1. Welcome Each of You to My Molecular Biology Class

2. 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

3. Part II: Maintenance of the Genome
Dedicated to the structure of DNA and the processes that propagate, maintain and alter it from one cell generation to the next

4. 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

5. CHAPTER 8: The replication of DNA
Molecular Biology Course
CHAPTER 8: The replication of DNA

6. Teaching Arrangement Watch animation-Understand replication
CHAPTER 8 The replication of DNA
Teaching Arrangement
Watch animation-Understand replication
Go through some structural tutorial-Experience the BEAUTY of the DNA polymerase
Lecture-comprehensive understanding and highlight Key points

7. The Chemistry of DNA Synthesis The Mechanism of DNA Polymerase
CHAPTER 8 The replication of DNA
The Chemistry of DNA Synthesis
The Mechanism of DNA Polymerase
The Specialization of DNA Polymerases
The Replication Fork
DNA Synthesis at the Replication Fork
Initiation of DNA Replication
Binding and Unwinding
Finishing Replication
Reaction & Catalyst
Process
Initiation &
Termination

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

9. 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

10. Figure 8-3 Substrates required for DNA synthesis

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

12. 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).

13. Distinguishing different dNTPs: kinetic selectivity
Figure 8-3

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

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

16. Thumb
Fingers
Palm
Figure 8-8

17. DNA Polymerase-palm domain
Contains two catalytic sites, one for addition of dNTPs and one for removal 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 hydrogen bond contacts with minor groove of the newly synthesized DNA.
Exonuclease site/proof reading site (See proofreading)

18. Figure 8-6
Figure 8-7

19. 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

20. 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.

21. 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 (varying from a few to >50,000 nucleotides).

22. 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.

23. Figure 8-9

24. 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

25. Figure 8-10

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

27. 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

28. Eukaryotic cells have multiple DNA polymerases
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***

29. 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. [p221 for details]
Eukaryotic sliding DNA clamp is PCNA

30. Figure 8-17

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

32. 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.

33. Box 8-4 ATP control of Protein Function: Loading a Sliding Clamp

34. 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.

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

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

37. 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
The born and death of a RNA primer: primase and RNase H/exonuclease/DNA Pol/ligase
Dealing the DNA structure (helicase, topoisomerase, SSB)

38. 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

39. 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

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

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

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

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

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

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

46. Figure 8-21*** Trombone model

51. DNA synthesis at the replication fork
Interactions between replication fork proteins form the E. coli replisome
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.

52. 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.

53. 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.

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

55. 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.

56. The replicon model of replication initiation---a general view
Initiation of DNA replication
The replicon model of replication initiation---a general view
Proposed by Jacob and Brenner in 1963
All the DNA replicated from a particular origin is a replicon
Two components, replicator and initiator, control the initiation of replication

57. Replicator: the entire site of cis-acting DNA sequences sufficient to direct the initiation of DNA replication
Initiator protein: specifically recognizes a DNA element in the replicator and activates the initiation of replication
Figure 8-23

58. Replicator sequences include initiator binding sites and easily unwound DNA

59. CHAPTER 8 The replication of DNA
Binding and Unwinding: origin selection and activation by the initiator protein

60. 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)

62. Binding and unwinding
Protein-protein and protein-DNA interactions direct the initiation process

63. Initiating replication in bacteria
DnaA recruits the DNA helicase DnaB and the helicase loader DnaC
DnaB interacts with primase to initiate RNA primer synthesis.

64. Figure 8-27*

65. Initiating replication in eukaryotes
Binding and unwinding
Initiating replication in eukaryotes
Eukaryotic chromosome are replicated exactly once per cell cycle, which is critical for these organisms

66. Pre-replicative complex (pre-RC) formation and activation directs the initiation of replication in eukaryotes
Initiation in eukaryotes requires two distinct steps:
1st step---Replicator selection: the process of identifying sequences for replication initiation (G1 phase), which is mediated by the formation of pre-RCs at the replicator region.

67. Figure 8-30 pre-RC formation

68. 2nd step---Origin activation: pre-RCs are activated by two protein kinases (Cdk and Ddk) that are active only when the cells enter S phase.

69. Figure 8-31: Activation of the pre-RC leads to the assembly of the eukaryotic replication fork.

70. 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 one opportunity for pre-RC activation.

71. Figure 8-32 Effect of Cdk activity on pre-RC formation and activation

72. Figure 8-33 Cell cycle regulation of Cdk activity and pre-RC formatin

73. Finishing replication
CHAPTER 8 The replication of DNA
Finishing replication

74. Finishing replication in bacteria:
Type II topoisomerases separate daughter DNA molecules
Finishing replication
Figure 8-34
Topoisomerase II catalyze the decatenation of replication products.

75. Finishing replication in eukaryotes:
The end replication problem
Telomere & telomerase: a link with cancer and aging
Finishing replication

76. What is the end replication problem?
Lagging strand synthesis is unable to copy the extreme ends of the linear chromosome
Figure 8-34

77. Telomerase is a novel DNA polymerase that does not require an exogenous template

78. How telomerase works? Telomerase extends the protruding 3’ end of the chromosome using its RNA component s as a template. (Figure 8-37)

79. 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.

80. Telomere -binding proteins regulate telomerase activity and telomere length
Figure 8-39: Telomere-binding proteins.

81. 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 telomerase activity.
Figure 8-40: Telomere length regulation by telomere-binding proteins.

82. 重点 Completely understand 三个Animations
CHAPTER 8 The replication of DNA 重点
Completely understand 三个Animations
DNA polymerization (Topics 1 & 2)
DNA replication (Topics 3－5)
Action of Telomerase (Topic 8)

83. The Chemistry of DNA Synthesis: substrate, direction and energy.
CHAPTER 8 The replication of DNA
The Chemistry of DNA Synthesis: substrate, direction and energy.
The Mechanism of DNA Polymerase: 1 polymerization mechanism, 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 the anti-parallel template strands are copied/replicated toward the replication fork. Replisome/protein interaction.

84. 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 cycle (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.

85. CHAPTER 8 The replication of DNA重点
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.

86. CHAPTER 8 The replication of DNA重点
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)

87. Topic 6-7: Initiation of DNA replication
Topic 6-7: Initiation of DNA replication. 重点掌握(1) 概念origin of replication, replicator, initiator (DnaA & ORC) , 图8－23, 26,27; （2）How the eukaryotic chromosomes are ensured to be replicated exactly once per cell cycle? 图30，图32。
注：图26和30把原核和真核细胞一个复制叉的复制起始和延伸整合起来了。

88. Topic 6-7: Initiation of DNA replication
Topic 6-7: Initiation of DNA replication. 重点掌握(1) 概念origin of replication, replicator, initiator (DnaA & ORC) , 图8－23，25, 26; （2）How the eukaryotic chromosomes are ensured to be replicated exactly once per cell cycle? 图30，图32。
注：图26和30把原核和真核细胞一个复制叉的复制起始和延伸整合起来了。