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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.

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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 structure 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 molecular 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 chemistry.

  • The fine art of DNA polymerase

  • The harmonious coordination between many protein factors.

  • The strict control of the initiation and the completeness of termination.


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 understandingand highlight Key points


CHAPTER 8 The replication of DNA

I. Reaction & Catalyst

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

II. Replication at a fork

III. Initiation & Termination


CHAPTER 8 The replication of DNA

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

  • 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


CHAPTER 8 The replication of DNA

The mechanism of DNA Polymerase (Pol)


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).


Distinguishing different dNTPs: kinetic selectivity

Figure 8-3


Distinguishing between rNTP and dNTP by steric exclusion of rNTPs from the active site.

The mechanism of DNA Pol

Figure 8-4


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


Thumb

Fingers

Palm

Figure 8-8


DNA Polymerase-palm domain

Contains two catalytic sites, one for addition of dNTPs and one for removal of the mispaireddNTP.

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)


Figure 8-6

Figure 8-7


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


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.


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).


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 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


Figure 8-10


CHAPTER 8 The replication of DNA

The specialization of DNA polymerases


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


  • 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***


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


Figure 8-17


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


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.


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


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.


CHAPTER 8 The replication of DNA

The replication fork

  • 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 replication fork

Leading strand

Okazaki fragment

Replication fork

Lagging strand

Figure 8-11


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)


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


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


Topoisomerase removes supercoils produced by DNA unwinding at the replication fork

The replication fork

Figure 8-15


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

  • Cooperativebinding

  • Sequence-independent manner

    (electrostatic interactions)

Figure 8-14


CHAPTER 8 The replication of DNA

DNA synthesis at the replication fork

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 P205-206

  • 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


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.


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.


CHAPTER 8 The replication of DNA

The third partfocuses 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


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.


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


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


Replicator sequences include initiator binding sites and easily unwound DNA


CHAPTER 8 The replication of DNA

Binding and Unwinding: origin selection and activation by the initiator protein


  • 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)


Binding and unwinding

Protein-protein and protein-DNA interactions direct the initiation process


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*


Binding and unwinding

Initiating replication in eukaryotes

Eukaryotic chromosome are replicated exactly once per cell cycle, which is critical for these organisms


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.


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 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 one 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


CHAPTER 8 The replication of DNA

Finishing replication


Finishing replication in bacteria:

Type II topoisomerasesseparate daughter 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?

Lagging 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. (Figure 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.


Telomere -binding proteins regulate telomerase activity and telomere length

Figure 8-39: 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.


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)


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.


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.


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.


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)


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。


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。


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