EDNAReplication/DNA復(fù)制Geneticinformationisalsopassedfromgenerationtogeneration.Acrucialpropertyoflifeisthateventhemostcomplexorganismsareabletobecopied.3’-5’phosphodiesterbond3’-5’磷酸二酯鍵PPi(pyrophosphate)

Substrates(底物)forDNAsynthesis:deoxynucleoside5’-triphosphates三磷酸脫氧核苷dNTPs(dATP,dGTP,dCTP&TTP)DNAsynthesisoccursbyaddingnucleotidesto3’-OHDNA/RNA:5’to3’Protein:NtoCPCR的原理PCR的反應(yīng)條件PCR的特點(diǎn)PCR的類型PCR的應(yīng)用PCR技術(shù)及其應(yīng)用PCR的原理引物引物DNA聚合酶DNA聚合酶特定DNA片段PCR反應(yīng)循環(huán)72℃94℃55℃PCR循環(huán)變性退火延伸

PCR的指數(shù)擴(kuò)增（2n）引物延伸延伸5’5’3’3’變性、退火變性、退火1）PCR反應(yīng)成分（1）模板單、雙鏈DNA均可。不能混有蛋白酶、核酸酶、DNA聚合酶抑制劑、DNA結(jié)合蛋白類。

DNA模板一般100ng/100L。模板濃度過(guò)高會(huì)導(dǎo)致反應(yīng)的非特異性增加。PCR反應(yīng)條件（2）引物濃度

0.1-0.5mol/L

濃度過(guò)高易導(dǎo)致模板與引物錯(cuò)配,反應(yīng)特異性下降。（3）TaqDNA聚合酶

0.5-2.5U/50l

酶量增加使反應(yīng)特異性下降;酶量過(guò)少影響反應(yīng)產(chǎn)量。（4）dNTP

dNTP濃度取決于擴(kuò)增片段的長(zhǎng)度四種dNTP濃度應(yīng)相等濃度過(guò)高易產(chǎn)生錯(cuò)誤堿基的摻入,濃度過(guò)低則降低反應(yīng)產(chǎn)量

dNTP可與Mg2+結(jié)合,使游離的Mg2+濃度下降,影響DNA聚合酶的活性。（5）Mg2+

Mg2+是DNA聚合酶的激活劑。適宜濃度為0.5-2.5mmol/L反應(yīng)體系。

Mg2+濃度過(guò)低會(huì)使Taq酶活性喪失、PCR產(chǎn)量下降；Mg2+過(guò)高影響反應(yīng)特異性。

Mg2+可與負(fù)離子結(jié)合,所以反應(yīng)體系中dNTP、EDTA等的濃度影響反應(yīng)中游離的Mg2+濃度。2）循環(huán)參數(shù)變性

使雙鏈DNA解鏈為單鏈

94℃，20-30秒(2)退火

溫度由引物長(zhǎng)度和GC含量決定。增加溫度能減少引物與模板的非特異性結(jié)合;降低溫度可增加反應(yīng)的靈敏性。（3）延伸

70-75℃,一般為72℃

延伸時(shí)間由擴(kuò)增片段長(zhǎng)度決定（4）循環(huán)次數(shù)

主要取決于模版DNA的濃度一般為25-35次次數(shù)過(guò)多：擴(kuò)增效率降低錯(cuò)誤摻入率增加PCR技術(shù)的特點(diǎn)1）高度的靈敏性30輪循環(huán)擴(kuò)增量達(dá)230個(gè)拷貝（109拷貝）PCR產(chǎn)物每輪循環(huán)增加一倍2）特異性引物引物

引物的序列及其與模板結(jié)合的特異性是決定PCR反應(yīng)結(jié)果的關(guān)鍵。引物設(shè)計(jì)的最大原則是最大限度地提高擴(kuò)增效率和特異性；同時(shí)盡可能減少非特異性擴(kuò)增。引物設(shè)計(jì)：（1)序列應(yīng)位于高度保守區(qū),與非擴(kuò)增區(qū)無(wú)同源序列。（2）引物長(zhǎng)度以15-40bp為宜。（3）堿基盡可能隨機(jī)分布,G+C占40-60%。（4）引物內(nèi)部避免形成二級(jí)結(jié)構(gòu)。（5）兩引物間避免有互補(bǔ)序列。（6）引物3’端為關(guān)鍵堿基；5’端無(wú)嚴(yán)格限制。3’5’3’5’限制性內(nèi)切酶的識(shí)別序列啟動(dòng)子序列定點(diǎn)突變探針標(biāo)記3）操作簡(jiǎn)便易行

利用PCR擴(kuò)增,只需要數(shù)小時(shí),就可以擴(kuò)增出可用電泳法檢出的DNA，可用于檢測(cè)基因組中僅含數(shù)個(gè)拷貝的模板序列。不對(duì)稱PCR反向PCR多重PCR標(biāo)記引物PCR錨定PCRPCR固相分析法原位PCRRT-PCR定量PCR-----PCR的類型ＲＴ－ＰＣＲ逆轉(zhuǎn)錄酶ＤＮＡ聚合酶mRNAcDNA雜化雙鏈PCR擴(kuò)增熒光定量PCR（real-timePCR)

通過(guò)熒光染料或熒光標(biāo)記的特異性的探針,對(duì)PCR產(chǎn)物進(jìn)行標(biāo)記跟蹤,實(shí)時(shí)在線監(jiān)控反應(yīng)過(guò)程,結(jié)合相應(yīng)的軟件可以對(duì)結(jié)果進(jìn)行分析,計(jì)算待測(cè)樣品的初始模板量。PCR技術(shù)的應(yīng)用1)基因克隆重組DNA質(zhì)粒DNA基因片段5’5’BamHIBamHIBamHIBamHIGTCCGGACCCTGCAGGGTCCGGACCCTGCAGGCCTGGGACGTCCCAGG質(zhì)粒DNA目的基因限制性核酸內(nèi)切酶2)基因檢測(cè)A’內(nèi)源性病變基因正常人A病人病原微生物基因正常人(-)病人(+)3)植物遺傳育種構(gòu)建遺傳圖譜、分離目的基因、基因定位分子標(biāo)記輔助育種了解不同品種間的親緣關(guān)系、系統(tǒng)進(jìn)化Vocabularysemi-conservativereplicationreplicationbubblereplicationforkbidirectionalreplicationsemi-discontinuousreplicationOkazakifragmentprimaseleadingstrandlaggingstrandhelicasesingle-strandbindingproteinslidingclampclamploaderprocessivity半保留復(fù)制復(fù)制泡復(fù)制叉雙向復(fù)制半不連續(xù)復(fù)制岡崎片段引發(fā)酶先導(dǎo)鏈后隨鏈解旋酶SSB,單鏈結(jié)合蛋白滑行夾滑行夾加載器持續(xù)合成能力Semi-Conservative

Replication

半保留復(fù)制Semi-conservativereplication:AstyleofDNAreplicationinwhichproducesaDNAwithonestrandfromtheparent,andonenewlysynthesizedstrand.Semi-Conservative

Replication

/

半保留復(fù)制每個(gè)新的雙鏈分子中都含有一條新的DNA鏈，但還保留了一條用做模板的母鏈?！ぜ?xì)胞生長(zhǎng)在15N標(biāo)記培養(yǎng)基中·轉(zhuǎn)入正常N源培養(yǎng)基中·分離各代DNA·分析各代DNA的浮力密度未標(biāo)記DNACsCL密度梯度離心浮力密度

N15－DNA1.742g/mlN15－N14－DNA1.717g/mlN14－DNA1.710g/mlReplicon(復(fù)制子):aunitofthegenomeinwhichDNAisreplicated;containsanorigin(復(fù)制起點(diǎn))forinitiationofreplicationandaterminus(終點(diǎn))tostopthereplication.復(fù)制子Repliconsmaybeunidirectional(單向的)orbidirectional(雙向的)Bidirectionalreplication(雙向復(fù)制)ofacircularbacterialrepliconOriginTerminusDaughterDNAstructureAllprokaryoticchromosomesandmanybacteriophageandviralDNAmoleculesarecircularandcomprisesinglerepliconsEukaryotic

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

原核生物細(xì)胞的基因組只有一個(gè)復(fù)制子。細(xì)菌內(nèi)的質(zhì)粒是一個(gè)自主復(fù)制的環(huán)狀DNA基因組，構(gòu)成一個(gè)獨(dú)立的復(fù)制子。真核生物的DNA分子上有多個(gè)復(fù)制子。MultipleeukaryoticrepliconsReplicationforkRe-initiation(重新啟動(dòng))ofbacterialreplicationatneworiginsTer(terminus)O(origin)RNApriming:DNAsynthesisisprimedbyRNAthatisthenremovedbeforefragmentsarejoined.DNApolymerase(DNA聚合酶)cannotinitiateDNAsynthesisdenovo(從頭開始)Semi-discontinuousreplication/半不連續(xù)復(fù)制Okazakifragment/岡崎片段Okazakifragments:IndividualpiecesofnewlysynthesizedDNAcreatedduringdiscontinuoussynthesis.DNA復(fù)制的起始SSB4個(gè)起始因子DnaA蛋白結(jié)合位點(diǎn)解旋酶E2細(xì)菌的DNA復(fù)制的起始E2細(xì)菌的DNA復(fù)制大腸桿菌起始點(diǎn)遺傳基因座OriC:oriCcontainsfour9bpbindingsitesfortheinitiatorproteinDnaA.SynthesisofDnaAiscoupledtogrowthratesothatinitiationofreplicationisalsocoupledtogrowthrate.DnaAformsacomplexof30-40molecules,facilitating促使

meltingofthree13bpAT-richrepeatsequenceforDnaBbinding.DnaBisahelicasethatusetheenergyofATPhydrolysistofurthermeltthedouble-strandedDNA.SSB(single-strandedbindingprotein)coatstheunwoundDNA(ssDNAbubble).DNAprimase引物酶loadtosynthesizeashortRNAprimerforsynthesisoftheleadingstrand.Primosome引發(fā)體:DnaBhelicaseandDNAprimaseHelicaseandSSBs/解旋酶與SSBHelicase:

Enzymethatseparatesthetwostrandsofthedoublehelixbybreakinghydrogenbondsbetweenthetwostrands.解旋酶：通過(guò)打斷兩條鏈之間的氫鍵而將雙螺旋的兩條鏈分開的酶。SSB:single-strandDNAbindingprotein單鏈DNA結(jié)合蛋白：在復(fù)制叉處與單鏈DNA結(jié)合的蛋白質(zhì)，它們保護(hù)單鏈防止它們互相重新結(jié)合。DNA聚合酶III包含七種不同的亞單位和9個(gè)亞基。生物活性形式為二聚體。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夾住thepolymerasetoDNA,e-subunit---3’

5’proofreading(校正);exonuclease(核酸外切酶)

;DNA聚合酶III包含七種不同的亞單位和9個(gè)亞基。生物活性形式為二聚體。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(岡崎片段)arelinkedbyDNAligase(連接酶).DNApolIIIDNApolI(5’3’exonulcleaseactivity)DNApolI(5’3’polymeraseactivity)DNAligase(NAD+)滯后鏈的復(fù)制TopoisomeraseI

/拓?fù)洚悩?gòu)酶ITopoisomeraseI:

Topoisomerasethatreleasesupercoilingbyintroducingsingle-strandedcutintheDNA.拓?fù)洚悩?gòu)酶I：通過(guò)在DNA中產(chǎn)生單鏈切口而釋放超螺旋的拓?fù)洚悩?gòu)酶。TopoisomeraseII

/拓?fù)洚悩?gòu)酶IITopoisomeraseII:

Topoisomerasethatreleasesupercoilingbyintroducingdouble-strandedcutintheDNA.拓?fù)洚悩?gòu)酶II：通過(guò)在DNA中產(chǎn)生雙鏈切口而釋放超螺旋的拓?fù)洚悩?gòu)酶。HowisE.coliDNAreplicationterminated?

大腸桿菌DNA復(fù)制是如何終止的？Themagicrings魔術(shù)套環(huán)Themagicringshavehidden(隱蔽的)openings.Butthebacteriaarefacedwiththerealcatenaneproblem.Howcanbacteriasolvethisproblem?TerminationofDNAreplicationinE.coli

大腸桿菌DNA復(fù)制終止為了解開兩條子代染色體這樣的連環(huán)分子，II型拓?fù)洚悩?gòu)酶在其中的一條染色體上產(chǎn)生一個(gè)雙鏈缺口，讓另一條染色體從缺口中穿過(guò)而將它釋放。在染色體被分開后，缺口又被重新封閉。Proofreading/校正3’

5’exonucleaseactivity3’

5’外切核酸酶活性5’→3’Synthesiscontinuesafterproofreading在校正后5’→3’合成繼續(xù)3’→5’Synthesisstopsafterproofreading在校正后3’→5’合成終止Whycan’tDNAsynthesisrunin3’→5’direction?

為什么DNA復(fù)制不能以3’→5’方向進(jìn)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（退繞）andDNAgyrase(旋轉(zhuǎn)酶):unwindingistoremovehelicalturnresultinginadditionalturnsintherestofthemoleculeintheformofpositivesupercoilingwhichisresolvedbyDNAgyrase(TypeIItopoisomerase).RelaxationbygyraseDuringreplication,theincreasedpositivesupercoilingshouldberelaxedUnwoundregionGyraseNicktranslation:theabilityofE.coliDNApolymeraseItouseanickasastartingpointfromwhichonestrandofaduplexDNAcanbedegradedandreplacedbyresynthesisofnewmaterial;isusedtointroduceradioactivelylabelednucleotidesintoDNAinvitro.Nicktranslation缺口平移（獲得探針）DNAPolI:singlepolypeptideof103kD.Trypsincleavge>68kDlargefragment(Klenow)

with5’-3’poland3’-5’exonucleaseactivity;smallfagment(35kD)with5’-3’nucleaseactivity.E3細(xì)胞周期G1checkpoint:fissionyeastcells:Startpoint；Animalcells:RestrictionPointcdc基因（celldivisioncyclegene，細(xì)胞分裂周期基因）是一類產(chǎn)物表達(dá)具細(xì)胞周期依賴性或直接參與細(xì)胞周期調(diào)控的基因，主要包括cyclin基因、Cdk基因、CKI基因等。此外，與DNA復(fù)制相關(guān)的DNA聚合酶、DNA連接酶基因也屬于cdc基因。Cyclin-dependentkinase(CDK):

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

Forexample,itcanphosphorylatelamina（核纖層）

proteintodisassemblenuclearskeletonandenvelope.Bythisdisassembly,thecellcyclecanbekeptforcontinueddivision.MPF=cdc2+CyclinB(maturation-promotingfactor)，細(xì)胞分裂促進(jìn)因子79ThecombinationofCyclinDandCDKpromotesRbtoreleaseoutthecombinedtranscriptionfactor,E2FCDK

inhibitor(CDKI):

ThereareCDK

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

真核生物DNA復(fù)制ProkaryoticDNAEukaryoticDNA~1000nt/sec~1000nt/minSmallanimalviruses(simian猿virusSV40,5kb)aregoodmammalianmodelsforelongation(replicationfork).Mammalianchromosomehasabout50000-100000replicons2. Yeast(Saccharomycescerevisiae):1.4x107bpin16chromosomes,400repliconsExperimentalsystems實(shí)驗(yàn)系統(tǒng)

3. Cell-freeextractpreparedfromXenopus(frog)eggscontaininghighconcentrationofreplicationproteinsandcansupportinvitroreplication.Multipleinitiationsites

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

真核生物復(fù)制起始S.cerevisiae（面包酵母）ARS:autonomouslyreplicatingsequence自主復(fù)制序列

OriCInitiation起始點(diǎn)與起始InitiationofeachrepliconwithinaninitiationzonemayoccuratrandomYeastorigin:theminimal11bpconcensus:

[A/T]TTTAT[A/G]TTT[A/T]Boundbyoriginrecognitioncomplex(ORC)activatedbyCDK(e.g.shortlivedcdc6).ARS:autonomouslyreplicatingsequence自動(dòng)復(fù)制序列,containingyeastOriKnowledgeabouteukaryoticreplicationoriginsislimited/

有關(guān)真核生物復(fù)制起點(diǎn)的知識(shí)并不多Inhighereukaryotes,ithas