1、Welcome Each of You to My Molecular Biology Class,Molecular Biology of the Gene, 5/E - Watson et al. (2004),Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods,Part II: Maintenance of the Genome,Dedicated to the str
2、ucture of DNA and the processes that propagate, maintain and alter it from one cell generation to the next,Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The replication of DNA Ch 9: The mutability and repair of DNA Ch 10: Homologous recombination at the m
3、olecular level Ch 11: Site-specific recombination and transposition of DNA,CHAPTER 8: The replication of DNA,Molecular Biology Course,How DNA molecules sustaining the beautiful living creatures are propagated from one cell generation to the next,Appreciate The simple beauty of the replication chemis
4、try. The fine art of DNA polymerase The harmonious coordination between many protein factors. The strict control of the initiation and the completeness of termination.,Teaching Arrangement,Watch animation-Understand replication Go through some structural tutorial-Experience the BEAUTY of the DNA pol
5、ymerase Lecture-comprehensive understanding and highlight Key points,CHAPTER 8 The replication of DNA,The Chemistry of DNA Synthesis The Mechanism of DNA Polymerase The Specialization of DNA PolymerasesThe Replication Fork DNA Synthesis at the Replication ForkInitiation of DNA Replication Binding an
6、d Unwinding Finishing Replication,CHAPTER 8 The replication of DNA,I. Reaction & Catalyst,II. Replication at a fork,III. Initiation & Termination,The first part describes the basic chemistry of DNA synthesis and the function of the DNA polymerase,CHAPTER 8 The replication of DNA,The Chemistry of DNA
7、,CHAPTER 8 The replication of DNA,DNA synthesis requires deoxynucleoside triphosphates and a primer:template junction DNA is synthesized by extending the 3 end of the primer Hydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis,Figure 8-3 Substrates required for DNA synthesis,The
8、mechanism of DNA Polymerase (Pol),CHAPTER 8 The replication of DNA,DNA Pol use a single active site to catalyze DNA synthesis,A single site to catalyze the addition of any of the four dNTPs. Recognition of different dNTP by monitoring the ability of incoming dNTP in forming A-T and G-C base pairs; i
9、ncorrect base pair dramatically lowers the rate of catalysis (kinetic selectivity).,The mechanism of DNA Pol,Figure 8-3,Distinguishing different dNTPs: kinetic selectivity,Distinguishing between rNTP and dNTP by steric exclusion of rNTPs from the active site.,The mechanism of DNA Pol,Figure 8-4,DNA
10、Pol resemble a hand that grips the primer-template junction,The mechanism of DNA Pol,Figure 8-5,Schematic of DNA pol bound to a primer:template junction,A similar view of the T7 DNA pol bound to DNA,Figure 8-8,Thumb,Fingers,Palm,Contains two catalytic sites, one for addition of dNTPs and one for rem
11、oval of the mispaired dNTP. The polymerization site: (1) binds to two metal ions that alter the chemical environment around the catalytic site and lead to the catalysis. (how? Figures 8-6, 8-7). (2) Monitors the accuracy of base-pairing for the most recently added nucleotides by forming extensive hy
12、drogen bond contacts with minor groove of the newly synthesized DNA. Exonuclease site/proof reading site (See proofreading),DNA Polymerase-palm domain,Figure 8-6,Figure 8-7,Binds to the incoming dNTP, encloses the correct paired dNTP to the position for catalysis Bends the template to expose the onl
13、y nucleotide at the template that ready for forming base pair with the incoming nucleotide Stabilization of the pyrophosphate,DNA Polymerase-finger domain,Not directly involved in catalysis Interacts with the synthesized DNA to maintain correct position of the primer and the active site, and to main
14、tain a strong association between DNA Pol and its substrate.,DNA Polymerase-thumb domain,DNA Pol are processive enzymes,The mechanism of DNA Pol,Processivity is a characteristic of enzymes that operate on polymeric substrates. The processivity of DNA Pol is the average number of nucleotides added ea
15、ch time the enzyme binds a primer:template junction (varying from a few to 50,000 nucleotides).,The rate of DNA synthesis is closely related to the polymerase processivity, because the rate-limiting step is the initial binding of polymerase to the primer-template junction.,Figure 8-9,Exonucleases pr
16、oofread newly synthesized DNA,The mechanism of DNA Pol,The occasional flicking of the bases into “wrong” tautomeric form results in incorrect base pair and mis-incorporation of dNTP. (10-5 mistake) The mismatched dNMP is removed by proofreading exonuclease, a part of the DNA polymerase.,How does the
17、 exonucleases work? Kinetic selectivity,Figure 8-10,The specialization of DNA polymerases,CHAPTER 8 The replication of DNA,DNA Pols are specialized for different roles in the cell,The specialization of DNA pol,Each organism has a distinct set of different DNA Pols Different organisms have different
18、DNA Pols DNA Pol III holoenzyme: a protein complex responsible for E. coli genome replication DNA Pol I: removes RNA primers in E. coli,Eukaryotic cells have multiple DNA polymerases. Three are essential to duplicate the genome: DNA Pol d, DNA Pol e and DNA Pol a/primase. (What are their functions?)
19、 Polymerase switching in Eukaryotes: the process of replacing DNA Pol a/primase with DNA Pol d or DNA Pol e.,Table 8-2*,Sliding clamps dramatically increase DNA polymerase activity,The specialization of DNA pol,Encircle the newly synthesized double-stranded DNA and the polymerase associated with the
20、 primer:template junction Ensures the rapid rebinding of DNA Pol to the same primer:template junction, and thus increases the processivity of Pol. p221 for details Eukaryotic sliding DNA clamp is PCNA,Figure 8-17,Figure 8-19 Sliding DNA clamps are found across all organism and share a similar struct
21、ure,Sliding clamps are opened and placed on DNA by clamp loaders,The specialization of DNA pol,Clamp loader is a special class of protein complex catalyzes the opening and placement of sliding clamps on the DNA, such a process occurs anytime a primer:template junction is present. Sliding clamps are
22、only removed from the DNA once all the associated enzymes complete their function.,Box 8-4 ATP control of Protein Function: Loading a Sliding Clamp,The second part describes how the synthesis of DNA occurs in the context of an intact chromosome at replication forks. An array of proteins are required
23、 to prepare DNA replication at these sites.,CHAPTER 8 The replication of DNA,The replication fork,CHAPTER 8 The replication of DNA,The junction between the newly separated template strands and the unreplicated duplex DNA,Both strands of DNA are synthesized together at the replication fork.,The repli
24、cation fork,Figure 8-11,Leading strand,Lagging strand,Okazaki fragment,Replication fork,The replication fork,Replication fork enzymes extend the range of DNA polymerase substrate,DNA Pol can not accomplish replication without the help of other enzymes The born and death of a RNA primer: primase and
25、RNase H/exonuclease/DNA Pol/ligase Dealing the DNA structure (helicase, topoisomerase, SSB),The initiation of a new strand of DNA require an RNA primer,The replication fork,Primase is a specialized RNA polymerase dedicated to making short RNA primers on an ssDNA template. Do not require specific DNA
26、 sequence. DNA Pol can extend both RNA and DNA primers annealed to DNA template,RNA primers must be removed to complete DNA replication,The replication fork,A joint efforts of RNase H, DNA polymerase & DNA ligase,Figure 8-12,The replication fork,Figure 8-15,Topoisomerase removes supercoils produced
27、by DNA unwinding at the replication fork,DNA helicases unwind the double helix in advance of the replication fork,The replication fork,Figure 8-13,Single-stranded binding proteins (SSBs) stabilize single-stranded DNA,The replication fork,Cooperative binding Sequence-independent manner (electrostatic
28、 interactions),Figure 8-14,DNA synthesis at the replication fork,CHAPTER 8 The replication of DNA,The leading strand and lagging strand are synthesized simultaneously.,At the replication, the leading strand and lagging strand are synthesized simultaneously. The biological relevance is listed in P205
29、-206 To coordinate the replication of both strands, multiple DNA Pols function at the replication fork. DNA Pol III holoenzyme is such an example.,Figure 8-20 The composition of the DNA Pol III holoenzyme,Figure 8-21* Trombone model,Interactions between replication fork proteins form the E. coli rep
30、lisome,DNA synthesis at the replication fork,Replisome is established by protein-protein interactionsDNA helicase & DNA Pol III holoenzyme, this interaction is mediated by the clamp loader and stimulates the activity of the helicase (10-fold)DNA helicase & primase, which is relatively week and stron
31、gly stimulates the primase function (1000-fold). This interaction is important for regulation the length of Okazaki fragments.,DNA Pol III holoenzyme, helicase and primase interact with each other to form replisome, a finely tuned factory for DNA synthesis with the activity of each protein is highly
32、 coordinated.,The third part focuses on the initiation and termination of DNA replication. DNA replication is tightly controlled in all cells and initiation is the step for regulation.,CHAPTER 8 The replication of DNA,CHAPTER 8 The replication of DNA,Initiation of DNA replication,CHAPTER 8 The repli
33、cation of DNA,Specific genomic DNA sequences direct the initiation of DNA replication,Origins of replication, the sites at which DNA unwinding and initiation of replication occur.,Initiation of DNA replication,The replicon model of replication initiation-a general view,Proposed by Jacob and Brenner
34、in 1963 All the DNA replicated from a particular origin is a replicon Two components, replicator and initiator, control the initiation of replication,Initiation of DNA replication,Replicator: the entire site of cis-acting DNA sequences sufficient to direct the initiation of DNA replication,Initiator
35、 protein: specifically recognizes a DNA element in the replicator and activates the initiation of replication,Figure 8-23,Replicator sequences include initiator binding sites and easily unwound DNA,Binding and Unwinding: origin selection and activation by the initiator protein,CHAPTER 8 The replicat
36、ion of DNA,Three different functions of initiator protein: (1) binds to replicator, (2) distorts/unwinds a region of DNA, (3) interacts with and recruits additional replication factors DnaA in E. coli (all 3 functions), origin recognition complex (ORC) in eukaryotes (functions 1 & 3),Protein-protein
37、 and protein-DNA interactions direct the initiation process,Binding and unwinding,Initiating replication in bacteria,DnaA recruits the DNA helicase DnaB and the helicase loader DnaC DnaB interacts with primase to initiate RNA primer synthesis.,Figure 8-27*,Initiating replication in eukaryotes Eukary
38、otic chromosome are replicated exactly once per cell cycle, which is critical for these organisms,Binding and unwinding,Pre-replicative complex (pre-RC) formation and activation directs the initiation of replication in eukaryotes,Initiation in eukaryotes requires two distinct steps: 1st step-Replica
39、tor selection: the process of identifying sequences for replication initiation (G1 phase), which is mediated by the formation of pre-RCs at the replicator region.,Figure 8-30 pre-RC formation,2nd step-Origin activation: pre-RCs are activated by two protein kinases (Cdk and Ddk) that are active only
40、when the cells enter S phase.,Figure 8-31: Activation of the pre-RC leads to the assembly of the eukaryotic replication fork.,Pre-RC formation and activation is tightly regulated to allow only a single round of replication during each cell cycle.,Only one opportunity for pre-RCs to form, and only on
41、e opportunity for pre-RC activation.,Figure 8-32 Effect of Cdk activity on pre-RC formation and activation,Figure 8-33 Cell cycle regulation of Cdk activity and pre-RC formatin,Finishing replication,CHAPTER 8 The replication of DNA,Finishing replication in bacteria: Type II topoisomerases separate d
42、aughter DNA molecules,Finishing replication,Figure 8-34 Topoisomerase II catalyze the decatenation of replication products.,Finishing replication in eukaryotes:The end replication problem Telomere & telomerase: a link with cancer and aging,Finishing replication,What is the end replication problem? L
43、agging strand synthesis is unable to copy the extreme ends of the linear chromosome,Figure 8-34,Telomerase is a novel DNA polymerase that does not require an exogenous template,How telomerase works? Telomerase extends the protruding 3 end of the chromosome using its RNA component s as a template. (F
44、igure 8-37),How the end problem is eventually resolved?,Figure 8-38,The extended 3 end allows the DNA polymerase to synthesize a new Okazaki fragment, which prevents the loss of genetic information at the chromosomal end.,Figure 8-39: Telomere-binding proteins.,Telomere -binding proteins regulate te
45、lomerase activity and telomere length,Figure 8-40: Telomere length regulation by telomere-binding proteins.,Short telomere is bound by few telomere-binding proteins, allowing the telomerase to extend telomere.,The extended telomere is bound by more telomere-binding proteins, which inhibits the telom
46、erase activity.,重点,CHAPTER 8 The replication of DNA,Completely understand 三个Animations DNA polymerization (Topics 1 & 2) DNA replication (Topics 35) Action of Telomerase (Topic 8),The Chemistry of DNA Synthesis: substrate, direction and energy. The Mechanism of DNA Polymerase: 1 polymerization mecha
47、nism, 2 different ways of discriminating substrates, 2 catalytic sites; 3 domains. The Specialization of DNA Polymerases The Replication Fork: the enzyme/proteins required to synthesize the leading and lagging strands. DNA Synthesis at the Replication Fork: Holoenzyme/trombone model to explain how t
48、he anti-parallel template strands are copied/replicated toward the replication fork. Replisome/protein interaction.,CHAPTER 8 The replication of DNA,Initiation of DNA Replication/binding and unwinding: the replicon model; initiation in bacteria; initiation control in eukaryotes-a link with cell cycl
49、e (pre-RC assembly and activiation). Finishing Replication: Finishing in bacteria; Finishing in eukaryotes-the end replication problem and resolution (telomere, telomerase, telomere binding proteins)- a link with cancer and aging.,CHAPTER 8 The replication of DNA,重点,CHAPTER 8 The replication of DNA,
50、Chemistry of DNA DNA polymerization (Topics 1 & 2): DNA polymerase: catalysis mechanism, catalytic sites, different ways to distinguish substrates, structure and function of three domains.,重点,CHAPTER 8 The replication of DNA,2.DNA replication (Topics 3 5):trumbone model, how the anti-parallel template strands are copied/replicated toward the replication fork.3.Action of Telomerase (Topic 8),
