现代分子生物学之DNA复制学习教案

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1、会计学1第一页，共70页。DNA polymerization Genome replication 1.The Chemistry of DNA Synthesis-化学反应化学反应2. The Mechanism of DNA Polymerase-催化反应的主要催化反应的主要(zhyo)酶酶3. The Replication Fork: the enzymes-催化反应的其他蛋白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细胞内的聚合酶多种用途2. How DNA pol III work in E. Co

2、li: the trombone model-原核的原核的POL III 3. Initiation of DNA Replication-复制复制(fzh)的起始（原核与真核）的起始（原核与真核）4. Finishing replication: Action of Telomerase-复制复制(fzh)的终止（原核与真核）的终止（原核与真核）第1页/共69页第二页，共70页。Annealed primertemplateG = - 3.5kcal/molG = - 7kcal/mol第2页/共69页第三页，共70页。Palm: for of dNTPs and for of mispai

3、red dNTP. inds to two metal ions - alter the chemical environment - catalysis. (2) H-bonds with minor groove of DNA - Monitors the accuracy of the new base-pairFinger: Binds to the new dNTP;Bends the template; Stabilize the phosphateThumb: Holds the primer-template junction in active site,reduces ra

4、te of dissociation第3页/共69页第四页，共70页。Nucleotide addition: catalyzed at the active site of DNA polymerase第4页/共69页第五页，共70页。 are held in place by interactions with two highly conserved aspartate residues. Nucleotide addition: catalyzed at the active site of DNA polymerase第5页/共69页第六页，共70页。Two metal ions a

5、re held in place by interactions with two highly conserved Asp. Metal ion A reduce the O and H association leaves a nucleophilic 3 O-. Metal ion B to neutralize their negative charge. After catalysis,Nucleotide addition: catalyzed at the active site of DNA polymerase第6页/共69页第七页，共70页。Finger domain bi

6、nds to the incoming dNTP and bends the template第7页/共69页第八页，共70页。Finger domain binds to the incoming dNTP and bends the template第8页/共69页第九页，共70页。Finger domain binds to the incoming dNTP and bends the template第9页/共69页第十页，共70页。Move to enclose dNTPFinger domain binds to the incoming dNTP and bends the t

7、emplate第10页/共69页第十一页，共70页。DNA polymerase “grips” the template and the incoming dNTP when a correct base pair is madeThe critical ,The base and deoxyribose of the dNTPThe primerTemplatestrandPhosphates第11页/共69页第十二页，共70页。How to avoid incorporation of wrong nucleotideswrong base pair lowers rate of cat

8、alysis (kinetic selectivity)准准-稳稳-快快-接；接； 错错-晃晃-慢慢-掉掉第12页/共69页第十三页，共70页。第13页/共69页第十四页，共70页。The occasional flicking of the bases into : Exonucleases proofread newly synthesized DNA第14页/共69页第十五页，共70页。varies from a few to 50,000 nucleotides第15页/共69页第十六页，共70页。第16页/共69页第十七页，共70页。 The junction between the

9、 newly separated template strands and the unreplicated duplex DNAProteins needed at the replication fork第17页/共69页第十八页，共70页。DNA helicases unwind double helix in advance of the replication forkHigh processivity because they encircle the DNAThis DNA helicase has a 5-3 polarity.Crystal structures of com

10、plexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism, Cell. 1999 Apr 2;97(1):75-84第18页/共69页第十九页，共70页。Single-stranded binding proteins (SSBs) stabilize single-stranded DNASequence-independent: electrostatic and stacking interactions 第19页/共69页第二十页，共70页。RNA primers must be re

11、moved: by RNase H, DNA polymerase & DNA ligase RNaseH can only cleave bonds between two ribonucleotides. The final ribonucleotide is removed by a 5 exonuclease第20页/共69页第二十一页，共70页。Topoisomerase removes supercoils produced by DNA unwinding at the replication fork第21页/共69页第二十二页，共70页。DNA Pol III holoenz

12、yme: a protein complex responsible for E. coli genome replicationDNA Pols are specialized for different roles in cell第22页/共69页第二十三页，共70页。第23页/共69页第二十四页，共70页。The DNA Pol /primase product is between 50100 bp, DNA Polsubstitutes DNA Pol /primase on the leading-strand template and DNA Pol substitutes on

13、 the lagging-strand template.and the further extension by them is 10010,000 nucleotides.第24页/共69页第二十五页，共70页。第25页/共69页第二十六页，共70页。DNA polymerization Genome replication 1.The Chemistry of DNA Synthesis-化学反应化学反应(huxu fnyng)2. The Mechanism of DNA Polymerase-催化反应的主要酶催化反应的主要酶3. The Replication Fork: the e

14、nzymes-催化反应的其他蛋白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细胞内的聚合酶多种用途(yngt)2. How DNA pol III work in E. Coli: the trombone model-原核的原核的POL III 3. Initiation of DNA Replication-复制的起始（原核与真核）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）复制的终止（原核与真核）第26页/共69

15、页第二十七页，共70页。DNA Pols are specialized for different roles in cell第27页/共69页第二十八页，共70页。第28页/共69页第二十九页，共70页。Ensures of DNA Pol to the make sure the pol associated with第29页/共69页第三十页，共70页。第30页/共69页第三十一页，共70页。 catalyzes the opening and placement of sliding clamps on the DNAOccurs when a junction is present

16、第31页/共69页第三十二页，共70页。Leading and lagging strands are synthesized at the replication fork: limiting the amount of ssDNAone pol to replicate the leading strand and the other two to replicate the lagging strand.Through the linker, 第32页/共69页第三十三页，共70页。 interact with each other to form interacts with and

17、activates (10 fold)第33页/共69页第三十四页，共70页。 interacts with weakly, but strongly stimulates its function (1000-fold) the length of Okazaki fragments.Primase: a specialized RNA polymerase, making short RNA primers on an ssDNA template, activated when associated with DNA helicase;第34页/共69页第三十五页，共70页。slidin

18、g clamp is loaded onto the newly primed lagging strandprimaseis released第35页/共69页第三十六页，共70页。 binds sliding clamp at the and begins to synthesize a new Okazaki fragment第36页/共69页第三十七页，共70页。 is released, after completion of an Okazaki fragment: the changing size of ssDNA loop formed between the DNA pol

19、(s) and the DNA helicase on the lagging-strand template.第37页/共69页第三十八页，共70页。DNA polymerization Genome replication 1.The Chemistry of DNA Synthesis2. The Mechanism of DNA Polymerase3. The Replication Fork: the enzymes1. The Specialization of DNA Polymerases 2. How DNA pol III work in E. Coli: the tro

20、mbone model 3. 4. Finishing replication: Action of Telomerase第38页/共69页第三十九页，共70页。Specific proteins recognizing a DNA element in replicator and activates the replication initiationThe DNA sequences to direct replication initiation: initiator binding sites and easily unwound DNAInitiation of genome re

21、plication: controlled by two components - and The specific sites where DNA and of replication occur第39页/共69页第四十页，共70页。 all of the DNA(RNA) replicated from a particular origin of replication第40页/共69页第四十一页，共70页。Homework: describe the figure(p290): Identification of Origins of Replication and Replicato

22、rs第41页/共69页第四十二页，共70页。Replicators of some genomes:Initiator-Binding Sites Easily Unwound DNA第42页/共69页第四十三页，共70页。Three functions of initiator protein: to replicator, a region of DNA additional replication factorsThe initiators: DnaA in E. coli (all 3 functions);Origin recognition complex (ORC) in euk

23、aryotes (functions 1 & 3)第43页/共69页第四十四页，共70页。oriCbound to ssDNA at the origin, the helicase loader Initial site of ssDNA formationHelicase recruits primase to the origin -interaction activity primer synthesis inactivates helicase, preventing it from acting at wrong sites. , , . activation of helicas

24、e.第44页/共69页第四十五页，共70页。oriCInitial site of ssDNA formation interacts with and activates (10 fold)第45页/共69页第四十六页，共70页。第46页/共69页第四十七页，共70页。第47页/共69页第四十八页，共70页。第48页/共69页第四十九页，共70页。1.The Chemistry of DNA Synthesis-化学反应化学反应(huxu fnyng)2. The Mechanism of DNA Polymerase-主要酶主要酶1) DNA pol active site-palm2)

25、DNA pol activity: synthesis/polymerization; avoid wrong nt inserting; proofreadingprocessivity3. The Replication Fork: the enzymes-其他蛋白其他蛋白1) Characteristics of replication fork: semi-conservative: template/primersemi-continuous:leading/ laggingdually directional2) elements at the replication fork:D

26、NA helicase; primer; primase; SSB; topoisomerase; okazaki fragments 1. The Specialization of DNA Polymerases -特化特化的聚合酶的聚合酶Prokaryotics: DNA pol I Prokaryotics: DNA pol III: core / holoenzyme clamp / clamp loaderEukaryotics: DNA pols for leading/lagging2. DNA pol III working: trombone modelreplisome:

27、 clamp loader-helicase-primase interactions 3. Initiation of DNA Replication-复制起始复制起始(q sh)replicator: origin + initiator binding site (consensus sequences)initiator: function and mechanismsrepliconinitiation and reinitiation process in prokaryoticsonly once initiation in eukaryotics-how?4. Finishin

28、g replication: Action of Telomerase-复制的终止（原核与真核）复制的终止（原核与真核）DNA polymerizationGenome replication 第49页/共69页第五十页，共70页。Replicators are by DNA replication. need to be activated to fully replicate a chromasome.第50页/共69页第五十一页，共70页。第51页/共69页第五十二页，共70页。origin recognition complex, with replicator. ORC recrui

29、ts helicase loaders, , and two copies of The proteins assembly triggers ATP hydrolysis by loading of a encircling the DNA Cdc6 and Cdt1 release. ATP hydrolysis by ORC release from Mcm2-7. Exchange of ATP for ADP allows a new round of helicase loading. is the 1st step in eukaryotes replication initia

30、tion第52页/共69页第五十三页，共70页。a head-to-head double hexamer (through N-termini interactions) encircling dsDNAinteraction between the two Mcm2-7 has been brokenen; encircling ssDNATwo kinases CDK and DDK are activated in S phase are phosphorylatedHelicase before activationHelicase after activation第53页/共69页

31、第五十四页，共70页。Two kinases CDK and DDK are activated in S phasePhosphorylated Sld2 & Sld3 bind to Dpb11 facilitate binding of helicase-activating proteins, the Cdc45 & GINS, to helicasethe () forms and .Before activation, loaded helicases encircle dsDNA and are in form of a head-to-head double hexamer (

32、through Mcm2-7 N-termini interactions)After activation, the Mcm2-7 in CMG complex encircle ssDNA, and the interaction between the two Mcm2-7 has been brokenThe leading-strand DNA pol is recruited to the helicase before DNA unwinding;DNA Pol a/primase and DNA Pol (for lagging strand) are only recruit

33、ed after DNA unwinding. Activation of loaded helicases assembly of eukaryotic replisomepre-RC第54页/共69页第五十五页，共70页。.第55页/共69页第五十六页，共70页。Cell cycle regulation of controls replication.ensures the in eukaryotic cell cycle Helicase activation and replisome assembly 第56页/共69页第五十七页，共70页。1.The Chemistry of D

34、NA Synthesis2. The Mechanism of DNA Polymerase3. The Replication Fork: the enzymes1. The Specialization of DNA Polymerases 2. DNA Synthesis at the Replication Fork 3. Initiation of DNA Replication4. Finishing replication: Action of Telomerase第57页/共69页第五十八页，共70页。Finishing replicationType II topoisome

35、rases separate daughter DNA molecules: decatenation of replication products.第58页/共69页第五十九页，共70页。第59页/共69页第六十页，共70页。 : Lagging strand synthesis is unable to copy the extreme ends of the linear chromosome第60页/共69页第六十一页，共70页。第61页/共69页第六十二页，共70页。Telomeres: the ends of eukaryotic chromosomes, composed of

36、 第62页/共69页第六十三页，共70页。第63页/共69页第六十四页，共70页。The end sequence of linear DNA molecules from Tetrahymena added to artificial minichromosomes allows their long-term stable maintenance in yeast.Conservation of the structure and function of chromosomal termini between very distant species is striking.第64页/共6

37、9页第六十五页，共70页。第65页/共69页第六十六页，共70页。第66页/共69页第六十七页，共70页。Telomerase: an enzyme with multiple protein subunits and an RNA componentas a template)第67页/共69页第六十八页，共70页。Telomere-binding proteins regulate telomerase activity and telomere lengthweak Inhibition第68页/共69页第六十九页，共70页。NoImage内容(nirng)总结会计学。The critical tyr,lys,arg。binds to replicator,。Helicase loading (G1)。weak Inhibition第七十页，共70页。