶間接誘變Indirectmutagenesis

轉(zhuǎn)移損傷DNA合成保持DNA的完整性但不保證序列的準(zhǔn)確性轉(zhuǎn)移損傷DNA合成有時也稱為“易錯修復(fù)”F2 DNA損傷DNAdamage

DNA損傷DNAlesions:氧化性損傷oxidativedamage烷基化Alkylation聚化加和物bulkyadductsF2-1 DNA損傷DNAlessions

損傷是DNA正常的化學(xué)或物理結(jié)構(gòu)的改變。AnalterationtothenormalchemicalorphysicalstructureoftheDNAThebiologicaleffectoftheunrepairedDNAlesionsLethal(celldeath)PhysicaldistortionofthelocalDNAstructureBlocksreplicationAnd/ortranscriptionMutagenicAllowedtoRemainedintheDNAAmutationcouldbecomefixedbydirectorindirectmutagenesisLivingcellAlteredchemistryofthebasesChemicalreactivityofbasesisresponsibleforsomeDNAlesiondeamination--ATGCTACG----TACGATGC----ATGUTACG----TACGATGC----ATGTACG----TACGATGC--

U--ATGCTACG----TACGATGC--UracilDNAglycosylaseCytosinedeaminationandrepairbackOxidationproducts在所以需氧細胞中，由于活性氧如超氧化物，氫過氧化物及羥基自由基的存在，在正常條件下會發(fā)生氧化損傷occursunderNORMALconditionsinallaerobiccellsduetothepresenceofreactiveoxygenspecies(ROS),suchassuperoxide,hydrogenperoxide,andthehydroxylradicals(?OH).電離輻射引起的水輻射分解產(chǎn)生的羥基自由基能提高這些氧化產(chǎn)物的水平ThelevelofthisdamagecanbeINCREEASEDbyhydroxylradicalsfromtheradiolysisofH2OcausedbyionizingradiationF2-3烷基化Alkylation烷化劑是可將烷基（如甲基）加入到核酸上各種位點的親電化學(xué)試劑，如甲基甲烷磺酸鹽和乙基亞硝基脲Nucleotidemodificationcausedbyelectrophilicalkylatingagentssuchasmethylmethanesulfonate（MMS）andethylnitrosourea(ENU)烷基堿基親電化學(xué)試劑加烷基到核酸上各種位點Electrophilicchemicalsaddsalkylgroupstovariouspositionsonnucleicacids不同于正常甲基化酶的甲基化位點Distinctfromthosemethylatedbynormalmethylatingenzymes.烷基劑Cyclobutanepyrimidinedimer(嘧啶二聚體)Guanineadductofbenzo[a]pyrene芳香烷化劑共價加合物backF3 DNA修復(fù)DNArepairPhotoreactivation(光活化作用)Alkyltransferase(烷基轉(zhuǎn)移酶)Exisionrepair(切割修復(fù))Mismatchrepair(錯配修復(fù))Hereditaryrepairdefects(遺傳修復(fù)缺陷)F3-1:光復(fù)活Photoreactivation在可見光存在情況下，DNA光解酶可將環(huán)丁烷嘧啶二聚體再分解成單體MonomerizationofcyclobutanepyrimidinedimersbyDNAphotolyasesinthepresenceofvisiblelight光復(fù)活是損傷被直接修復(fù)的例子，是無差錯修復(fù)Directreversalofalesionandiserror-freeF3-2:烷基轉(zhuǎn)移酶Alkyltransferase烷基轉(zhuǎn)移酶可以直接從突變的O6-烷基鳥嘌呤（可與T配對）上去除烷基。烷基轉(zhuǎn)移到該蛋白并使之失活，故每個烷基轉(zhuǎn)移酶只能用1次。RemovesthealkylgroupfrommutagenicO6-alkylguaninewhichcanbase-pairwithT.Thealkylgroupistransferredtotheproteinitselfandinactivateit.光復(fù)活是損傷被直接修復(fù)的例子，是無差錯修復(fù)Directreversalofalesionandiserror-free這種修復(fù)對烷化劑具有適應(yīng)性：E.coli中低劑量烷化劑會誘發(fā)這一反應(yīng)并提高了機體對高劑量的誘變效應(yīng)和致死的保護能力。TheresponseisadaptivebecauseitisinducedinE.colibylowlevelsofalkylatingagentsandgivesincreasedprotectionagainstthelethalandmutageniceffectsofthehighdosesF3-3:切除修復(fù)Excisionrepair包括核苷酸切除修復(fù)和堿基切除修復(fù)兩種形式Includs

nucleotideexcisionrepair(NER)andbaseexcisionrepair(BER).是一種普遍存在的修復(fù)機制Isaubiquitousmechanismrepairingavarietyoflesions.是無差錯修復(fù)Error-freerepairNucleotideexcisionrepair內(nèi)切酶在損傷部位兩邊各切除精確數(shù)目的堿基AnendonucleasecleavesDNAaprecisenumberofbasesonbothsidesofthelesions(UvrABCendonulceaseremovespyrimidinedimers)包含損傷的DNA大片段被切除移去Excisedlesion-DNAfragmentisremoved缺口被填補和連接ThegapisfilledbyDNApolymeraseIandsealedbyligaseBaseexcisionrepairDNAglycolasescleavesapurinicorpyrimidinesiteDNApolymeraseDNAligasecleavesN-glycosylicbondAPendonuclease3’5’cleavageand&5’3’synthesisF3-3:錯配修復(fù)Mismatchrepair錯配修復(fù)是切除修復(fù)的一種特殊形式，用于修復(fù)在復(fù)制中錯配并漏過校正檢驗的任何堿基。AspecializedformofexcisionrepairwhichdealswithanybasemispairsproducedduringreplicationandwhichhaveescapedproofreadingF1

誘變MutagenesisF2 損傷DNAdamageMutation:replicationfidelity,mutagens,mutagenesisF3 修復(fù)DNArepair

DNAlesions:oxidativedamage,alkylation,bulkyadductsPhotoreaction,alkyltransferase,excisionrepair,mismatchrepairsummarySectionG基因操作GenemanipulationG1-1DNA克隆DNAcloningG1-2宿主和載體HostsandvectorsG1-3亞克隆SubcloningG1-4DNA文庫DNAlibrariesG1-5篩選文庫ScreeninglibrariesG1-6克隆分析Analysisofaclone基因操作GenemanipulationG1 DNA克隆概述

DNAcloning:anoverview基因操作Genemanipulation基因克隆的重要環(huán)節(jié)就是把一個外源基因?qū)肷锛毎?，并使它得到擴增。然而一個外源DNA片段是很難進入受體細胞的，即使進入細胞，一般也不能進行復(fù)制和功能的表達(是因為所得到的外源DNA片斷一般不帶有復(fù)制子系統(tǒng)及在新的受體細胞中進行功能表達的調(diào)控系統(tǒng))。在基因工程中，通常是把外源DNA片斷利用運載工具送入生物細胞。攜帶外源基因進入受體細胞的這種工具叫做載體(Vector)

G1-1DNAcloning(definition)

DNA克隆定義：將基因組中攜有某一目的基因或其他相關(guān)序列的較小片段連接到一段可以自主復(fù)制的DNA（即載體）上，從而形成可以在另一宿主中進行復(fù)制的重組DNA，這種復(fù)制是獨立于原初基因組的。帶有重組DNA的宿主細胞的增殖構(gòu)成了一群具有遺傳一致性的個體（或稱為單克隆)。這一系列操作過程就被稱作DNA克隆。

DNAcloningistoplacearelativelyshortfragmentofagenome,whichmightcontainthegeneorothersequenceofinterest,inanautonomouslyreplicatingpieceofDNA,knownasavector,formingrecombinantDNA,whichcanbereplicatesindependentlyoftheoriginalgenome,andnormallyinotherhostspeciesaltogether.PropagationofthehostorganismcontainingtherecombinantDNAformsasetofgeneticallyidenticalorganism,oraclone.ThisprocessiscalledDNAcloning.G1DNAcloning:AnOverview遺傳工程范疇DNA序列分析，以及由此派生的蛋白質(zhì)序列分析(參見J2)。對基因啟動子及其他調(diào)控序列的分離與分析(參見J4)。通過大量正常和突變形式的產(chǎn)物研究來了解這些蛋白質(zhì)/酶/RNA的功能(參見J5)。突變的鑒定，例如由于基因缺陷導(dǎo)致形成的疾病(參見J6)。生物技術(shù)，如蛋白質(zhì)及其他重要生物功能的分子如人胰島素和生長素的大規(guī)模工業(yè)化生產(chǎn)(參見J6)。工程動物、工程植物以及基因治療(參見J6)。改變了特性的工程蛋白質(zhì)(參見J6)。

G1-2宿主和載體Hostsandvectors宿主細胞Hostorganism/cell:

wheretheplasmidsgetmultipliedandpropagatedfaithfully,whichiscrucialforDNAcloning.用于DNA克隆的宿主HostsforDNAcloningvector

Prokaryotichost:E.coli(mostcases) Eukaryotichost:Yeast

Saccharomycescerevisiae(largefragmentsofhumangenome)G1DNAcloning:AnOverview載體具備的特性GeneralfeaturesofaVector能夠獨立于宿主細胞基因組之外進行自我復(fù)制與分離

autonomouslyreplicatingDNA

independentofhost’sgenome.容易從宿主細胞中分離純化Easilytobeisolated

fromthehostcell大部分是環(huán)形的Mostarecircular,somearelinear含有至少一個選擇標(biāo)記（即某個基因能賦予宿主對某種毒素的抗性，或使宿主細胞能在特定生長條件下生存，從而能使含有該基因的載體的宿主可從那些不含該載體的細胞中被篩選出來）Containsatleastone

selectivemarker,whichallowshostcellscontainingthevectortobeselectedamongstthosewhichdonot.含有一個多克隆位點

Containsa

multiplecloningsite(MCS)G1DNAcloning:AnOverview載體的類型Typesofvectors克隆載體Cloningvectors表達載體Expressionvectors整合載體IntegrationvectorsG1DNAcloning:AnOverview克隆載體Cloningvectors:

allowingtheexogenousDNAtobeinserted,stored,andmanipulatedatDNAlevel.E.colicloningvector:plasmids,bacteriophages(landM13),plasmid-bacteriophagelhybrids(cosmids).Yeastcloningvector:yeastartificialchromosomes(YACs)MCS表達載體Expressionvectors:

allowingtheexogenousDNAtobeinsertedandexpressed.啟動子和終止子是必需的Promoter

andterminatorforRNAtranscriptionarerequired.bacterialexpressionvectorsyeastexpressionvectorsmammalianexpressionvectorsG1DNAcloning:AnOverview整合載體Integrationvectors:allowingtheexogenousDNAtobeinsertedandintegratedintoachromosomalDNAafteratransformation.Theintegrationisconductedbyhomologousrecombinationbetweenthehomologoussequencesharedbytheplasmidandthegenomeoftherecipientcells.

細菌整合載體bacterialintegrationvectors(Agrobacterium

tumefaciensTiplasmidisusedtointegrateDNAintoplantgenome)

酵母整合載體yeastintegrationvectors

哺乳類整合載體Mammalianintegrationvector:virusbased

G1DNAcloning:AnOverview

G1-3亞克隆Subcloning定義：將已克隆的DNA片段從一載體向另一載體的轉(zhuǎn)移，這一過程被稱為亞克隆

TransferofafragmentofclonedDNAfromonevectortoanother.亞克隆的意義亞克隆可用來對較大的克隆片段上較短區(qū)段進行仔細研究

Enablesustoinvestigateashortregionofalargeclonedfragmentinmoredetail.可將基因轉(zhuǎn)移到能在特定物種中表達的另一載體上

Totransferagenefromoneplasmidtoavectordesignedtoexpressitinaparticularspecies.G1DNAcloning:AnOverviewPreparationofplasmidscontainingaclonedDNAfragment(insert)Plasmidpreparation(vector)Restrictiondigestion(trimmingtheDNAends)

Ligation(jointheinsertandthevector)Transformation&selectionoftransformants(introducetheplasmidsintohostcells)AssayoftherecombinantsDNASubcloning:aflowchartRestrictionendonuclease瓊脂糖電泳AgroseGelElectrophoresis:

CheckyourDNAateachstepSeparationandPurificationofDNAfragmentsofinterestsAnalysisofrecombinantplasmidsladderRestrictionanalysisofaplasmidG1DNAcloning:AnOverviewG1-4DNA文庫DNAlibrariesDNA文庫定義：DNA文庫是一套DNA克隆，每個克隆都是插入了不同片段的載體在宿主中擴增后的產(chǎn)物。

DNAlibraries

aresetsofDNAclones,eachofwhichhasbeenderivedfromtheinsertionofadifferentfragmentintoavectorfollowedbypropagationinthehost.

一個克隆是一個遺傳上獨特的個體或一群相同的個體。

Aclone

isageneticallydistinctindividualorsetofidenticalindividuals基因組文庫和cDNA文庫

Genomiclibrariesand

cDNAlibrariesG1DNAcloning:AnOverview基因組文庫是用基因組DNA的隨機片段制備成的。然而用基因組文庫來克隆某一基因是一種效率極低的方法，特別是對于龐大的真核生物基因組，其中大量DNA是非編碼的

Genomiclibraries：preparedformrandomfragmentsofgenomicDNA,whichmaybeinefficienttofindagenebecauseofthehugeabundanceofthenon-codingDNAcDNA文庫是指用來自表達目的基因細胞或組織的mRNA作為來源構(gòu)建成的文庫。經(jīng)反轉(zhuǎn)錄將mRNA合成cDNA(DNA拷貝)后，插入載體構(gòu)建成cDNA文庫。用DNA文庫來克隆目的基因是有效的，但克隆的僅是其編碼區(qū)，而不包含編碼區(qū)以外的基因組序列。cDNAlibraries：DNAcopies(cDNA)synthesizedfromthemRNAbyreversetranscription

areinsertedintoavectortoformacDNAlibrary.Muchmoreefficientinidentifyingagene,butdonotcontainDNAcodingfunctionalRNAornoncodingsequence.G1DNAcloning:AnOverviewG1-5篩選文庫

Screeninglibraries

通常篩選方法：是用一段與目的基因序列的某一區(qū)段互補或部分互補的放射性或熒光標(biāo)記的DNA探針，通過雜交來檢測出該基因，用雜交來鑒定目的基因HybridizationtoidentifytheinterestedDNAoritsRNAproduct放射性探針Radiolabeledprobeswhichiscomplementarytoaregionoftheinterestedgene探針Probes:

根據(jù)蛋白產(chǎn)物序列推測出的一段寡核昔酸

Anoligonucleotidederivedfromthesequenceofaproteinproductofthegene可以來自另一物種中的某一相關(guān)基因

ADNAfragment/oligofromarelatedgeneofanotherspecies點在膜上BlottingtheDNAorRNAonamembrane探針與DNA膜雜交HybridizethelabeledprobewithDNAmembrane(Southern)orRNA(Northern)membrane找基因SearchingthegenesofinterestinaDNAlibrary

G1DNAcloning:AnOverview另一篩選方法是基于文庫中被克隆的編碼區(qū)表達后:1)對其蛋白產(chǎn)物進行活性鑒定2)或是用特異抗體對其進行鑒定

IdentifytheproteinproductofaninterestedgeneProteinactivityWesternblottingusingaspecificantibodyG1DNAcloning:AnOverviewG1-6克隆分析Analysisofaclone

限制性酶切圖譜

Restrictionmapping:

digestionofthewithrestrictionenzymes.測序Sequencing

theclonedDNATable1須記憶YoumustfullyunderstandthefunctionandapplicationofalltheenzymeslistedinTable1beforethefinalexam.G1DNAcloning:AnOverviewG2質(zhì)粒DNA的制備PreparationofplasmidDNAG2-1質(zhì)粒載體PlasmidasvectorsG2-2質(zhì)粒的小量制備PlasmidminipreparationG2-3堿裂解AlkalinelysisG2-4酚抽提PhenolextractionG2-5乙醇沉淀EthanolprecipitationG2-6氯化銫梯度

Cesiumchloridegradient(purification)GenemanipulationG2-1質(zhì)粒載體Plasmidasvectors質(zhì)粒：質(zhì)粒為染色體外的小型環(huán)狀分子，大小約為2－200kb，通常以多拷貝（可多達幾百個）形式存在于宿主細胞大腸桿菌中。Plasmids:small,extrachromosomalcircularmolecules,from2to~200kbinsize,whichexistinmultiplecopieswithinthehostcells.質(zhì)粒含有復(fù)制起點，用以保證質(zhì)粒的自主復(fù)制

Containanoriginofreplicationandreplicateindependently質(zhì)粒一般僅含攜有幾個基因

，其中之一會賦予細菌對抗生素物質(zhì)的抗性如amp抗性基因。

Usuallycarryafewgenes,oneofwhichmayconferresistancetoantibacterialsubstance.Example:

amp抗性基因，編碼可以降解青霉素類抗生素如氨芐青霉素的β-內(nèi)酰胺酶。amprgeneencodingtheenzymeb-lactamsewhichdegradespenicillinantibioticssuchasampicillin.G2PreparationofplasmidDNAG2-2質(zhì)粒的小量制備PlasmidminipreparationfromE.coli質(zhì)粒Plasmids質(zhì)粒遠遠小于大腸桿菌染色體DNA，可以用物理化學(xué)方法將它們相分離，例如堿裂解法

~2-20kbinlengththatmuchsmallerthanE.colichromosomalDNA(4600kb),andindependentlysupercoiledResistanttoshearingforceandchemicaldenaturation,thuscanbeisolatedfromthechromosomalDNAeasilysuchasalkalinelysis.小量制備Minipreparation(miniprep)從幾毫升菌液中提取出足夠量的質(zhì)粒DNA供初步操作所需是完全可以的。這樣的提取操作通常稱為小量制備。

IsolationofplasmidDNAfromafewmililiters(ml)ofbacterialculture.G2PreparationofplasmidDNA小量制備Miniprep培養(yǎng)Growthofthecellscontainingplasmids離心收集細胞Collect

thecellsbycentrifugation堿性裂解Alkalinelysis

resuspensionalkalinelysisneutralization用酚去蛋白Phenolextractiontogetridoftheproteincontaminants乙醇沉淀收集核酸Ethanolprecipitationtoconcentratethenucleicacidsremained.(PleasenotedthatRNaseAisverybadforthelabworkingwithRNA)G2PreparationofplasmidDNAG2-3堿裂解Alkalinelysis

菌體沉淀重懸于懸浮緩沖液Resuspendthecellsinabuffersolution用溶菌酶降解菌體細胞壁Lysozymetodigestthecellwall(optional)細胞裂解液即含去污劑十二烷基硫酸鈉(SDS)的堿性NaOH溶液

CelllysisinlysisbuffercontainingSDS(disruptscellmembraneanddenaturesproteins)and

NaOH(denaturesDNA)用高濃度pH5的乙酸鉀溶液中和。其作用是沉淀已變性蛋白質(zhì)和染色體DNA及大部分去污劑（十二烷基硫酸鉀不溶于水）。將溶液再次離心，上清(裂解液)中含有質(zhì)粒DNA。

NeutralizationbuffercontainingKOAc(pH5):renaturationofplasmidDNA(supercoiled)andprecipitationofdenaturedproteinsandchromosomalDNAwhichcannotberenaturedbecauseofitssizeandphysicalpropertyofeasilybeingsheared.G2PreparationofplasmidDNAGrowthecellHarvestthecellbycentrifugationAlkalinelysisofthecellResuspendthecellpelletneutralizationPhenolextractionEthanolprecipitationCsClgradientpurificationG2-6氯化銫梯度離心Cesiumchloridegradientcentrifugation氯化銫梯度離心可被用來作為最終純化步驟

CsClgradientpurificationisthelaststepoflargescaleplasmidDNApurification步驟雖有點費力

Laborious獲得極純的超螺旋質(zhì)粒DNA的最佳方法

BestfortheproductionofverypuresupercoiledplasmidDNA粗裂解液經(jīng)含有溴化乙錠的的CsCl分級分離后.各種成分會完全分離

Thepresenceofethidiu