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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Teaching Arrangement
CHAPTER 8 The replication of DNA

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

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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
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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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Figure 8-3
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Distinguish 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
The mechanism of DNA Pol
Schematic of DNA pol bound to a primertemplate
junction
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 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)

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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 (a few50,000).
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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 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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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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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
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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.
• 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 primer-template junction is present.
• Sliding clamps are only removed from the DNA once
  all the associated enzymes complete their
  function.

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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. Note that 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
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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Initiation of DNA replication
Replicator sequences include initiator binding
sites and easily unwound DNA
3/18/05
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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

• DnaA recruits the DNA helicase DnaB and the
  helicase loader DnaC
• DnaB interacts with primase to initiate RNA
  primer synthesis, see replisome part for more
  details.

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Figure 8-26
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Binding and unwinding
Eukaryotic chromosome are replicated exactly once
per cell cycle, which is critical for these
organims
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Pre-replicative complex (pre-RC) formation
directs the initiation of replication in
eukaryotes
Binding and unwinding

• Initiation in eukaryotes requires two distinct
  steps
• 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 8-29 pre-RC formation
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Origin activation pre-RCs are activated by two
protein kinases (Cdk and Ddk) that are active
only when the cells enter S phase.
Figure 8-30 pre-RC activation assembly of the
replication fork in eukaryotes
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Binding and unwinding
Pre-RC formation and activation is 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 8-31 Effect of Cdk activity on pre-RC
formation and activation
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Figure 8-32 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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Type II topoisomerases are required to separate
daughter DNA molecules
Finishing replication
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Lagging strand synthesis is unable to copy the
extreme ends of the linear chromosome
Finishing replication
The End replication problem (Figure 8-34)
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Telomerase is a novel DNA polymerase that does
not require an exogenous template
Finishing replication
How telomerase works? (Figure 8-36 How the end
problem is eventually resolved? (Figure 8-37)
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CHAPTER 8 The replication of DNA
T1The Chemistry of DNA Synthesis T2 The
Mechanism of DNA Polymerase T3 The Replication
Fork (?) T4 The Specialization of DNA
Polymerases (?) T5 DNA Synthesis at the
Replication Fork (?) T6 Initiation of DNA
Replication T7 Binding and Unwinding T8
Finishing Replication
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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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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???????????????????????????