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
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
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
Process
Initiation &
Termination
CHAPTER 8 The replication of DNA
Reaction
& Catalyst
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
CHAPTER 8 The replication 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
The mechanism of DNA
Polymerase (Pol)
CHAPTER 8 The replication of DNA
DNA Pol use a single active
site to catalyze DNA synthesis
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).
Th
e me
ch
an
ism
of
D
NA
P
ol
Figure 8-3
Distinguishing different dNTPs,kinetic selectivity
Distinguishing between rNTP and
dNTP by steric exclusion of rNTPs
from the active site.
Th
e me
ch
an
ism
of
D
NA
P
ol
Figure 8-4
DNA Pol resemble a hand that
grips the primer-template junction
Th
e m
ec
ha
nis
m
of
D
NA
P
ol Figure 8-5
Schematic of DNA pol
bound to a
primer:template junction
A similar view of
the T7 DNA pol
bound to DNA
Figure 8-8
Thumb
Fingers
Palm
1,Contains two catalytic sites,one for addition of
dNTPs and one for removal of the mispaired
dNTP,
2,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,
3,Exonuclease site/proof reading site (See
proofreading)
DNA Polymerase-palm domain
Figure 8-6 Figure 8-7
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-finger 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 Polymerase-thumb domain
DNA Pol are processive enzymes T
he
me
ch
an
ism
of
D
NA
P
ol
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 me
ch
an
ism
of
D
NA
P
ol
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
The specialization of DNA
polymerases
CHAPTER 8 The replication of DNA
DNA Pols are specialized for
different roles in the cellThe
sp
ec
ial
iza
tio
n
of
D
NA
po
l
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 activityThe
sp
ec
ial
iza
tio
n
of
D
NA
po
l
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 loadersThe
sp
ec
ial
iza
tio
n
of
D
NA
po
l
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
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
CHAPTER 8 The replication of DNA
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
re
pli
ca
tio
n
fo
rk
Figure 8-11
Leading strand
Lagging strand
Okazaki fragment
Replication fork
Th
e r
ep
lic
ati
on
fo
rk
Replication fork enzymes extend the
range of DNA polymerase substrate
1,DNA Pol can not accomplish
replication without the help of
other enzymes
2,The born and death of a RNA
primer,primase and RNase
H/exonuclease/DNA Pol/ligase
3,Dealing the DNA structure
(helicase,topoisomerase,SSB)
The initiation of a new strand of DNA
require an RNA primerThe
re
pli
ca
tio
n
fo
rk
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 replicationThe
re
pli
ca
tio
n
fo
rk
A joint efforts of
RNase H,DNA
polymerase &
DNA ligase
Figure 8-12
Th
e r
ep
lic
ati
on
fo
rk
Figure 8-15
Topoisomerase removes supercoils
produced by DNA unwinding at the
replication fork
DNA helicases unwind the double
helix in advance of the replication
fork
Th
e r
ep
lic
ati
on
fo
rk
Figure 8-13
Single-stranded binding proteins
(SSBs) stabilize single-stranded
DNA
Th
e r
ep
lic
ati
on
fo
rk
Cooperative binding
Sequence-independent manner
(electrostatic interactions)
Figure 8-14
DNA synthesis at the
replication fork
CHAPTER 8 The replication of DNA
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
Interactions between replication fork
proteins form the E,coli replisome
DN
A
sy
nt
he
sis
at
th
e r
ep
lic
ati
on
fo
rk
Replisome is established by protein-protein
interactions
1,DNA helicase & DNA Pol III holoenzyme,
this interaction is mediated by the clamp
loader and stimulates the activity of the
helicase (10-fold)
2,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.
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,
CHAPTER 8 The replication of DNA
Initiation of DNA replication
CHAPTER 8 The replication of DNA
Specific genomic DNA
sequences direct the initiation of
DNA replication
Origins of replication,the sites
at which DNA unwinding and
initiation of replication occur,
Ini
tia
tio
n
of
D
NA
re
pli
ca
tio
n
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
Ini
tia
tio
n
of
D
NA
re
pli
ca
tio
n
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
Binding and Unwinding,origin
selection and activation by the
initiator protein
CHAPTER 8 The replication of DNA
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)
Protein-protein and protein-DNA
interactions direct the initiation
process
Bi
nd
ing
an
d u
nw
ind
ing
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*
Initiating replication in
eukaryotes
Eukaryotic chromosome are replicated
exactly once per cell cycle,which is
critical for these organisms
Bi
nd
ing
an
d u
nw
ind
ing
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
Finishing replication
CHAPTER 8 The replication of DNA
Finishing replication in bacteria:
Type II topoisomerases separate daughter
DNA molecules
Fin
ish
ing
re
pli
ca
tio
n
Figure 8-34
Topoisomerase
II catalyze the
decatenation of
replication
products.
Finishing replication in
eukaryotes:
1,The end replication problem
2,Telomere & telomerase,a link
with cancer and aging
Fin
ish
ing
re
pli
ca
tio
n
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.
Figure 8-39,Telomere-binding proteins.
Telomere -binding proteins regulate
telomerase activity and telomere length
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,
重点
CHAPTER 8 The replication of DNA
Completely understand 三个 Animations
1,DNA polymerization (Topics 1 & 2)
2,DNA replication (Topics 3－ 5)
3,Action of Telomerase (Topic 8)
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
CHAPTER 8 The replication of DNA
Chemistry of DNA
1,DNA polymerization (Topics 1 & 2),
DNA polymerase,catalysis mechanism,
catalytic sites,different ways to
distinguish substrates,structure and
function of three domains,
重点
CHAPTER 8 The replication of DNA
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。
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
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
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
Process
Initiation &
Termination
CHAPTER 8 The replication of DNA
Reaction
& Catalyst
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
CHAPTER 8 The replication 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
The mechanism of DNA
Polymerase (Pol)
CHAPTER 8 The replication of DNA
DNA Pol use a single active
site to catalyze DNA synthesis
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).
Th
e me
ch
an
ism
of
D
NA
P
ol
Figure 8-3
Distinguishing different dNTPs,kinetic selectivity
Distinguishing between rNTP and
dNTP by steric exclusion of rNTPs
from the active site.
Th
e me
ch
an
ism
of
D
NA
P
ol
Figure 8-4
DNA Pol resemble a hand that
grips the primer-template junction
Th
e m
ec
ha
nis
m
of
D
NA
P
ol Figure 8-5
Schematic of DNA pol
bound to a
primer:template junction
A similar view of
the T7 DNA pol
bound to DNA
Figure 8-8
Thumb
Fingers
Palm
1,Contains two catalytic sites,one for addition of
dNTPs and one for removal of the mispaired
dNTP,
2,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,
3,Exonuclease site/proof reading site (See
proofreading)
DNA Polymerase-palm domain
Figure 8-6 Figure 8-7
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-finger 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 Polymerase-thumb domain
DNA Pol are processive enzymes T
he
me
ch
an
ism
of
D
NA
P
ol
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 me
ch
an
ism
of
D
NA
P
ol
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
The specialization of DNA
polymerases
CHAPTER 8 The replication of DNA
DNA Pols are specialized for
different roles in the cellThe
sp
ec
ial
iza
tio
n
of
D
NA
po
l
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 activityThe
sp
ec
ial
iza
tio
n
of
D
NA
po
l
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 loadersThe
sp
ec
ial
iza
tio
n
of
D
NA
po
l
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
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
CHAPTER 8 The replication of DNA
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
re
pli
ca
tio
n
fo
rk
Figure 8-11
Leading strand
Lagging strand
Okazaki fragment
Replication fork
Th
e r
ep
lic
ati
on
fo
rk
Replication fork enzymes extend the
range of DNA polymerase substrate
1,DNA Pol can not accomplish
replication without the help of
other enzymes
2,The born and death of a RNA
primer,primase and RNase
H/exonuclease/DNA Pol/ligase
3,Dealing the DNA structure
(helicase,topoisomerase,SSB)
The initiation of a new strand of DNA
require an RNA primerThe
re
pli
ca
tio
n
fo
rk
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 replicationThe
re
pli
ca
tio
n
fo
rk
A joint efforts of
RNase H,DNA
polymerase &
DNA ligase
Figure 8-12
Th
e r
ep
lic
ati
on
fo
rk
Figure 8-15
Topoisomerase removes supercoils
produced by DNA unwinding at the
replication fork
DNA helicases unwind the double
helix in advance of the replication
fork
Th
e r
ep
lic
ati
on
fo
rk
Figure 8-13
Single-stranded binding proteins
(SSBs) stabilize single-stranded
DNA
Th
e r
ep
lic
ati
on
fo
rk
Cooperative binding
Sequence-independent manner
(electrostatic interactions)
Figure 8-14
DNA synthesis at the
replication fork
CHAPTER 8 The replication of DNA
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
Interactions between replication fork
proteins form the E,coli replisome
DN
A
sy
nt
he
sis
at
th
e r
ep
lic
ati
on
fo
rk
Replisome is established by protein-protein
interactions
1,DNA helicase & DNA Pol III holoenzyme,
this interaction is mediated by the clamp
loader and stimulates the activity of the
helicase (10-fold)
2,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.
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,
CHAPTER 8 The replication of DNA
Initiation of DNA replication
CHAPTER 8 The replication of DNA
Specific genomic DNA
sequences direct the initiation of
DNA replication
Origins of replication,the sites
at which DNA unwinding and
initiation of replication occur,
Ini
tia
tio
n
of
D
NA
re
pli
ca
tio
n
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
Ini
tia
tio
n
of
D
NA
re
pli
ca
tio
n
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
Binding and Unwinding,origin
selection and activation by the
initiator protein
CHAPTER 8 The replication of DNA
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)
Protein-protein and protein-DNA
interactions direct the initiation
process
Bi
nd
ing
an
d u
nw
ind
ing
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*
Initiating replication in
eukaryotes
Eukaryotic chromosome are replicated
exactly once per cell cycle,which is
critical for these organisms
Bi
nd
ing
an
d u
nw
ind
ing
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
Finishing replication
CHAPTER 8 The replication of DNA
Finishing replication in bacteria:
Type II topoisomerases separate daughter
DNA molecules
Fin
ish
ing
re
pli
ca
tio
n
Figure 8-34
Topoisomerase
II catalyze the
decatenation of
replication
products.
Finishing replication in
eukaryotes:
1,The end replication problem
2,Telomere & telomerase,a link
with cancer and aging
Fin
ish
ing
re
pli
ca
tio
n
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.
Figure 8-39,Telomere-binding proteins.
Telomere -binding proteins regulate
telomerase activity and telomere length
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,
重点
CHAPTER 8 The replication of DNA
Completely understand 三个 Animations
1,DNA polymerization (Topics 1 & 2)
2,DNA replication (Topics 3－ 5)
3,Action of Telomerase (Topic 8)
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
CHAPTER 8 The replication of DNA
Chemistry of DNA
1,DNA polymerization (Topics 1 & 2),
DNA polymerase,catalysis mechanism,
catalytic sites,different ways to
distinguish substrates,structure and
function of three domains,
重点
CHAPTER 8 The replication of DNA
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。
