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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 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,
