DNA synthesis at the replication fork
Interactions between replication fork
proteins form the E. coli replisome
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 (10fold)
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.
PTER 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.
PTER 8 The replication of DNA
PTER 8 The replication of DNA
Initiation of DNA replication
Initiation of DNA replication
Specific genomic DNA
sequences direct the initiation of
DNA replication
Origins of replication, the sites
at which DNA unwinding and
initiation of replication occur.
Initiation of DNA replication
The replicon model of replication
initiation---a general view
 Proposed by Jacob and Brenner
in 1963
 All the DNA replicated from a
particular origin is a replicon
 Two components, replicator and
initiator, control the initiation of
replication
Replicator: the entire site of
cis-acting DNA sequences
sufficient to direct the
initiation of DNA replication
Initiator protein: specifically
recognizes a DNA element in
the replicator and activates the
initiation of replication
Figure 8-23
Replicator sequences include
initiator binding sites and easily
unwound DNA
PTER 8 The replication of DNA
Binding and Unwinding: origin
selection and activation by the
initiator protein
 Three different functions of
initiator protein: (1) binds to
replicator, (2) distorts/unwinds a
region of DNA, (3) interacts with
and recruits additional replication
factors
 DnaA in E. coli (all 3 functions),
origin recognition complex (ORC) in
eukaryotes (functions 1 & 3)
Binding and unwinding
Protein-protein and protein-DNA
interactions direct the initiation
process
Initiating replication in
bacteria
 DnaA recruits the DNA helicase
DnaB and the helicase loader DnaC
 DnaB interacts with primase to
initiate RNA primer synthesis.
Figure 8-27*
Binding and unwinding
Initiating replication in
eukaryotes
Eukaryotic chromosome are replicated
exactly once per cell cycle, which is
critical for these organisms
Pre-replicative complex (pre-RC) formation and
activation directs the initiation of replication in
eukaryotes
Initiation in eukaryotes requires two distinct
steps:
1st step---Replicator selection: the process of
identifying sequences for replication
initiation (G1 phase), which is mediated by
the formation of pre-RCs at the replicator
region.
Figure 8-30 preRC 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
PTER 8 The replication of DNA
Finishing replication
Finishing replication in bacteria:
Type II topoisomerases separate daughter
DNA molecules
Finishing replication
Figure 8-34
Topoisomerase II
catalyze the
decatenation of
replication products.
Finishing replication in
eukaryotes:
Finishing replication
1.The end replication problem
2.Telomere & telomerase: a link
with cancer and aging
What is the end replication problem?
Lagging strand synthesis is unable to copy
the extreme ends of the linear chromosome
Figure 8-34
Telomerase is a novel DNA polymerase that
does not require an exogenous template
How telomerase works?
Telomerase extends the
protruding 3’ end of the
chromosome using its RNA
component s as a template.
(Figure 8-37)
How the end problem is eventually resolved?
Figure 8-38
The extended 3’ end
allows the DNA
polymerase to
synthesize a new
Okazaki fragment, which
prevents the loss of
genetic information at
the chromosomal end.
Telomere -binding proteins regulate
telomerase activity and telomere length
Figure 8-39: Telomere-binding proteins.
Short telomere is bound by few telomere-binding proteins,
allowing the telomerase to extend telomere.
The extended telomere is bound by more telomere-binding
proteins, which inhibits the telomerase activity.
Figure 8-40: Telomere length regulation by
telomere-binding proteins.
CHAPTER 8 The replication of DNA
重点
Completely understand 三个Animations
1. DNA polymerization (Topics 1 & 2)
2. DNA replication (Topics 3－5)
3. Action of Telomerase (Topic 8)
CHAPTER 8 The replication of DNA

The Chemistry of DNA Synthesis:
substrate, direction
and energy.

The Mechanism of DNA Polymerase:

The Specialization of DNA Polymerases

The Replication Fork: the enzyme/proteins required to
1 polymerization
mechanism, 2 different ways of discriminating substrates, 2 catalytic
sites; 3 domains.
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.
重点
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.
PTER 8 The replication of DNA
重点
2.DNA replication (Topics 3－
5):trumbone model, how the antiparallel 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。