分子生物学：E DNA Replication DNA复制

EDNAReplication/DNA复制Geneticinformationisalsopassedfromgenerationtogeneration.Acrucialpropertyoflifeisthateventhemostcomplexorganismsareabletobecopied.3’-5’phosphodiesterbond3’-5’磷酸二酯键PPi(pyrophosphate)

Substrates(底物)forDNAsynthesis:deoxynucleoside5’-triphosphates三磷酸脱氧核苷dNTPs(dATP,dGTP,dCTP&TTP)DNAsynthesisoccursbyaddingnucleotidesto3’-OHDNA/RNA:5’to3’Protein:NtoCPCR的原理PCR的反应条件PCR的特点PCR的类型PCR的应用PCR技术及其应用PCR的原理引物引物DNA聚合酶DNA聚合酶特定DNA片段PCR反应循环72℃94℃55℃PCR循环变性退火延伸

PCR的指数扩增（2n）引物延伸延伸5’5’3’3’变性、退火变性、退火1）PCR反应成分（1）模板单、双链DNA均可。不能混有蛋白酶、核酸酶、DNA聚合酶抑制剂、DNA结合蛋白类。

DNA模板一般100ng/100L。模板浓度过高会导致反应的非特异性增加。PCR反应条件（2）引物浓度

0.1-0.5mol/L

浓度过高易导致模板与引物错配,反应特异性下降。（3）TaqDNA聚合酶

0.5-2.5U/50l

酶量增加使反应特异性下降;酶量过少影响反应产量。（4）dNTP

dNTP浓度取决于扩增片段的长度四种dNTP浓度应相等浓度过高易产生错误碱基的掺入,浓度过低则降低反应产量

dNTP可与Mg2+结合,使游离的Mg2+浓度下降,影响DNA聚合酶的活性。（5）Mg2+

Mg2+是DNA聚合酶的激活剂。适宜浓度为0.5-2.5mmol/L反应体系。

Mg2+浓度过低会使Taq酶活性丧失、PCR产量下降；Mg2+过高影响反应特异性。

Mg2+可与负离子结合,所以反应体系中dNTP、EDTA等的浓度影响反应中游离的Mg2+浓度。2）循环参数变性

使双链DNA解链为单链

94℃，20-30秒(2)退火

温度由引物长度和GC含量决定。增加温度能减少引物与模板的非特异性结合;降低温度可增加反应的灵敏性。（3）延伸

70-75℃,一般为72℃

延伸时间由扩增片段长度决定（4）循环次数

主要取决于模版DNA的浓度一般为25-35次次数过多：扩增效率降低错误掺入率增加PCR技术的特点1）高度的灵敏性30轮循环扩增量达230个拷贝（109拷贝）PCR产物每轮循环增加一倍2）特异性引物引物

引物的序列及其与模板结合的特异性是决定PCR反应结果的关键。引物设计的最大原则是最大限度地提高扩增效率和特异性；同时尽可能减少非特异性扩增。引物设计：（1)序列应位于高度保守区,与非扩增区无同源序列。（2）引物长度以15-40bp为宜。（3）碱基尽可能随机分布,G+C占40-60%。（4）引物内部避免形成二级结构。（5）两引物间避免有互补序列。（6）引物3’端为关键碱基；5’端无严格限制。3’5’3’5’限制性内切酶的识别序列启动子序列定点突变探针标记3）操作简便易行

利用PCR扩增,只需要数小时,就可以扩增出可用电泳法检出的DNA，可用于检测基因组中仅含数个拷贝的模板序列。不对称PCR反向PCR多重PCR标记引物PCR锚定PCRPCR固相分析法原位PCRRT-PCR定量PCR-----PCR的类型ＲＴ－ＰＣＲ逆转录酶ＤＮＡ聚合酶mRNAcDNA杂化双链PCR扩增荧光定量PCR（real-timePCR)

通过荧光染料或荧光标记的特异性的探针,对PCR产物进行标记跟踪,实时在线监控反应过程,结合相应的软件可以对结果进行分析,计算待测样品的初始模板量。PCR技术的应用1)基因克隆重组DNA质粒DNA基因片段5’5’BamHIBamHIBamHIBamHIGTCCGGACCCTGCAGGGTCCGGACCCTGCAGGCCTGGGACGTCCCAGG质粒DNA目的基因限制性核酸内切酶2)基因检测A’内源性病变基因正常人A病人病原微生物基因正常人(-)病人(+)3)植物遗传育种构建遗传图谱、分离目的基因、基因定位分子标记辅助育种了解不同品种间的亲缘关系、系统进化Vocabularysemi-conservativereplicationreplicationbubblereplicationforkbidirectionalreplicationsemi-discontinuousreplicationOkazakifragmentprimaseleadingstrandlaggingstrandhelicasesingle-strandbindingproteinslidingclampclamploaderprocessivity半保留复制复制泡复制叉双向复制半不连续复制冈崎片段引发酶先导链后随链解旋酶SSB,单链结合蛋白滑行夹滑行夹加载器持续合成能力Semi-Conservative

Replication

半保留复制Semi-conservativereplication:AstyleofDNAreplicationinwhichproducesaDNAwithonestrandfromtheparent,andonenewlysynthesizedstrand.Semi-Conservative

Replication

/

半保留复制每个新的双链分子中都含有一条新的DNA链，但还保留了一条用做模板的母链。·细胞生长在15N标记培养基中·转入正常N源培养基中·分离各代DNA·分析各代DNA的浮力密度未标记DNACsCL密度梯度离心浮力密度

N15－DNA1.742g/mlN15－N14－DNA1.717g/mlN14－DNA1.710g/mlReplicon(复制子):aunitofthegenomeinwhichDNAisreplicated;containsanorigin(复制起点)forinitiationofreplicationandaterminus(终点)tostopthereplication.复制子Repliconsmaybeunidirectional(单向的)orbidirectional(双向的)Bidirectionalreplication(双向复制)ofacircularbacterialrepliconOriginTerminusDaughterDNAstructureAllprokaryoticchromosomesandmanybacteriophageandviralDNAmoleculesarecircularandcomprisesinglerepliconsEukaryotic

chromosomes:Multiplereplicons,eachwithitsownorigin.Mammaliancellhas50000-100000repliconswith50-200kbNote

原核生物细胞的基因组只有一个复制子。细菌内的质粒是一个自主复制的环状DNA基因组，构成一个独立的复制子。真核生物的DNA分子上有多个复制子。MultipleeukaryoticrepliconsReplicationforkRe-initiation(重新启动)ofbacterialreplicationatneworiginsTer(terminus)O(origin)RNApriming:DNAsynthesisisprimedbyRNAthatisthenremovedbeforefragmentsarejoined.DNApolymerase(DNA聚合酶)cannotinitiateDNAsynthesisdenovo(从头开始)Semi-discontinuousreplication/半不连续复制Okazakifragment/冈崎片段Okazakifragments:IndividualpiecesofnewlysynthesizedDNAcreatedduringdiscontinuoussynthesis.DNA复制的起始SSB4个起始因子DnaA蛋白结合位点解旋酶E2细菌的DNA复制的起始E2细菌的DNA复制大肠杆菌起始点遗传基因座OriC:oriCcontainsfour9bpbindingsitesfortheinitiatorproteinDnaA.SynthesisofDnaAiscoupledtogrowthratesothatinitiationofreplicationisalsocoupledtogrowthrate.DnaAformsacomplexof30-40molecules,facilitating促使

meltingofthree13bpAT-richrepeatsequenceforDnaBbinding.DnaBisahelicasethatusetheenergyofATPhydrolysistofurthermeltthedouble-strandedDNA.SSB(single-strandedbindingprotein)coatstheunwoundDNA(ssDNAbubble).DNAprimase引物酶loadtosynthesizeashortRNAprimerforsynthesisoftheleadingstrand.Primosome引发体:DnaBhelicaseandDNAprimaseHelicaseandSSBs/解旋酶与SSBHelicase:

Enzymethatseparatesthetwostrandsofthedoublehelixbybreakinghydrogenbondsbetweenthetwostrands.解旋酶：通过打断两条链之间的氢键而将双螺旋的两条链分开的酶。SSB:single-strandDNAbindingprotein单链DNA结合蛋白：在复制叉处与单链DNA结合的蛋白质，它们保护单链防止它们互相重新结合。DNA聚合酶III包含七种不同的亚单位和9个亚基。生物活性形式为二聚体。它既有5’→3’方向聚合酶活性，也有3’→5’核酸外切酶活性。DNApolymeraseIIIholoenzyme(全酶):adimercomplex,onehalfsynthesizingtheleadingstrandandtheotherlaggingstrand.EnsuressynthesisofbothstrandsatthesamerateBothpolymerasescontainana-subunit,polymerase;b-subunit,clamp夹住thepolymerasetoDNA,e-subunit---3’

5’proofreading(校正);exonuclease(核酸外切酶)

;DNA聚合酶III包含七种不同的亚单位和9个亚基。生物活性形式为二聚体。它既有5’→3’方向聚合酶活性，也有3’→5’核酸外切酶活性。RemovalofRNAprimer(引物),andgapfillingwithDNApolILigationofOkazakifragments(冈崎片段)arelinkedbyDNAligase(连接酶).DNApolIIIDNApolI(5’3’exonulcleaseactivity)DNApolI(5’3’polymeraseactivity)DNAligase(NAD+)滞后链的复制TopoisomeraseI

/拓扑异构酶ITopoisomeraseI:

Topoisomerasethatreleasesupercoilingbyintroducingsingle-strandedcutintheDNA.拓扑异构酶I：通过在DNA中产生单链切口而释放超螺旋的拓扑异构酶。TopoisomeraseII

/拓扑异构酶IITopoisomeraseII:

Topoisomerasethatreleasesupercoilingbyintroducingdouble-strandedcutintheDNA.拓扑异构酶II：通过在DNA中产生双链切口而释放超螺旋的拓扑异构酶。HowisE.coliDNAreplicationterminated?

大肠杆菌DNA复制是如何终止的？Themagicrings魔术套环Themagicringshavehidden(隐蔽的)openings.Butthebacteriaarefacedwiththerealcatenaneproblem.Howcanbacteriasolvethisproblem?TerminationofDNAreplicationinE.coli

大肠杆菌DNA复制终止为了解开两条子代染色体这样的连环分子，II型拓扑异构酶在其中的一条染色体上产生一个双链缺口，让另一条染色体从缺口中穿过而将它释放。在染色体被分开后，缺口又被重新封闭。Proofreading/校正3’

5’exonucleaseactivity3’

5’外切核酸酶活性5’→3’Synthesiscontinuesafterproofreading在校正后5’→3’合成继续3’→5’Synthesisstopsafterproofreading在校正后3’→5’合成终止Whycan’tDNAsynthesisrunin3’→5’direction?

为什么DNA复制不能以3’→5’方向进行？Primers/引物PrimerforOkazakifragmentPrimerRemoval/引物去除DNApolymeraseI:

AprokaryoticDNApolymerasewithaspecial5’

3’exonucleaseactivity,usedtoremoveprimers.DNAreplication(1/7)DNAisreplicatedbycontinuoussynthesisofaleadingstrandanddiscontinuoussynthesisofalaggingstrand.DNAreplication(2/7)CoordinationbetweenleadingandlaggingstrandsynthesisisachievedbythedimerizationofDNApolymerasemoleculesatthereplicationfork.DNAreplication(3/7)TheDNApolymerasedimermoveswiththereplicationfork.ThepolymeraseattheleadingstrandtemplateremainsattachedtotheDNA,continuouslysynthesizingtheleadingstrand.DNAreplication(4/7)ThelaggingstrandpolymeraseinitiatesDNAsynthesisatthefork,fromanRNAprimermadebyaprimasecomplex.DNAreplication(5/7)Thepolymeraseelongatesthelaggingstrandinadirectionoppositethefork,butstaysboundatthefork.Asaresult,newlysynthesizedlaggingstrandfragmentloopsoutbetweenthepolymeraseandthefork.DNAreplication(6/7)OncethepolymerasecompletesanOkazakifragment,itdissociatesfromtheDNAtemplate.Anewprimerisproducedatthefork.Thepolymerasereassociateswiththetemplateatthisposition,tocontinuesynthesisofthelaggingstrand.DNAreplication(7/7)Bythismechanism,thetwopolymerasescanaddnucleotidestothegrowingstrandsatthesametime,andatratesupto1000bp/s.Unwinding（退绕）andDNAgyrase(旋转酶):unwindingistoremovehelicalturnresultinginadditionalturnsintherestofthemoleculeintheformofpositivesupercoilingwhichisresolvedbyDNAgyrase(TypeIItopoisomerase).RelaxationbygyraseDuringreplication,theincreasedpositivesupercoilingshouldberelaxedUnwoundregionGyraseNicktranslation:theabilityofE.coliDNApolymeraseItouseanickasastartingpointfromwhichonestrandofaduplexDNAcanbedegradedandreplacedbyresynthesisofnewmaterial;isusedtointroduceradioactivelylabelednucleotidesintoDNAinvitro.Nicktranslation缺口平移（获得探针）DNAPolI:singlepolypeptideof103kD.Trypsincleavge>68kDlargefragment(Klenow)

with5’-3’poland3’-5’exonucleaseactivity;smallfagment(35kD)with5’-3’nucleaseactivity.E3细胞周期G1checkpoint:fissionyeastcells:Startpoint；Animalcells:RestrictionPointcdc基因（celldivisioncyclegene，细胞分裂周期基因）是一类产物表达具细胞周期依赖性或直接参与细胞周期调控的基因，主要包括cyclin基因、Cdk基因、CKI基因等。此外，与DNA复制相关的DNA聚合酶、DNA连接酶基因也属于cdc基因。Cyclin-dependentkinase(CDK):

Cdc2cangetkinaseactivitywhenitcombinestocyclin（细胞周期蛋白）,so,cdc2iscalledascyclin-dependentkinase(CDK).ActivatedCDKcanphosphorylateproteinstotakefunctions.

Forexample,itcanphosphorylatelamina（核纤层）

proteintodisassemblenuclearskeletonandenvelope.Bythisdisassembly,thecellcyclecanbekeptforcontinueddivision.MPF=cdc2+CyclinB(maturation-promotingfactor)，细胞分裂促进因子79ThecombinationofCyclinDandCDKpromotesRbtoreleaseoutthecombinedtranscriptionfactor,E2FCDK

inhibitor(CDKI):

ThereareCDK

inhibitors(CDKI)incellsthatcanregulatecellcyclenegatively(inhibiting).TwofamiliesofCDKIhavebeenfoundsofar:①Ink4(Inhibitorofcdk4）②Kip(Kinaseinhibitionprotein）TheactivationofCDK1needsitsThr14andTyr15dephosphorylatedandTyr161phosphorylatedCDKandPCNAareinhibitedbyP21cip1E4DNAReplicationinEukaryotes

真核生物DNA复制ProkaryoticDNAEukaryoticDNA~1000nt/sec~1000nt/minSmallanimalviruses(simian猿virusSV40,5kb)aregoodmammalianmodelsforelongation(replicationfork).Mammalianchromosomehasabout50000-100000replicons2. Yeast(Saccharomycescerevisiae):1.4x107bpin16chromosomes,400repliconsExperimentalsystems实验系统

3. Cell-freeextractpreparedfromXenopus(frog)eggscontaininghighconcentrationofreplicationproteinsandcansupportinvitroreplication.Multipleinitiationsites

多重起始位点eukaryoticDNApolymerasesEukaryoticDNApolymerasesPolα(Synthesisoflaggingstrand)Polδ(Synthesisofleadingstrand)Polε(Functionunknown)Polβ(DNArepair)Polγ(ReplicationofmitochondrialDNA)ReplicationinitiationinEukaryotes

真核生物复制起始S.cerevisiae（面包酵母）ARS:autonomouslyreplicatingsequence自主复制序列

OriCInitiation起始点与起始InitiationofeachrepliconwithinaninitiationzonemayoccuratrandomYeastorigin:theminimal11bpconcensus:

[A/T]TTTAT[A/G]TTT[A/T]Boundbyoriginrecognitioncomplex(ORC)activatedbyCDK(e.g.shortlivedcdc6).ARS:autonomouslyreplicatingsequence自动复制序列,containingyeastOriKnowledgeabouteukaryoticreplicationoriginsislimited/

有关真核生物复制起点的知识并不多Inhighereukaryotes,ithas