1、n34 34 DNADNA的復(fù)制和修復(fù)的復(fù)制和修復(fù)DNA replication DNA replication and repair and repairnDNA replication DNA replicates in a semi- conservative fashion The origin and fashion of DNA replication Enzymes of DNA polymeric reaction Semidiscontinuous replication of DNA DNA Replication process DNA Replication in Eu

2、karyotesnDNA Damage and repairnDNA mutationoverviewoverviewCENTRAL DOGMADNAmRNAProteintranscriptionrevers transcriptiontranslation Prokaryotes and EukaryotesnPlasmid in Prokaryotes, Organelles in Eukaryotesn Virus 34.1 34.1 DNA replicationDNA replication 34.1.1 34.1.1 DNA replicates in a semi- DNA r

3、eplicates in a semi- conservative fashionconservative fashionA C C C T G G S S P S S P S S P P S S S P S P S S P S S S S S S S S T T T A A P P P S P S P P P P P S S P P S S 3 Hydrogen bonds Covalent bonds Beginning of unwinding New double strands; replication is complete. 5 3 5 3 5 3 5 S P P P P P P

4、 S S S S S SPPP SPPP SPPP SPPP PPPS PPPS PPPS PPPS S C C C C C A G G G G S P S S P P P S S S P S S P P P S S T T A A C C G G T T T A T A A C G C G C G C G G T A C G G T A C G T A P P S S P S P P S S P S P S S P S S P S S S S S P P P P P P S P S P S S P S S S P S S P S S S S S P P P S P S S S S P P P

5、 S P T T A A C C G G G Copyright 1997, by John Wiley & Sons, Inc. All rights reserved. n實驗實驗1：1958年 Meselson 和 Stahl利用氮的同位素15N標(biāo)記大腸桿菌DNA，首先證明了DNA的半保留復(fù)制。nE.coliE.coli，1515NHNH4 4ClCl為唯一氮源為唯一氮源，培養(yǎng)，培養(yǎng)1212代，代，從而使所有從而使所有E.coliE.coli DNA DNA標(biāo)記上標(biāo)記上1515NN。n1515N-DNA N-DNA 比比 1414N-DNAN-DNA的密度大。的密度大。n氯化銫密

6、度梯度離心氯化銫密度梯度離心DNA replicates in a semi- conservative fashionDNA replicates in a semi- conservative fashion 親代親代 F1 15NDNA replicates in a semi- conservative fashionDNA replicates in a semi- conservative fashion F2 DNA replicates in a semi- conservative fashionDNA replicates in a semi- conservative f

7、ashionDNA replicates in a semi- conservative fashionDNA replicates in a semi- conservative fashionn實驗實驗2：1963年Cairns用放射自顯影（autoradiograph）的方法第一次觀察到完整的正在復(fù)制的大腸桿菌染色體DNA.n3 3H-dTTPH-dTTP標(biāo)記大腸桿菌標(biāo)記大腸桿菌DNADNA，經(jīng)過將近經(jīng)過將近兩代時間兩代時間n溶菌酶消化細(xì)胞壁溶菌酶消化細(xì)胞壁n放射自顯影放射自顯影DNA replicates in a semi- conservative fashionDNA repli

8、cates in a semi- conservative fashion ABcDNA replicates in a semi- conservative fashionDNA replicates in a semi- conservative fashionDaughter strand (H3+H3)Parents strand (H3+H)圖像解讀圖像解讀DNA replicates in a semi- conservative fashionDNA replicates in a semi- conservative fashionn34.1.1 34.1.1 The orig

9、in and fashion of The origin and fashion of DNA replicationDNA replicationnrepliconnorigin,terminusnreplication forkThe origin and fashion of DNA replicationThe origin and fashion of DNA replicationnEukaryote multirepliconmultireplicon相對基因頻率（放射活性）ilvtactrphistyrmalAthrilvDNA復(fù)制起點的測定（分子雜交法）thr0/100lac

10、att25trptre終點his50tyrcycCmalA75起點oriCilv復(fù)制起點和終點的位置The origin and fashion of DNA replicationThe origin and fashion of DNA replicationDNA replication in different fashions DNA replication in different fashions Rolling circle(X174)n34.1.3 34.1.3 Enzymes of DNA polymeric Enzymes of DNA polymeric reactio

11、nreactionnPolymerase and DNA ligase34.1.3.1 34.1.3.1 DNA polymeric reaction and PolymeraseDNA polymeric reaction and Polymerase DNA polymeric reactionDNA polymeric reactionnn1dATP dAMPn + nn2dGTP dGMPn + + DNA 聚合酶聚合酶 -DNA+(n1+n2+n3+n4)PPinn3dCTP dCMPn +nn4dTTP dTMP鏈的延長鏈的延長OHOHHHCH2H堿基O引物OHOHHHHCH2H堿

12、基O堿基堿基模板鏈POHOPPiPPiDNA聚合酶催化的鏈延長反應(yīng)OHOHHHHCH2H堿基O引物堿基堿基模板鏈OHOHHHHCH2H堿基OPOHOPOO-OPOOO-5353核苷酸加入到引物鏈的3末端鏈的合成方向模板鏈引物鏈DNA酶促合成的引物鏈和模板鏈鏈的延長鏈的延長34.1.3.2 DNA Polymerases in E. ColinDNA polymerase、53核酸外切酶35核酸外切酶聚合酶小片段大片段蛋白酶NCDNA聚合酶的酶切片段nN-核酸外切酶核酸外切酶(5 3 ) 核酸外切酶核酸外切酶(3 5 ) 聚合酶聚合酶-CnDNADNA聚合酶聚合酶nComparing of DN

13、A Polymerases in Comparing of DNA Polymerases in E.ColiE.Colin n DNA聚合酶聚合酶 DNA聚合酶聚合酶 DNA聚合酶聚合酶 n結(jié)構(gòu)基因結(jié)構(gòu)基因 pol A pol B pol Cn不同種類亞基數(shù)不同種類亞基數(shù) 1 7 10n相對分子質(zhì)量相對分子質(zhì)量 103 000 88 000 830 000n35核酸外切酶活力核酸外切酶活力 + + +n53核酸外切酶活力核酸外切酶活力 + - -n功能功能 切除引物，修復(fù)切除引物，修復(fù) 修復(fù)修復(fù) 復(fù)制復(fù)制n34.1.1 34.1.1 DNADNA ligaseligasen催化雙鏈催化雙鏈D

14、NA切口處的切口處的5-磷酸基和磷酸基和3-羥基生成磷酸二酯鍵。羥基生成磷酸二酯鍵。34.1.1 34.1.1 Semidiscontinuous replication of DNASemidiscontinuous replication of DNAn n岡崎片段（岡崎片段（Okazaki fragment）。34.1.1 34.1.1 DNADNA的半不連續(xù)復(fù)制的半不連續(xù)復(fù)制n前導(dǎo)鏈前導(dǎo)鏈（leading strand）n滯后鏈滯后鏈（lagging strand）lgyrase 旋轉(zhuǎn)酶（旋轉(zhuǎn)酶（topoisomerase ）lHelicase 解螺旋酶解螺旋酶n34.1.6 34.1

15、.6 DNADNA replication progressreplication progressn 大腸桿菌起點與復(fù)制有關(guān)的酶與輔助因子大腸桿菌起點與復(fù)制有關(guān)的酶與輔助因子n34.1.634.1.6 DNADNA Replication in EukaryoteReplication in EukaryoteDNA Polymerases in Mammal DNA Polymerases in Mammal DNADNA Replication in EukaryoteReplication in Eukaryoten端粒端粒 (telomere)n端粒酶端粒酶 (telomerase)

16、n真核生物的細(xì)胞周期真核生物的細(xì)胞周期 (cell cycle)端粒和端粒酶端粒和端粒酶n1970年代年代 Elizabeth Blackburn 和她的合作和她的合作者發(fā)現(xiàn)了在染色體兩段的端粒結(jié)構(gòu)者發(fā)現(xiàn)了在染色體兩段的端粒結(jié)構(gòu)重重復(fù)的復(fù)的DNA序列，序列，Jack Szostak 和她合作，和她合作，先是合成擁有端粒序列的染色體，然后注先是合成擁有端粒序列的染色體，然后注入酵母細(xì)胞，證明端粒的存在可以在酵母入酵母細(xì)胞，證明端粒的存在可以在酵母細(xì)胞分裂的過程中，保護(hù)染色體不會丟失，細(xì)胞分裂的過程中，保護(hù)染色體不會丟失，因此而發(fā)現(xiàn)了端粒的作用。這是在因此而發(fā)現(xiàn)了端粒的作用。這是在1982年，年，

17、2年后她當(dāng)時的博士生年后她當(dāng)時的博士生Carol Greider發(fā)現(xiàn)了發(fā)現(xiàn)了細(xì)胞內(nèi)合成端粒的端粒酶細(xì)胞內(nèi)合成端粒的端粒酶(telomerase)。今。今年年(2009年年)的諾貝爾生理醫(yī)學(xué)獎就是頒給的諾貝爾生理醫(yī)學(xué)獎就是頒給這這3人。人。端粒和端粒酶端粒和端粒酶端粒和端粒酶端粒和端粒酶talomerase MCB1204.MOVn34.1 34.1 DNADNA Damage and Repair Damage and Repair nDNA在復(fù)制時可能產(chǎn)生錯配在復(fù)制時可能產(chǎn)生錯配nDNA重組、病毒基因的整合，破壞重組、病毒基因的整合，破壞DNA的結(jié)的結(jié)構(gòu)構(gòu)n某些理化因子如紫外線、電離輻射、和

18、化學(xué)某些理化因子如紫外線、電離輻射、和化學(xué)誘變劑都能作用于誘變劑都能作用于DNA引起引起DNA結(jié)構(gòu)與功能結(jié)構(gòu)與功能的破壞的破壞n目前已知的修復(fù)系統(tǒng)目前已知的修復(fù)系統(tǒng)n錯配修復(fù)（錯配修復(fù)（mismatch repair）n直接修復(fù)直接修復(fù) （direct repair）n切除修復(fù)切除修復(fù) （excision repair）n重組修復(fù)重組修復(fù) （recombination repair）n易錯修復(fù)（易錯修復(fù)（error-prone repair） DNADNA Damage and RepairDamage and Repair DNADNA Damage and RepairDamage and Repair DNADNA Damage and RepairDamage and Repair DNADNA Damage and RepairDamage and Repair n34.1 34.1 DNA MutationDNA Mutationn34.3.1 34.3.1 突變