Chapter 11 Lecture Outline Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
INTRODUCTION
• DNA replication is the process by which the
genetic material is copied
– The original DNA strands are used as
templates for the synthesis of new strands
• It occurs very quickly, very accurately and
at the appropriate time in the life of the cell
– This chapter examines how!
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11-2
11.1 STRUCTURAL OVERVIEW OF
DNA REPLICATION
• DNA replication relies on the complementarity of
DNA strands
– The AT/GC rule or Chargaffʼs rule
• The process can be summarized as such
– The two complementary DNA strands come apart
– Each serves as a template strand for the synthesis of
new complementary DNA strands
– The two newly-made DNA strands = daughter strands
– The two original DNA strands = parental strands
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11-3
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5′
3′
5′
C G
A T
C G
A T
C G
C G
A pairs with T and G pairs with C
during synthesis of a new strand
Replication"
fork
A T
A
T
C
3′
G
A
G
A T
T A
C G
T A
Leading"
strand
G C
C G
T
A
3′
C
Identical
base sequences
T A
Lagging"
A T
strand
T A
C G
Incoming"
T A
nucleotides
Original"
Newly"
(template)" synthesized"
strand
daughter strand
(a) The mechanism of DNA replication
3′
3′
C G
A T
C G
T A
GC
GC
T A
A T
C G
A T
GC
GC
T A
C
G
C
5′
G
A
5′
Original"
(template)"
strand
3′
Figure 11.1
5′
T
C
G
T A
3′
C G
A T
C G
T A
GC
GC
T A
A T
C G
A T
GC
GC
T A
3′
C G
A T
C G
T A
GC
GC
T A
A T
C G
A T
GC
GC
T A
5′
3′
5′
(b) The products of replication
11-4
Experiment 11A: Which Model of
DNA Replication is Correct?
• In the late 1950s, three different mechanisms
were proposed for the replication of DNA
– Conservative model
• Both parental strands stay together after DNA replication
– Semiconservative model
• The double-stranded DNA contains one parental and one
daughter strand following replication
– Dispersive model
• Parental and daughter DNA are interspersed in both strands
following replication
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11-5
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Original"
double"
helix
First round of"
replication
Second round"
of replication
(a) Conservative model
Figure 11.2
(b) Semiconservative model
(c) Dispersive model
11-6
• In 1958, Matthew Meselson and Franklin Stahl
devised a method to investigate these models
– They found a way to experimentally distinguish
between daughter and parental strands
• Their experiment can be summarized as such
– Grow E. coli in the presence of 15N (a heavy isotope of
Nitrogen) for many generations
• The population of cells had heavy-labeled DNA
– Switch E. coli to medium containing only 14N (a light
isotope of Nitrogen)
– Collect sample of cells after various times
– Analyze the density of the DNA by centrifugation using
a CsCl gradient
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11-7
The Hypothesis
– Based on Watsonʼs and Crickʼs ideas, the
hypothesis was that DNA replication is
semiconservative.
Testing the Hypothesis
n
Refer to Figure 11.3
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11-8
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Conceptual level
Experimental level
1. Add an excess of 14N-containing"
compounds to the bacterial cells so"
all of the newly made DNA will contain"
14N.
Generation
0
14N"
solution
Add 14N
1
Suspension of"
bacterial"
cells labeled"
with 15N
2. Incubate the cells for various lengths of"
time. Note: The 15N-labeled DNA is"
shown in purple and the 14N-labeled"
DNA is shown in blue.
3. Lyse the cells by the addition of"
lysozyme and detergent, which"
disrupt the bacterial cell wall and"
cell membrane, respectively.
2
37°C
Up to 4 generations
Lyse"
cells
DNA
Cell wall
4. Load a sample of the lysate onto a CsCl"
gradient. (Note: The average density of"
DNA is around 1.7 g/cm3, which is well"
isolated from other cellular"
macromolecules.)
Lysate
CsCl"
gradient
Cell membrane
Density centrifugation
5. Centrifuge the gradients until the DNA"
molecules reach their equilibrium"
densities.
Light"
DNA
Half-heavy"
DNA
6. DNA within the gradient can be"
observed under a UV light.
Figure 11.3
UV"
light
Heavy"
DNA
(Result shown here is"
after 2 generations.)
11-9
Interpreting the Data
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Generations After 14N Addition
4.1
3.0
2.5
1.9
1.5
1.1
1.0
0.7
0.3
Light
Half-heavy
Heavy
*Data from: Meselson, M. and Stahl, F.W. (1958) The Replication of DNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 44: 671−682
After one generation,
DNA is “half-heavy”
After ~ two generations, DNA is of
two types: “light” and “half-heavy”
This is consistent with both semiconservative and dispersive models
This is consistent with only
the semi-conservative model
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11-11
11.2 BACTERIAL
DNA REPLICATION
• Figure 11.4 presents an overview of the process
of bacterial chromosomal replication
– DNA synthesis begins at a site termed the origin of
replication
• Each bacterial chromosome has only one origin of replication
– Synthesis of DNA proceeds bidirectionally around the
bacterial chromosome
– The two replication forks eventually meet at the
opposite side of the bacterial chromosome
• This ends replication
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11-12
Figure 11.4
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Origin of"
replication
Replication"
forks
Site where"
replication"
ends
(a) Bacterial chromosome replication
Replication"
fork
Replication"
fork
From Cold Spring Harbor Symposia of Quantitative Biology, 28, p. 43 (1963).
Copyright holder is Cold Spring Habour Laboratory Press.
0.25 μm
(b) Autoradiograph of an E. coli chromosome in the act of replication
11-13
Initiation of Replication
n
The origin of replication in E. coli is termed oriC
n
n
origin of Chromosomal replication
Three types of DNA sequences in oriC are
functionally significant
n
AT-rich region
DnaA boxes
GATC methylation sites
n
Refer to Figure 11.5
n
n
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11-14
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E. coli!
chromosome
oriC
AT-rich region
5′–GGA T CC T GGGT A T T A A A A AGA AGA T C T A T T T A T T T AGAGA T C T G T T C T A T
CC T AGGACCC AT A AT T T T T C T T C T AGAT A A A T A A AT C T C T AGAC A AGAT A
1
50
DnaA box
T G T GA T C T C T T A T T AGGA T CGC AC T GCCC T GT GGA T A ACA AGGA T CGGC T
AC AC T AGAGA A T A AT CC T AGCGT GACGGGAC ACC T AT T GT T CC T AGCCGA
51
100
DnaA box
T T T A AGA T CA ACA ACC T GGA A AGGA T C A T T A AC T G T GA A T GA T CGG T GA T
A A AT T C T AGT T GT T GGACC T T T CC T AGT A AT T GAC AC T T AC T AGCC AC T A
101
150
DnaA box
CC T GGACCGT A T A AGC T GGGA T C AGA A T GAGGGT T A T ACA CAGC T C A A A A
GGACC T GGC A T AT T CGACCC T AGT C T T AC T CCC A AT AT GT GT CGAGT T T T
151
200
DnaA box
AC T GA AC AA CGGT T GT T C T T T GGA T A AC T ACCGGT T GA T CCA AGC T T CC T
T GAC T T GT T GCC A AC A AGA A ACC T AT T GAT GGCC A AC T AGGT T CGA AGGA
201
250
DnaA box
Figure 11.5
GAC AGAGT T A T CCA CAGT AGA T CGC –3′
C T GT C T C A AT AGGT GT C AT C T AGC G
251
275
11-15
Figure 11.6
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3′
5′
5′"
3′"
AT-rich region
n
DnaA boxes
Other proteins such as HU and
IHF also bind.
n
This causes the DNA to wrap
around the DnaA proteins
causing the separation of the
AT-rich region
AT- "
rich"
region
5′"
3′"
n
DNA replication is initiated by the binding of
DnaA proteins to the DnaA box sequences
n
This binding stimulates the cooperative
binding of additional ATP-bound DnaA
proteins to form a large complex
DnaA protein
3′
5′
DNA helicase (DnaB protein) binds to the"
origin. DnaC protein (not shown) assists"
this process.
11-16
Figure 11.6
Composed of six subunits
Travels along the DNA in
the 5ʼ to 3ʼ direction
Uses energy from ATP
Helicase
5′"
3′"
3′
5′
DNA helicase separates the DNA in both"
directions, creating 2 replication forks.
3′
5′"
3′"
Fork
Fork
Bidirectional replication
is initiated
5′
11-17
n
n
DNA helicase separates the two DNA strands by
breaking the hydrogen bonds between them
This generates positive supercoiling ahead of each
replication fork
n
n
n
DNA gyrase travels ahead of the helicase and alleviates
these supercoils
Single-strand binding proteins bind to the separated
DNA strands to keep them apart
Then short (10 to 12 nucleotides) RNA primers are
synthesized by DNA primase
n
These short RNA strands start, or prime, DNA synthesis
n
n
The leading strand has a single primer, the lagging strand needs
multiple primers
They are eventually removed and replaced with DNA
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11-18
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Functions of key proteins involved with DNA replication
• DNA helicase breaks the hydrogen"
bonds between the DNA strands.
Origin
Single-strand"
binding protein
• Topoisomerase alleviates positive"
supercoiling.
DNA helicase
• Single-strand binding proteins keep"
the parental strands apart.
Topoisomerase
Leading"
strand
• Primase synthesizes an RNA"
primer.
• DNA polymerase III synthesizes a"
daughter strand of DNA.
• DNA polymerase I excises the"
RNA primers and fills in with"
DNA (not shown).
3′
5′
DNA polymerase III
RNA"
primer
RNA primer
DNA polymerase III
Replication fork
Okazaki fragment
Primase
5′
3′
Lagging strand
Parental DNA
DNA"
ligase
3′
5′
• DNA ligase covalently links the"
Okazaki fragments together.
Direction of fork movement
Figure 11.7
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Linked Okazaki"
fragments
11-19
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11-20
DNA Polymerases
n
n
DNA polymerases are the enzymes that catalyze
the attachment of nucleotides to synthesize a new
DNA strand
In E. coli there are five proteins with polymerase
activity
n
DNA pol I, II, III, IV and V
n
DNA pol I and III
n
n
Normal replication
DNA pol II, IV and V
n
DNA repair and replication of damaged DNA
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11-21
DNA Polymerases
n
DNA pol I
n
n
n
Composed of a single polypeptide
Removes the RNA primers and replaces them with DNA
DNA pol III
n
n
Responsible for most of the DNA replication
Composed of 10 different subunits (Table 11.2)
n
n
n
The α subunit catalyzes bond formation between adjacent
nucleotides (DNA synthesis)
The other 9 fulfill other functions
The complex of all 10 subunits is referred to as the DNA
pol III holoenzyme
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11-22
11-23
n
Bacterial DNA polymerases may vary in their
subunit composition
n
However, they all have the same type of catalytic subunit
Structure resembles a
human right hand
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DNA polymerase"
catalytic site
Template DNA is
threaded through the
palm;
Thumb
Thumb and fingers
wrapped around the DNA
3′ exonuclease"
site
3′
5′
Fingers
Figure 11.8
3′
5′
Palm
Template"
strand
Incoming"
deoxyribonucleoside triphosphates"
(dNTPs)
(a) Schematic side view of DNA polymerase III
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11-24
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DNA polymerases cannot
initiate DNA synthesis
Unable to"
covalently link"
the 2 individual"
nucleotides together
Problem is overcome by
the RNA primers
synthesized by primase
Able to"
covalently link"
together
(a)
DNA polymerases can
attach nucleotides only in
the 5ʼ to 3ʼ direction
Cannot link nucleotides "
in this direction
5′
3′
3′
5′
5′
3′
3′
Problem is overcome by
synthesizing the new
strands both toward, and
away from, the replication
fork
Can link nucleotides"
in this direction
5′
(b)
Figure 11.9
Unusual features of DNA polymerase function
11-25
n
The two new daughter strands are synthesized in
different ways
n Leading strand
n
n
n
One RNA primer is made at the origin
DNA pol III attaches nucleotides in a 5ʼ to 3ʼ direction
as it slides toward the opening of the replication fork
Lagging strand
n
Synthesis is also in the 5ʼ to 3ʼ direction
n
n
n
However it occurs away from the replication fork
Many RNA primers are required
DNA pol III uses the RNA primers to synthesize small
DNA fragments (1000 to 2000 nucleotides each)
n
These are termed Okazaki fragments after their discoverers
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11-26
n
DNA pol I removes the RNA primers and fills the
resulting gap with DNA
n
n
n
It uses a 5ʼ to 3ʼ exonuclease activity to digest the RNA
and 5ʼ to 3ʼ polymerase activity to replace it with DNA
After the gap is filled a covalent bond is still
missing
DNA ligase catalyzes the formation of a
phosphodiester bond
n
Thereby connecting the DNA fragments
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11-27
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DNA strands separate at"
origin, creating 2 replication"
forks.
Origin of replication
Replication"
forks
Primers are needed to initiate DNA synthesis."
The synthesis of the leading strand occurs in"
the same direction as the movement of the"
replication fork. The first Okazaki"
Leading"
fragment of the lagging strand is"
strand
made in the opposite direction.
Primer
3′
5′
5′
3′
Direction of"
replication fork
3′
5′
Primer
The leading strand elongates,"
and a second Okazaki fragment"
is made.
3′
5′
First Okazaki fragment"
of the lagging strand
3′
5′
5′
3′
3′
5′
Second"
Okazaki"
fragment
First"
Okazaki"
fragment
3′
5′
The leading strand continues to"
elongate. A third Okazaki"
fragment is made, and the first"
and second are connected"
together.
3′
5′
3′
3′
Third"
Okazaki"
fragment
5′
Figure 11.10
First and second Okazaki"
fragments have been"
connected to each other.
5′
3′
5′
11-28
The synthesis of leading and lagging strands from a single origin of replication
Origin of replication
Leading!
strand
Lagging!
strand
5′
3′
3′
Replication fork
Lagging!
strand
Replication fork
5′
Leading!
strand
Figure 11.11
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11-29
The Reaction of DNA Polymerase
n
DNA polymerases catalyzes the formation of a
covalent (ester) bond between the
n
n
n
Innermost phosphate group of the incoming
deoxyribonucleoside triphosphate
n AND
3ʼ-OH of the sugar of the previous deoxynucleotide
In the process, the last two phosphates of the
incoming nucleotide are released
n
n
In the form of pyrophosphate (PPi)
Refer to figure 11.12
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11-30
Figure 11.12
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
New DNA strand
Original DNA strand
5′ end
3′ end
O
O
P
O
5′
CH2
O–
Innermost
phosphate
O
3′
O
Cytosine
Guanine
O–
O
H 2C
O
O
O
P
O
CH2
O
Guanine
Cytosine
3′
O–
O
H 2C
O
OH
O
O
O
–
O
P
O
O
–
O
P
5′
O CH 2
P
O
–
Thymin
e
Adenine
3′
O
O
OH
5′
CH2
O–
O
H 2C
5′
P
O
O–" P
O–
O
O
P
P
Guanine
O–
O
H 2C
O
+
O–
Pyrophosphate (PPi)
P
O
O
O
CH2
O
Guanine
Cytosine
O–
O
H 2C
O
P
O
O
O
CH2
O–
O–
O
3′
O
O
O
O
3′ end
Cytosine
O–
New"
ester"
bond
P
5′ end
O
O
O
O–
O
5′ end
P
P
O
O
–
Incoming nucleotide"
(a deoxyribonucleoside"
triphosphate)
O
O
O
O–
O
P
O
Thymine
3′
OH
3′ end
Adenine
O–
O
O
H 2C
5′
P
O
O
5′ end
11-31
DNA Polymerase III is a Processive Enzyme
n
n
DNA polymerase III remains attached to the
template as it is synthesizing the daughter strand
This processive feature is due to several different
subunits in the DNA pol III holoenzyme
n
β subunit forms a dimer in the shape of a ring around
template DNA
n
n
n
n
It is termed the clamp protein
Once bound, the β subunits can freely slide along dsDNA
Promotes association of holoenzyme with DNA
γ complex catalyzes β dimer clamping to the DNA
n
n
It is termed the clamp-loader complex
Includes γ , δ, δʼ, χ and ψ subunits
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11-32
DNA Polymerase III is a
Processive Enzyme
n
The effect of processivity is quite remarkable
n
In the absence of the β subunit
n
n
n
DNA pol III falls off the DNA template after about 10
nucleotides have been polymerized
Its rate is ~ 20 nucleotides per second
In the presence of the β subunit
n
n
DNA pol III stays on the DNA template long enough to
polymerize up to 500,000 nucleotides
Its rate is ~ 750 nucleotides per second
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11-33
Termination of Replication
n
On the opposite side of the chromosome to oriC is
a pair of termination sequences called ter
sequences
n
These are designated T1 and T2
n
n
The protein tus (termination utilization substance)
binds to the ter sequences
n
n
T1 stops counterclockwise forks, T2 stops clockwise forks
tus bound to the ter sequences stops the movement of
the replication forks
Refer to Figure 11.13
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11-34
Figure 11.13
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
oriC!
Prevents advancement of fork moving
left-to-right (clockwise fork)
Tus
(T1)
ter
Fork
Fork
Fork
Fork
ter
(T2)
oriC
Tus
Prevents advancement of fork moving right-to-left
(counterclockwise fork)
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11-35
Termination of Replication
n
n
n
DNA replication ends when oppositely advancing
forks meet (usually at T1 or T2)
Finally DNA ligase covalently links the two daughter
strands
DNA replication often results in two intertwined
molecules
n
n
Intertwined circular molecules are termed catenanes
These are separated by the action of topoisomerase
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11-36
Figure 11.14
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Catenanes
Replication
Catalyzed by
DNA topoisomerase
Decatenation via"
topoisomerase
11-37
DNA Replication Complexes
n
DNA helicase and primase are physically bound to
each other to form a complex called the primosome
n
n
n
This complex leads the way at the replication fork
The primosome is physically associated with two
DNA polymerase holoenzymes to form the replisome
Figure 11.15 provides a three-dimensional view of
DNA replication
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11-38
Figure 11.15
11-39
DNA Replication Complexes
n
Two DNA pol III proteins act in concert to replicate
both the leading and lagging strands
n
n
The two proteins form a dimeric DNA polymerase that
moves as a unit toward the replication fork
DNA polymerases can only synthesize DNA in the
5ʼ to 3ʼ direction
n
So synthesis of the leading strand is continuous
n
And that of the lagging strand is discontinuous
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11-40
DNA Replication Complexes
n
Lagging strand synthesis is summarized as such:
n
The lagging strand is looped
n
n
n
Upon completion of an Okazaki fragment, the enzyme
releases the lagging template strand
n
n
n
This allows the attached DNA polymerase to synthesize the
Okazaki fragments in the normal 5ʼ to 3ʼ direction
The polymerase synthesizing the lagging strand is also moving
toward the replication fork
The clamp loader complex then reloads the polymerase at the
next RNA primer
Another loop is then formed
This processed is repeated over and over again
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11-41
Proofreading Mechanisms
n
DNA replication exhibits a high degree of fidelity
n
Mistakes during the process are extremely rare
n
n
DNA pol III makes only one mistake per 108 bases made
There are several reasons why fidelity is high
n
n
n
1. Instability of mismatched pairs
2. Configuration of the DNA polymerase active site
3. Proofreading function of DNA polymerase
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11-42
Proofreading Mechanisms
n
1. Instability of mismatched pairs
n
n
Complementary base pairs have much higher stability
than mismatched pairs
Stability of base pairs only accounts for part of the fidelity
n
n
Error rate for mismatched base pairs is 1 per 1,000 nucleotides
2. Configuration of the DNA polymerase active site
n
n
DNA polymerase is unlikely to catalyze bond formation
between mismatched pairs
This induced-fit phenomenon decreases the error rate to
a range of 1 in 100,000 to 1 million
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11-43
Proofreading Mechanisms
n
3. Proofreading function of DNA polymerase
n
n
n
DNA polymerases can identify a mismatched nucleotide
and remove it from the daughter strand
The enzyme uses a 3ʼ to 5ʼ exonuclease activity to
digest the newly made strand until the mismatched
nucleotide is removed
DNA synthesis then resumes in the 5ʼ to 3ʼ direction
n
Refer to figure 11.16
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11-44
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Mismatch causes DNA polymerase to pause,"
leaving mismatched nucleotide near the 3′ end.
3′ exonuclease"
site
T
3′
Template"
strand
5′
C
5′
Base pair "
mismatch "
near the "
3′ end
3′
The 3′ end enters the"
exonuclease site.
Site where DNA
backbone is cut
3′
3′
5′
5′
At the 3′ exonuclease site,"
the strand is digested in"
the 3′ to 5′ direction until the"
incorrect nucleotide is"
removed.
Incorrect"
nucleotide"
removed
3′
5′
5′
Figure 11.16 A schematic drawing of proofreading
11-45
Bacterial DNA Replication is
Coordinated with Cell Division
n
Bacterial cells can divide into two daughter cells at
an amazing rate
n
n
n
E. coli à 20 to 30 minutes
It is critical that DNA replication take place only when a
cell is about to divide
Bacterial cells regulate the DNA replication process
by controlling the initiation of replication at oriC
n
E. coli does this via two different mechanisms
n
Refer to Figures 11.17 and 11.18
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-46
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Able to start DNA "
replication due to"
a sufficient amount"
of DnaA protein:
DnaA "
proteins
DnaA boxes
DnaA hydrolyzes ATP after
initiation of replication. DnaAADP has a lower affinity for
DnaA boxes.
DNA
After DNA replication, "
an insufficient"
amount of DnaA"
protein is available," DNA
and some may be"
degraded or stuck"
to the membrane:
After replication, the number of
DnaA boxes doubles and there
is insufficient DnaA to bind
them all.
DnaA "
boxes"
DnaA proteins
Figure 11.17
The amount of DnaA protein provides a way to regulate DNA replication
11-47
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
HNH
N
N
Adenine
N
Recognizes the
5ʼ – GATC – 3ʼ
sequence and
attaches a
methyl group
onto the
adenine
N
Dam"
methylase
DNA adenine
methyltransferase
HN CH3
N
N
N
N
N 6-methyladenine"
(Ame)
(a) Structure of adenine "
and methyladenine"
Figure 11.18 Methylation of GATC sites in oriC
11-48
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5ʹ′
Before replication, GATC sites are
methylated on both strands
3ʹ′
G
Ame T
This full methylation facilitates
initiation of DNA replication
T Ame
C
3ʹ′
Following replication, GATC
sites are not methylated on the
daughter strands
5ʹ′
3ʹ′
G
This state of half-methylation
inhibits initiation of replication
Several minutes will pass
before Dam methylase will
methylate the GATC sites in
the daughter strands
3ʹ′
Figure 11.18 Methylation of GATC sites in oriC
C
G
5ʹ′
5ʹ′
3ʹ′
C
G
C
Ame T
A
T
T
A
T Ame
C
G
C
5ʹ′
G
3ʹ′
5ʹ′
(b) Results immediately after "
DNA replication
11-49
Experiment 11B: DNA Replication
Can Be Studied In Vitro
• The in vitro study of DNA replication was
pioneered by Arthur Kornberg in the 1950s
– He received a Nobel Prize for his efforts in 1959
• Kornberg hypothesized that deoxyribonucleoside
triphosphates are the precursors of DNA
synthesis
• He also knew that these nucleotides are soluble in
an acidic solution while long DNA strands are not
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-50
Experiment 11B: DNA Replication
Can Be Studied In Vitro
• In this experiment, Kornberg mixed the following
– An extract of proteins from E. coli
– Template DNA
– Radiolabeled nucleotides
• These were incubated for sufficient time to allow
the synthesis of new DNA strands
– Addition of acid will precipitate these DNA strands
– Centrifugation will separate them from the radioactive
nucleotides
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-51
The Hypothesis
– DNA synthesis can occur in vitro if all the
necessary components are present
Testing the Hypothesis
n
Refer to Figure 11.19
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-52
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Conceptual level
Experimental level
1.Mix together the extract of E. coli
proteins, template DNA that is not
radiolabeled, and 32P-radiolabeled
deoxyribonucleoside triphosphates. This
is expected to be a complete system that
contains everything necessary for DNA
synthesis. As a control, a second sample
is made in which the template DNA was
omitted from the mixture.
32P-labeled
deoxyribonucleoside
triphosphates (dNTPs)
E. coli
proteins
Template
DNA
E. coli
proteins
Template
DNA
32P
32P
G
T
32P
32P
Control
32P
Complete
system
A
C
C
32P-radiolabeled
deoxyribonucleoside
triphosphates
2.Incubate the mixture for 30 minutes at
37°C.
37°C
3.Add perchloric acid to precipitate DNA.
(It does not precipitate free nucleotides.)
4.Centrifuge the tube.
Note: The radiolabeled deoxyribonucleoside
triphosphates that have not
been incorporated into DNA will remain
in the supernatant.
37°C
Add
perchloric
acid.
Free labeled
nucleotides
32P
32P
A
Supernatant
Pellets
32P
32P
32P
G
32P
A
C
C
32P
32P
T
G
5.Collect the pellet, which contains
precipitated DNA and proteins. (The
control pellet is not expected to
contain DNA.)
32
A
32P
32P
32P
G
C
C
32P
P
32P
6.Count the amount of radioactivity in
the pellet using a scintillation counter.
(See the Appendix.)
Figure 11.19
32
32
P
P
C GA T T T C
GC T A A A G
DNA with 32P-labeled
nucleotides in new strand
11-53
Interpreting the Data
E. coli proteins + nonlabeled
template DNA + radiolabled
nucleotides
= Radiolabeled product
n
Taken together, these results indicate that
this technique can be used to measure
the synthesis of DNA in vitro
Expected result because
the template is
necessary for replication
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-55
11.3 EUKARYOTIC
DNA REPLICATION
• Eukaryotic DNA replication is not as well
understood as bacterial replication
– The two processes do have extensive similarities,
• The types of bacterial enzymes described in Table 11.1 have
also been found in eukaryotes
– Nevertheless, DNA replication in eukaryotes is more
complex
• Large linear chromosomes
• Chromatin is tightly packed within nucleosomes
• More complicated cell cycle regulation
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-62
Multiple Origins of Replication
n
Eukaryotes have long linear chromosomes
n
They therefore require multiple origins of replication
n
n
In 1968, Huberman and Riggs provided evidence
for multiple origins of replication
n
n
To ensure that the DNA can be replicated in a reasonable amount
of time
Refer to Figure 11.21
DNA replication proceeds bidirectionally from many
origins of replication
n
Refer to Figure 11.22
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-63
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
100 μm
Radiolabeled segments
© This article was published in Journal of Molecular Biology. Mar 14;32(2). Huberman JA. Riggs AD. “Links On the mechanism of DNA replication in mammalian chromosomes." 327-41. Copyright Elsevier, 1968. Reprinted with permission.
Figure 11.21
11-64
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Chromosome
Sister chromatids
Origin
Origin
Origin
Centromere
Origin
Origin
Figure 11.22
Before S phase
During S phase
End of S phase
11-65
Multiple Origins of Replication
n
The origins of replication found in eukaryotes have
some similarities to those of bacteria
n
Origins of replication in Saccharomyces cerevisiae are
termed ARS elements (Autonomously Replicating
Sequence)
n
n
n
They are about 50 bp in length
They have a high percentage of A and T
ARS consensus sequence (ACS)
n
ATTTAT(A or G)TTTA
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-66
Multiple Origins of Replication
n
Replication begins with assembly of the
prereplication complex (preRC)
n
n
n
Consists of at least 14 different proteins
An important part of the preRC is the Origin
recognition complex (ORC)
n A six-subunit complex that acts as the initiator of
eukaryotic DNA replication
Other preRC proteins include MCM Helicase
n
Binding of MCM completes DNA replication licensing
n
n
The origin becomes capable of initiating DNA synthesis
Binding of at least 22 additional proteins is required to
initiate synthesis during S phase
11-67
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Eukaryotes Contain Several
Different DNA Polymerases
n
Mammalian cells contain well over a dozen different
DNA polymerases
n
n
Refer to Table 11.4
Four: alpha (α), delta (δ), epsilon (ε) and gamma (γ)
have the primary function of replicating DNA
n
n
α, δ and ε ◊ Nuclear DNA
γ à Mitochondrial DNA
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-68
*The designations are those of mammalian enzymes."
†Many DNA polymerases have dual functions. For example, DNA polymerases α, δ, and ε are involved in the replication of normal DNA and
also play a role in DNA repair. In cells of the immune system, certain genes that encode antibodies (i.e., immunoglobulin genes) undergo a"
phenomenon known as hypermutation. This increases the variation in the kinds of antibodies the cells can make. Certain polymerases in this
list, such as η, may play a role in hypermutation of immunoglobulin genes. DNA polymerase σ may play a role in sister chromatid cohesion, a"
topic discussed in Chapter 10 ."
11-69
n
DNA pol α is the only polymerase to associate with
primase
n
The DNA pol α/primase complex synthesizes a short
RNA-DNA hybrid primer
n
n
n
10 RNA nucleotides followed by 20 to 30 DNA nucleotides
This hybrid primer is used by DNA pol ε or δ for the
processive elongation of the leading and lagging strands,
respectively
The exchange of DNA pol α for ε or δ is required for
elongation of the leading and lagging strands.
n This is called a polymerase switch
n
It occurs only after the RNA-DNA hybrid is made
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-70
n
DNA polymerases also play a role in DNA
repair
n
n
DNA pol β is not involved in DNA replication
It plays a role in base-excision repair
n
n
Removal of incorrect bases from damaged DNA
Recently, more DNA polymerases have been
identified
n
Lesion-replicating polymerases
n
n
Involved in the replication of damaged DNA
They can synthesize a complementary strand over the
abnormal region
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-71
n
Flap Endonuclease Removes RNA Primers
n
Polymerase δ runs into primer of adjacent
Okazaki fragment
n
n
n
n
Pushes portion of primer into short flap
Flap endonuclease removes the primer
Long flaps are removed by Dna2 nuclease/helicase
Refer to figure 11.23
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-72
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5′
3′
3′
5′
DNA polymerase δ elongates the"
left Okazaki fragment and causes"
a short flap to occur on the right"
Okazaki fragment.
Flap
5′
3′
3′
5′
Flap endonuclease"
removes the flap.
5′
3′
3′
5′
DNA polymerase δ"
continues to elongate and"
causes a second flap.
5′
3′
3′
5′
Flap endonuclease"
removes the flap.
5′
3′
3′
5′
Process continues until the"
entire RNA primer is removed."
DNA ligase seals the two"
fragments together.
Figure 11.23
5′
3′
3′
5′
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-73
Telomeres and DNA Replication
n
n
Linear eukaryotic chromosomes have telomeres at
both ends
The term telomere refers to the complex of
telomeric DNA sequences and bound proteins
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-74
n
Telomeric sequences consist of
n
n
Moderately repetitive tandem arrays
3ʼ overhang that is 12-16 nucleotides long
Figure 11.24
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Telomeric repeat sequences
3′"
T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG
A A TCCCA A TCCCAA TCCCA A TCCCAA TCCCAA TCCCA A TCCCAA T
Overhang
5′
n
Telomeric sequences typically consist of
n
n
n
Several guanine nucleotides
Many thymine nucleotides
Refer to Table 11.5
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
11-75
11-76
n
DNA polymerases possess two unusual features
n
n
n
1. They synthesize DNA only in the 5ʼ to 3ʼ direction
2. They cannot initiate DNA synthesis
These two features pose a problem at the 3ʼ ends
of linear chromosomes-the end of the strand cannot
be replicated!
Figure 11.25
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA polymerase cannot link"
these two nucleotides together"
without a primer.
3′
No place"
for a"
primer
5′
11-77
n
Therefore if this problem is not solved
n
n
n
n
The linear chromosome becomes progressively shorter
with each round of DNA replication
Indeed, the cell solves this problem by adding DNA
sequences to the ends of telomeres
This requires a specialized mechanism catalyzed
by the enzyme telomerase
Telomerase contains protein and RNA
n
The RNA is complementary to the DNA sequence found
in the telomeric repeat
n
n
This allows the telomerase to bind to the 3ʼ overhang
The lengthening mechanism is outlined in Figure 11.26
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11-78
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Telomere
5′
Telomerase reverse
transcriptase (TERT)
activity
3′
3′
5′
Eukaryotic"
chromosome
Repeat unit
T T A G G GT T A GG G T T A G GG T TA G G G 3
Step 1 = Binding
C CC A A U C C C
A A T C C CAA T
Telomerase synthesizes"
a 6-nucleotide repeat.
The bindingpolymerizationtranslocation cycle can
occurs many times
This greatly lengthens
one of the strands
RNA
3′
5′
Telomerase
G
G
T T A G G GT T A GG G T T A G GG T T A G G GT T A G
A A T C C CAA T
Step 2 = Polymerization
C CC A A U C C C
Telomerase moves 6"
nucleotides to the right and"
begins to make another repeat.
T
T
T T A G G GT T A GG G T T A G GG T T A G G GT T A G G G
A A T C C CAA T
C CC A A U C C C
The complementary"
strand is made by primase,"
DNA polymerase, and ligase.
T T A G G G T T A G G G T T A G G G T T A G G G T T A G G G T T A G G G 3′
A A T C C CAA T C CC A A T C C C A AT C C C A AU C CC A A U
Figure 11.26
Step 3 = Translocation
The end is now
lengthened
RNA primer
RNA primer
11-79