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1、Section E DNA Replication Molecular Biology Course E4 Eukaryotic DNA replicationE4 Eukaryotic DNA replication Molecular Biology Course E1 DNA Replication An OverviewE1 DNA Replication An Overview Replicons semi conservative semi discontinous RNA priming E2 Bacterial DNA replicationE2 Bacterial DNA r
2、eplication Experimental system initiation unwinding elongation termination segregation E3 The cell cycle Experimental system cell cycle initiation replication forks nuclear matrix telomere repl E3 The cell cycle Cell cycle Overlapped with cell biology DNA replication E1 DNA Replication An Overview E
3、1 DNA Replication An Overview 1 Replicons 2 semi conservative mechanism 3 semi discontinous replication 4 RNA priming 1 复制 replication 1 复制 replication 是指以亲代DNA为模板合成子代DNA的过程 即遗传物质的传代 是指以亲代DNA为模板合成子代DNA的过程 即遗传物质的传代 replication Parental DNA Progeny DNA 2 DNA复制的方式 特征 semi conservative mechanism半保留复制 se
4、mi discontinous replication半不连续复制 A G G T A C T G C C A C T G G T C C A T G A C G G T G A C C C C A C T G G G G T G A C C A G G T A C T G T C C A T G A C T C C A T G A C A G G T A C T G A G G T A C T G C C A C T G G T C C A T G A C G G T G A C C A G G T A C T G C C A C T G G T C C A T G A C G G T G
5、A C C Parental Parental DNA Replication forkReplication fork复 制叉 复 制叉 Progeny Progeny DNA semi conservative semi conservative 半保留复制半保留复制 Replicon is any piece of DNA which replicates as a single unit It contains an origin and sometimes a terminus Origin is the DNA sequence where a replicon initiates
6、 its replication usually AT rich Why Terminus is the DNA sequence where a replicon usually stops its replication DNA replication E1 1 Replicons 复制子复制子 Prokaryotic genome a single circular DNA a single replicon Eukaryotic genome multiple linear chromosomes multiple replicons on each chromosome DNA re
7、plication Bidirectional replication of a circular bacterial replicon All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circlular and comprise single replicons There is a single termination site roughly 180o opposite the unique origin DNA replication Linear viral DNA mole
8、cules usually have a single origin replication details see Section R In all the cases the origin is a complex region where the initiation of DNA replication and the control of the growth cycle of the organism are regulated and co ordinated DNA replication The long linear DNA molecules of eukaryotic
9、chromosomes consist of multiple regions each with its own origin A typical mammalian cell has 50000 100000 replicons with a size range of 40 200 kb When replication forks 复制叉复制叉 from adjacent replication bubbles meet they fuse to form the completely replicated DNA No distinct termini are required Mu
10、ltiple eukaryotic replicons and replication bubbles 复制泡复制泡 DNA replication Replication bubbles DNA replication E1 2 Replication is Semi conservative Semi conservative mechanism 15N labeling experiment 1 15N labeling grow cells in N15 2 Cells transferred to medium containing only N14 then collect DNA
11、 1 Separation method DGC 2 Result interpretation 15N labeled DNA unlabeled DNA DNA replication Starting DNA Heavy Heavy 1st generation All Heavy Light 2nd generation Two Heavy Light Two Light Light 3rd generation Two Heavy Light Six Light Light The Meselson Stahl Experiment DNA replication E1 3 Repl
12、ication is Semi discontinuous 解链方向解链方向 半不连续性复制半不连续性复制 复制叉 复制叉 3 5 3 5 3 5 3 5 3 5 领头链 leading strand 随从链 lagging strand 领头链 leading strand 随从链 lagging strand 冈崎片段冈崎片段 Okazaki fragment LigationDNA ligase Semi discontinuous replication Discovery of Okazaki fragments Evidence for semi discontinuous rep
13、lication 3H thymidine pulse chase labeling experiment 1 Grow E coli 2 Add 3H thymidine in the medium for a few second spin down and break the cell to stop labeling analyze found a large fraction of nascent DNA 1000 2000 nt Okazaki fragments 3 Grow the cell in regular medium then analyze the small fr
14、agments join into high molecular weight DNA Ligation of the Okazaki fragments DNA replication The physical direction of both leading and lagging strand synthesis is the same since the lagging strand is looped by 180 at the replication fork DNA replication E1 4 RNA priming RNA 引导引导 The first few nucl
15、eotides at the 5 end of Okazaki fragments are ribonucleotides Hence DNA synthesis is primed by RNA that is then removed before fragments are joined Crucial for high fidelity of replication 复制的高保真性复制的高保真性 1 template 模板 解开成单链的DNA母链 2 substrate 底物 或称原料 dATP dGTP dCTP dTTP 3 primer 引物 复制需要一小段RNA作引物 4 En
16、ergy 需ATP供能 原料dNTP本身也是高能化合物 5 Enzymes and protein factors DNA聚合酶 polymerase 等 参与DNA复制的物质及作用参与DNA复制的物质及作用 DNA replication E2 Bacterial DNA replication E2 Bacterial DNA replication 1 Experimental system 2 initiation 3 unwinding 4 elongation 5 termination segregation E2 1 In vitro 离体离体 experimental systems 1 Purified DNA smaller and simpler bacteriophage and plasmid DNA molecules X174 5 Kb 2 All the proteins and other factors for its complete replications rely on host replication factors DNA replic
