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Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome

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 molecular level Ch 11: Site-specific recombination and transposition of DNA.

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Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome

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  1. 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 molecular level Ch 11: Site-specific recombination and transposition of DNA

  2. CHAPTER 8: The replication of DNA • Molecular Biology Course

  3. CHAPTER 8 The replication of DNA Teaching Arrangement • Watch animation-Understand replication • Go through structural tutorial-Experience the BEAUTY of the nature • Lecture-comprehensive understanding of Chapter 8 • Summary and highlight Key points

  4. CHAPTER 8 The replication of DNA The Chemistry of DNA Synthesis • The Mechanism of DNA Polymerase • The Replication Fork • The Specialization of DNA Polymerases • DNA Synthesis at the Replication Fork • Initiation of DNA Replication • Binding and Unwinding • Finishing Replication General Detailed

  5. CHAPTER 8 The replication of DNA The first part describes the basic chemistry of DNA synthesis and the function of the DNA polymerase

  6. CHAPTER 8 The replication of DNA The Chemistry 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

  7. Figure 8-3 Substrates required for DNA synthesis

  8. CHAPTER 8 The replication of DNA The mechanism of DNA Polymerase (Pol)

  9. DNA Pol use a single active site to catalyze DNA synthesis The mechanism of DNA Pol 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; incorrect base pair dramatically lowers the rate of catalysis (kinetic selectivity).

  10. Figure 8-3

  11. Distinguish between rNTP and dNTP by steric exclusion of rNTPs from the active site. The mechanism of DNA Pol Figure 8-4

  12. DNA Pol resemble a hand that grips the primer-template junction The mechanism of DNA Pol Schematic of DNA pol bound to a primer:template junction A similar view of the T7 DNA pol bound to DNA Figure 8-5

  13. Thumb Fingers Palm Figure 8-8

  14. DNA Polymerase-palm domain Contains two catalytic sites, one for addition of dNTPs and one for removal of the mispaired dNTP. The polymerization site binds to two metal ions that alter the chemical environment around the catalytic site and lead to the catalysis. (how?) Monitors the accuracy of base-pairing for the most recently added nucleotides by forming extensive hydrogen bond contacts with minor groove of the newly synthesized DNA. (See proofreading)

  15. DNA Polymerase-finger domain Binds to the incoming dNTP, encloses the correct paired dNTP to the position for catalysis Bends the template to expose the only nucleotide at the template that ready for forming base pair with the incoming nucleotide Stabilization of the pyrophosphate

  16. DNA Polymerase-thumb domain Not directly involved in catalysis Interacts with the synthesized DNA to maintain correct position of the primer and the active site, and to maintain a strong association between DNA Pol and its substrate.

  17. 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 each time the enzyme binds a primer:template junction (a few~50,000).

  18. 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.

  19. Figure 8-9

  20. Exonucleases proofread 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

  21. Figure 8-10

  22. CHAPTER 8 The replication of DNA 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 to prepare DNA replication at these sites.

  23. CHAPTER 8 The replication of DNA The replication fork • The junction between the newly separated template strands and the unreplicated duplex DNA

  24. Both strands of DNA are synthesized together at the replication fork. The replication fork Leading strand Okazaki fragment Replication fork Lagging strand Figure 8-11

  25. 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 sequence. • DNA Pol can extend both RNA and DNA primers annealed to DNA template

  26. 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

  27. Topoisomerase removes supercoils produced by DNA unwinding at the replication fork The replication fork Figure 8-15

  28. DNA helicases unwind the double helix in advance of the replication fork The replication fork Figure 8-13

  29. Single-stranded binding proteins (SSBs) stabilize single-stranded DNA The replication fork • Cooperative binding • Sequence-independent manner (electrostatic interactions) Figure 8-14

  30. Replication fork enzymes extend the range of DNA polymerase substrate The replication fork DNA Pol can not accomplish replication without the help of other enzymes DNA helicase, SSB, primase, DNA topoisomerase

  31. CHAPTER 8 The replication of DNA The specialization of DNA polymerases

  32. 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 DNA Pols DNA Pol III holoenzyme: a protein complex responsible for E. coli genome replication DNA Pol I: removes RNA primers in E. coli

  33. 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?) • Polymerase switching in Eukaryotes: the process of replacing DNA Pol a/primase with DNA Pol d or DNA Pol e. Table 8-2***

  34. 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 primer:template junction • Ensures the rapid rebinding of DNA Pol to the same primer:template junction, and thus increases the processivity of Pol. • Eukaryotic sliding DNA clamp is PCNA

  35. Figure 8-17

  36. Figure 8-19 Sliding DNA clamps are found across all organism and share a similar structure

  37. 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 only removed from the DNA once all the associated enzymes complete their function.

  38. CHAPTER 8 The replication of DNA DNA synthesis at the replication fork: the leading strand and lagging strand are synthesized simultaneously.

  39. At the replication, the leading strand and lagging strand are synthesized simultaneously. The biological relevance is listed in P205-206 • To coordinate the replication of both strands, multiple DNA Pols function at the replication fork. DNA Pol III holoenzyme is such an example.

  40. Figure 8-20 The composition of the DNA Pol III holoenzyme

  41. Figure 8-21*** Trombone model

  42. DNA synthesis at the replication fork Interactions between replication fork proteins form the E. colireplisome Replisome is established by protein-protein interactions DNA 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 strongly stimulates the primase function (1000-fold). This interaction is important for regulation the length of Okazaki fragments.

  43. 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 coordinated.

  44. CHAPTER 8 The replication of DNA The third part focuses on the initiation and termination of DNA replication. Note that DNA replication is tightly controlled in all cells and initiation is the step for regulation.

  45. CHAPTER 8 The replication of DNA Initiation of DNA replication

  46. Initiation of DNA replication Specific genomic DNA sequences direct the initiation of DNA replication Origins of replication, the sites at which DNA unwinding and initiation of replication occur.

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