Welcome Each of You to My Molecular Biology Class

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Welcome Each of You to My Molecular Biology Class
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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
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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
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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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• Molecular Biology Course

• CHAPTER 8 The replication of DNA

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• How DNA molecules sustaining the beautiful living
  creatures are propagated from one cell generation
  to the next

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• 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.

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Teaching Arrangement
CHAPTER 8 The replication of DNA

• Watch animation-Understand replication
• Go through some structural tutorial-Experience
  the BEAUTY of the DNA polymerase
• Lecture-comprehensive understanding and highlight
  Key points

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

• DNA synthesis requires deoxynucleoside
  triphosphates and a primertemplate junction
• DNA is synthesized by extending the 3 end of the
  primer
• Hydrolysis of pyrophosphate (PPi) is the driving
  force for DNA synthesis

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Figure 8-3 Substrates required for DNA synthesis
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CHAPTER 8 The replication of DNA
The mechanism of DNA Polymerase (Pol)
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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).
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Distinguishing different dNTPs kinetic
selectivity
Figure 8-3
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Distinguishing between rNTP and dNTP by steric
exclusion of rNTPs from the active site.
The mechanism of DNA Pol
Figure 8-4
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DNA Pol resemble a hand that grips the
primer-template junction
Schematic of DNA pol bound to a primertemplate
junction
The mechanism of DNA Pol
A similar view of the T7 DNA pol bound to DNA
Figure 8-5
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Thumb
Fingers
Palm
Figure 8-8
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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)

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Figure 8-6
Figure 8-7
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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
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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.
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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
primertemplate junction (varying from a few to
gt50,000 nucleotides).
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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.
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Figure 8-9
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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
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Figure 8-10
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CHAPTER 8 The replication of DNA
The specialization of DNA polymerases
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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

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• 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
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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
  primertemplate junction
• Ensures the rapid rebinding of DNA Pol to the
  same primertemplate junction, and thus increases
  the processivity of Pol. p221 for details
• Eukaryotic sliding DNA clamp is PCNA

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Figure 8-17
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Figure 8-19 Sliding DNA clamps are found across
all organism and share a similar structure
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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 primertemplate junction is present.
• Sliding clamps are only removed from the DNA once
  all the associated enzymes complete their
  function.

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Box 8-4 ATP control of Protein Function Loading
a Sliding Clamp
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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.
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CHAPTER 8 The replication of DNA
The replication fork

• The junction between the newly separated template
  strands and the unreplicated duplex DNA

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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
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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)

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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

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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
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Topoisomerase removes supercoils produced by DNA
unwinding at the replication fork
The replication fork
Figure 8-15
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DNA helicases unwind the double helix in advance
of the replication fork
The replication fork
Figure 8-13
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Single-stranded binding proteins (SSBs) stabilize
single-stranded DNA
The replication fork

• Cooperative binding
• Sequence-independent manner
• (electrostatic interactions)

Figure 8-14
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CHAPTER 8 The replication of DNA
DNA synthesis at the replication fork
The leading strand and lagging strand are
synthesized simultaneously.
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• 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.

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Figure 8-20 The composition of the DNA Pol III
holoenzyme
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Figure 8-21 Trombone model
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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.

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

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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
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Replicator sequences include initiator binding
sites and easily unwound DNA
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CHAPTER 8 The replication of DNA
Binding and Unwinding origin selection and
activation by the initiator protein
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• 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)

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Binding and unwinding
Protein-protein and protein-DNA interactions
direct the initiation process
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Initiating replication in bacteria

• DnaA recruits the DNA helicase DnaB and the
  helicase loader DnaC
• DnaB interacts with primase to initiate RNA
  primer synthesis.

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Figure 8-27
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Binding and unwinding
Initiating replication in eukaryotes Eukaryotic
chromosome are replicated exactly once per cell
cycle, which is critical for these organisms
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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.
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Figure 8-30 pre-RC formation
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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.
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Figure 8-31 Activation of the pre-RC leads to
the assembly of the eukaryotic replication fork.
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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.
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Figure 8-32 Effect of Cdk activity on pre-RC
formation and activation
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Figure 8-33 Cell cycle regulation of Cdk activity
and pre-RC formatin
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CHAPTER 8 The replication of DNA
Finishing replication
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Finishing replication in bacteria Type II
topoisomerases separate daughter DNA molecules
Finishing replication
Figure 8-34 Topoisomerase II catalyze the
decatenation of replication products.
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• Finishing replication in eukaryotes
• The end replication problem
• Telomere telomerase a link with cancer and
  aging

Finishing replication
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What is the end replication problem? Lagging
strand synthesis is unable to copy the extreme
ends of the linear chromosome
Figure 8-34
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Telomerase is a novel DNA polymerase that does
not require an exogenous template
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How telomerase works?Telomerase extends the
protruding 3 end of the chromosome using its RNA
component s as a template. (Figure 8-37)
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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.
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Telomere -binding proteins regulate telomerase
activity and telomere length
Figure 8-39 Telomere-binding proteins.
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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.
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CHAPTER 8 The replication of DNA
??

• Completely understand ??Animations
• DNA polymerization (Topics 1 2)
• DNA replication (Topics 3-5)
• Action of Telomerase (Topic 8)

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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.

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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.

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CHAPTER 8 The replication of DNA
??

• 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.

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CHAPTER 8 The replication of DNA
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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)
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Topic 6-7 Initiation of DNA replication. ????(1)
??origin of replication, replicator, initiator
(DnaA ORC) , ?8-23, 26,27 (2)How the
eukaryotic chromosomes are ensured to be
replicated exactly once per cell cycle?
?30,?32? ??26?30???????????????????????????
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Topic 6-7 Initiation of DNA replication. ????(1)
??origin of replication, replicator, initiator
(DnaA ORC) , ?8-23,25, 26 (2)How the
eukaryotic chromosomes are ensured to be
replicated exactly once per cell cycle?
?30,?32? ??26?30???????????????????????????