现代分子生物学之DNA复制.ppt_第1页
现代分子生物学之DNA复制.ppt_第2页
现代分子生物学之DNA复制.ppt_第3页
现代分子生物学之DNA复制.ppt_第4页
现代分子生物学之DNA复制.ppt_第5页
已阅读5页,还剩64页未读 继续免费阅读

下载本文档

版权说明:本文档由用户提供并上传,收益归属内容提供方,若内容存在侵权,请进行举报或认领

文档简介

1、分子生物学的主要脉络:中心法则 课程目标:掌握主要脉络、熟悉基本概念、了解细枝末节,基因的物质状态:DNA上的一个片段 DNA本身的结构(为什么可以作为遗传物质) DNA在细胞中的存在形式(染色质、染色体、基因组的特点) DNA的传代方式:复制 DNA在传代及储存过程中可能遇到的问题及其解决方式 DNA并非静态存储: 同源重组是修复方式和减数分裂之必须; CSSR与转座是不同DNA间的剪切融合-DNA分子大尺度改变的基础 基因表达:DNA序列影响细胞结构功能 RNA的合成:合成工具、基本过程、原核真核异同、后处理 蛋白质的合成:密码、合成工具、基本过程、原核真核异同 基因表达的调控:基本的原则

2、和方法、原核真核异同 基因研究技术,问题: DNA作为遗传物质如何随着细胞分裂而传代?在原核与真核之间的区别与联系?,DNA replication,DNA polymerization Genome replication,1.The Chemistry of DNA Synthesis-化学反应 2. The Mechanism of DNA Polymerase-催化反应的主要酶 3. The Replication Fork: the enzymes-催化反应的其他蛋白,1. The Specialization of DNA Polymerases -细胞内的聚合酶多种用途 2. H

3、ow DNA pol III work in E. Coli: the trombone model-原核的POL III 3. Initiation of DNA Replication-复制的起始(原核与真核) 4. Finishing replication: Action of Telomerase-复制的终止(原核与真核),Annealed primer,The Chemistry of DNA Synthesis,template,G = - 3.5kcal/mol,G = - 7kcal/mol,The Mechanism of DNA Polymerase,Palm: for

4、addition of dNTPs and for removal of mispaired dNTP. (1) Binds to two metal ions - alter the chemical environment - catalysis. (2) H-bonds with minor groove of DNA - Monitors the accuracy of the new base-pair,Finger: Binds to the new dNTP; Bends the template; Stabilize the pyrophosphate,Thumb: Holds

5、 the primer-template junction in active site,reduces rate of dissociation,template strand,primer strand,dNTP,Nucleotide addition: catalyzed at the active site of DNA polymerase,H- bond contacts between minor groove (new BP) and palm,Two metal ions are held in place by interactions with two highly co

6、nserved aspartate residues.,Nucleotide addition: catalyzed at the active site of DNA polymerase,H- bond contacts between minor groove (new BP) and palm,Two metal ions are held in place by interactions with two highly conserved Asp. Metal ion A interacts with the 3-OH reduce the O and H association l

7、eaves a nucleophilic 3 O-. Metal ion B interacts with TPs of the new dNTP to neutralize their negative charge. After catalysis, the PPi is stabilized by interacting with metal ion B,Nucleotide addition: catalyzed at the active site of DNA polymerase,H- bond contacts between minor groove (new BP) and

8、 palm,Finger domain binds to the incoming dNTP and bends the template,Finger domain binds to the incoming dNTP and bends the template,Finger domain binds to the incoming dNTP and bends the template,Move to enclose dNTP,Finger domain binds to the incoming dNTP and bends the template,DNA polymerase “g

9、rips” the template and the incoming dNTP when a correct base pair is made,metal ions,The critical tyr,lys,arg,The base and deoxyribose of the dNTP,The primer,Template strand,Phosphates,Steric constraints空间位阻prevents DNA pol from using rNTP precursors.,How to avoid incorporation of wrong nucleotides,

10、wrong base pair lowers rate of catalysis (kinetic selectivity) 准-稳-快-接; 错-晃-慢-掉,Each bases has its preferred tautomeric form,The occasional flicking of the bases into “wrong” tautomeric form: 10-5 mistake,Exonucleases proofread newly synthesized DNA,The average number of nucleotides added each time

11、the enzyme binds a primer-template junction,The processivity of DNA Polymerase,varies from a few to 50,000 nucleotides,Replication fork: The junction between the newly separated template strands and the unreplicated duplex DNA,Proteins needed at the replication fork,DNA helicase Single strand DNA bi

12、nding protein (SSB) Primase: to synthesize a short RNA DNA polymerase DNA topoisomerase,DNA helicases unwind double helix in advance of the replication fork,High processivity because they encircle the DNA,This DNA helicase has a 5-3 polarity.,Crystal structures of complexes of PcrA DNA helicase with

13、 a DNA substrate indicate an inchworm mechanism, Cell. 1999 Apr 2;97(1):75-84,Single-stranded binding proteins (SSBs) stabilize single-stranded DNA,Sequence-independent: electrostatic and stacking interactions Cooperative binding: they bind to DNA, also bind to each other,RNA primers must be removed

14、: by RNase H, DNA polymerase and it has many subunites,3 3 3 3,Holoenzyme,Sliding clamps increase DNA polymerase activity,Ensures rapid rebinding of DNA Pol to the same primer-template junction,Encircle new dsDNA make sure the pol associated with primer-template junction,Sliding clamps are removed f

15、rom the DNA only when all the associated enzymes complete their function,Similar structure from different specieses,Clamp loader catalyzes the opening and placement of sliding clamps on the DNA,Occurs when a primer:template junction is present,Leading and lagging strands are synthesized simultaneous

16、ly at the replication fork: limiting the amount of ssDNA,one pol to replicate the leading strand and the other two to replicate the lagging strand.,Through the linker, the pols can move in independent manner,DNA Pol III holoenzyme, helicase and primase interact with each other to form replisome,Repl

17、isome is established by protein-protein interactions: Clamp loader interacts with and activates helicase (10 fold),DNA helicase interacts with primase weakly, but strongly stimulates its function (1000-fold) the length of Okazaki fragments.,Primase: a specialized RNA polymerase, making short RNA pri

18、mers on an ssDNA template, activated when associated with DNA helicase;,sliding clamp is loaded onto the newly primed lagging strand,primase is released,The “second” lagging-strand DNA pol binds sliding clamp at the primer: template junction and begins to synthesize a new Okazaki fragment,The “first

19、” lagging-strand DNA pol is released, after completion of an Okazaki fragment,The “trombone model” : the changing size of ssDNA loop formed between the DNA pol(s) and the DNA helicase on the lagging-strand template.,DNA replication,DNA polymerization Genome replication,1.The Chemistry of DNA Synthes

20、is 2. The Mechanism of DNA Polymerase 3. The Replication Fork: the enzymes,1. The Specialization of DNA Polymerases 2. How DNA pol III work in E. Coli: the trombone model 3. Initiation of DNA Replication(where, when, how) 4. Finishing replication: Action of Telomerase,Initiator: Specific proteins re

21、cognizing a DNA element in replicator and activates the replication initiation,Replicator: The DNA sequences to direct replication initiation: initiator binding sites and easily unwound DNA,Initiation of genome replication: controlled by two components -replicator and initiator,Origins of replicatio

22、n-The specific sites where DNA unwinding and initiation of replication occur,replicator,Replicon: all of the DNA(RNA) replicated from a particular origin of replication,Homework: describe the figure(p290): Identification of Origins of Replication and Replicators,Replicators of some genomes: Initiato

23、r-Binding Sites(It nucleates the assembly of the replication initiation machinery) Easily Unwound DNA(A stretch of AT-rich DNA that unwinds readily, not spontaneously),Three functions of initiator protein:,binds to replicator, distorts/unwinds a region of DNA interacts with and recruits additional r

24、eplication factors,The initiation process is directed by protein-protein and protein-DNA interactions,The initiator protein is the only sequence-specific DNA binding protein involved in replication initiation. The remaining proteins are recruited 招募 to replicator through proteinprotein interactions

25、and affinity for specific DNA structures (ssDNA or a primer:template junction). All of the initiator proteins are regulated by ATP binding and hydrolysis,The initiators: DnaA in E. coli (all 3 functions); Origin recognition complex (ORC) in eukaryotes (functions 1 avoid wrong nt inserting; proofread

26、ing processivity 3. The Replication Fork: the enzymes-其他蛋白 1) Characteristics of replication fork: semi-conservative: template/primer semi-continuous:leading/ lagging dually directional 2) elements at the replication fork: DNA helicase; primer; primase; SSB; topoisomerase; okazaki fragments,1. The S

27、pecialization of DNA Polymerases -特化的聚合酶 Prokaryotics: DNA pol I Prokaryotics: DNA pol III: core / holoenzyme clamp / clamp loader Eukaryotics: DNA pols for leading/lagging 2. DNA pol III working: trombone model replisome: clamp loader-helicase-primase interactions 3. Initiation of DNA Replication-复

28、制起始 replicator: origin + initiator binding site (consensus sequences) initiator: function and mechanisms replicon initiation and reinitiation process in prokaryotics only once initiation in eukaryotics-how? 4. Finishing replication: Action of Telomerase-复制的终止(原核与真核),DNA polymerization,Genome replica

29、tion,Replicators are inactivated by DNA replication.,Not all, but enough replicators need to be activated to fully replicate a chromasome.,Helicase loading and activation occur during different cell cycle stages in eukaryotic cells,ATP-bound ORC(origin recognition complex, the initiator) bind with r

30、eplicator. ORC recruits helicase loaders, Cdt1, Cdc6 and two copies of Mcm2-7(the helicase) The proteins assembly triggers ATP hydrolysis by Cdc6 loading of a head-to-head dimer of Mcm2-7 complex encircling the DNA Cdc6 and Cdt1 release. ATP hydrolysis by ORC release from Mcm2-7. Exchange of ATP for

31、 ADP allows a new round of helicase loading.,Helicase loading (G1 phase) is the 1st step in eukaryotes replication initiation,a head-to-head double hexamer (through N-termini interactions) encircling dsDNA interaction between the two Mcm2-7 has been brokenen; encircling ssDNA,Two kinases CDK and DDK

32、 are activated in S phase,helicase-activating proteins are phosphorylated,Mcm2-7 helicase activity is activated in S phase,Helicase before activation,Helicase after activation,Two kinases CDK and DDK are activated in S phase Phosphorylated Sld2 DNA Pol a/primase and DNA Pol (for lagging strand) are

33、only recruited after DNA unwinding.,Activation of loaded helicases assembly of eukaryotic replisome,pre-RC,.,Eukaryotic helicase loading and activation occur during different cell cycle stages.,Cell cycle regulation of CDK activity controls replication.,ensures the only-once-replication in eukaryoti

34、c cell cycle,Helicase loading (G1),Helicase activation and replisome assembly (S phase),DNA replication,DNA polymerization DNA replication,1.The Chemistry of DNA Synthesis 2. The Mechanism of DNA Polymerase 3. The Replication Fork: the enzymes,1. The Specialization of DNA Polymerases 2. DNA Synthesis at the Replication Fork 3. Initiation of DNA Replication 4. Finishing replication: Action of Telomerase,Finishing replication,Finishing replication in bacteria: Type

温馨提示

  • 1. 本站所有资源如无特殊说明,都需要本地电脑安装OFFICE2007和PDF阅读器。图纸软件为CAD,CAXA,PROE,UG,SolidWorks等.压缩文件请下载最新的WinRAR软件解压。
  • 2. 本站的文档不包含任何第三方提供的附件图纸等,如果需要附件,请联系上传者。文件的所有权益归上传用户所有。
  • 3. 本站RAR压缩包中若带图纸,网页内容里面会有图纸预览,若没有图纸预览就没有图纸。
  • 4. 未经权益所有人同意不得将文件中的内容挪作商业或盈利用途。
  • 5. 人人文库网仅提供信息存储空间,仅对用户上传内容的表现方式做保护处理,对用户上传分享的文档内容本身不做任何修改或编辑,并不能对任何下载内容负责。
  • 6. 下载文件中如有侵权或不适当内容,请与我们联系,我们立即纠正。
  • 7. 本站不保证下载资源的准确性、安全性和完整性, 同时也不承担用户因使用这些下载资源对自己和他人造成任何形式的伤害或损失。

评论

0/150

提交评论