DNA Replication

1
DNA Replication
? The basic rules for DNA replication
? DNA Polymerases
? Initiation of replication
? DNA synthesis at the replication fork
? Termination of replication
? Regulation of re-initiation
? Other modes of DNA replication
2
? Initiation of replication
? Common features of replication origins
? Common events of initiation
? Priming
3
? The replicon model of replication initiation
(proposed by F. Jacob, S. Brenner and J. Cuzin,
1963)

• All the DNA replicated from a particular
• origin as a replicon.

• Binding of the initiator to the replicator
• stimulates initiation of replication.

4
Replicator
Initiator
Replicator the entire set of cis-acting DNA
sequences that is sufficient
to direct the initiation of DNA replication
(the origin of replication is part
of replicator)
Initiator the DNA-binding protein that
specifically recognizes a DNA
element in the replicator and activates the
initiation of replication
5
Initiator binding site
Easily melted region
Replicator
(Origin)
DNA binding
Initiator
DNA unwinding
The functions of initiator
Protein recruitment
Priming and DNA synthesis
6
E. coli
oriC
DnaA
DNA binding
DNA unwinding
Protein recruitment
DnaB/DnaC
Priming and DNA synthesis
7
oriC the origin of replication in E. coli
Figure 14.26
Easily melted (AT rich)
Initiator (DnaA) binding site
8
oriC
L, M, R repeats (13 bp)
14 repeats (9 bp)
Consensus sequence
Consensus sequence
GATCTNTTNTTTT CTAGANAANAAAA
TTATNCANA AATANGTNT
9
The replicator (origin) of S. cerevisiae
ARS (Autonomously replicating sequence)
Origin recognition complex (ORC) (Initiation
complex)
ACS ARS consensus sequence
????Cooper, G. M. (1997) The cell a molecular
approach. ASM Press. Fig. 5.17
10
Figure 13.20
Mutation in ARS
?Mutations in B elements reduce origin function.
?Mutations in core consensus abolish origin
function.
11
E. coli
oriC
DnaA
DNA binding
DNA unwinding
Protein recruitment
DnaB/DnaC
Priming and DNA synthesis
12
DnaA.ATP
DNA helicase (DnaB)
DNA helicase Loader (DnaB)
Figure 14.27
Watson, J. D. et al. (2004) Molecular Biology of
the gene. 5th ed. CSHL Press. Fig. 8-26.
13
DnaA?ATP
HU ATP
(histone like protein)
14
DNA helicase (DnaB)
DNA helicase Loader (DnaB)
15
Figure 14.10
16
Two types of function are needed to convert
dsDNA to the single-stranded state

• Helicases separate the strands of
• DNA, usually using the hydrolysis of
• ATP to provide the necessay energy

2. Single-strand binding proteins bind to the
ssDNA, preventing it from reforming the
duplex state
17
DNA helicase (DnaB)
DNA helicase Loader (DnaB)
Single-strand binding proteins
Primase
18
For DNA replication, a primase is required to
catalyze the synthesis of RNA primer.
Primase in E. coli
? An RNA polymerase
? Synthesizing short stretches of RNA
? Encoded by the dnaG gene
19
Figure 14.14
20
Summary
Protein required to initiate replication at the
E. coli origin
21
DNA Replication
? The basic rules for DNA replication
? DNA Polymerases
? Initiation of replication
? DNA synthesis at the replication fork
? Termination of replication
? Regulation of re-initiation
? Other modes of DNA replication
22
? DNA synthesis at the replication fork
? Proteins at the replication forks

• Coordinating synthesis of the lagging
• and leading strands

? in E. coli
? in eukaryotic cells
23
? DNA replication is semidiscontinuous.
Figure 14.9
Okazaki fragments
1000-2000 nt in prokaryotes 100-400 nt in
eukaryotes
24
Proteins required at the replication forks
Primer removal enzyme
DNA ligase
SSB Single-strand binding proteins
25
Different replicase units are required to
synthesize the leading and lagging strands.

• In E. coli both units contain the same catalytic
• subunit of DNA Pol III.

• In other organisms, different catalytic subunits
• may be required for each strand.

26

• The helicase creating
• the replication fork
• is connected to two
• DNA polymerase
• catalytic subunits.

• Each polymerase
• catalytic subunit is
• held on DNA by a
• sliding clamp.

Figure 14.19
27

• The polymerase that
• synthesizes the lagging
• strand dissociates at
• the end of Okazaki
• fragment and then
• reassociates with a
• primer in the single-
• stranded template loop
• to synthesize the next
• fragment.

• The polymerase that
• synthesizes the
• leading strand moves
• continuously.

Figure 14.19
28
In E. coli
DnaB
DNA Pol III holoenzyme
DnaG
29
E. coli DNA Polymerase III holoenzyme
Based on Figure 14.17
30
proofreading
e
e
polymerization
polymerization
a
a
q
q
Core enzyme
t
t
Core enzyme dimerization
31
Clamp loader
ATP
ATP
b
b
Sliding clamp
ADP
ATP
Pi
ATP
hydrolysis
32
Processivity
Core enzyme 10 15
Holoenzyme gt500000
b converts Pol III from a distributive enzyme to
a highly processive enzyme.
Figure 14.18
33
y
d
c
g
d
Lagging strand synthesis
Leading strand synthesis
e
e
a
a
q
q
t
t
t subunits maintain dimeric structure of Pol III
and interact with DnaB
DnaB (helicase)
34

• DnaB (helicase) is
• responsible for
• forward movement
• at the replication
• fork.

• Each catalytic core
• of polymerase III
• synthesizes a
• daughter strand.

Figure 14.20
35
What happens to the loop when the Okazaki
fragment is completed?
Figure 14.21
36
5. Reassociation of core
1
4
Reassociation of b clamp
Initiation of Okazaki fragment
2
3
Termination of Okazaki fragment
Dissociation of core and b clamp
Figure 14.20
37
Each Okazaki fragment is synthesized as a
discrete unit.
Lagging strand
Primase synthesizes RNA primer.
Leading strand
DNA Pol III extends primer into Okazaki fragment.
Next Okazaki fragment is synthesized.
38
Okazaki fragments are linked together.
5
3
3
5
RNA primer
DNA Pol I uses nick translation to replace RNA
primer with DNA.
Ligase seals the nick.
Figure 14.22
39
? 5?3 Exonuclease activity of DNA Pol I
Figure 14.5
40
Mechanism of the DNA ligase reaction

Adenylylation of DNA ligase
1
NMN (or PPi)
41
Figure 14.23
O
2

Ligase
O
P
Ribose
Adenine
NH2
O-
Activation of 5 phosphate in nick
Ligase

3
42
O
O-
Ribose
Adenine
P
3
O-
O
O
The 3-hydroxyl group attacks the phosphate and
displaces AMP, producing a phosphodiester bond.
P
O
O
3
43
Eukaryotic cell
Pold/e
Pold
PCNA
Pola/primase
(RFC)
44
? Eukaryotes have many DNA polymerases.
Figure 14.24
45
Eukaryotic DNA polymerases for replication in
nucleus

46
DNA polymerase a (Pola/primase)
2 subunits Pol a
DNA synthesis
2 subunits primase
RNA synthesis
3
5
5
3
OH
DNA (iDNA)
RNA
10 bp
20-30 bp
47
DNA polymerase switching during eukaryotic DNA
replication
DNA Pol a/ primase
RNA primer synthesis by primase
a
P
Watson, J. D. et al. (2004) Molecular Biology of
the gene. 5th ed. CSHL Press. Fig. 8-16.
DNA synthesis by Pol a
RNA
iDNA
48
iDNA
Sliding clamp

Pola/primase
DNA Pol e (or d)
49
R-FC binds to the 3 end of iDNA and displaces
pol a/primase

R-FC attracts PCNA
PCNA binds pol d or e
RF-C Clamp loader
PCNA Sliding clamp
50
PCNA

• Proliferating
• cell nuclear
• antigen

(trimer)
????Voet, D., Voet, J. G. and Pratt, C.W. (1999)
Fundamentals of Biochemistry. John Wiley Sons,
Inc. Fig. 24-1
51
Summary
Proteins required at the replication forks
Figure 14.25
DNA topoisomerases are also required!
52
(No Transcript)
53
There are two ways to think of the relative
motion of the DNA and replication machinery

• The replication machinery moves along
• the DNA.
• (similar to a train moving along its track)

2. The DNA moves while the replication
machinery is static. (similar to film moving
into a movie projector)
54
The two replisomes of E. coli are linked together
and tethered to one point on the bacterial inner
membrane.
Pol III holoenzyme
Pol III holoenzyme
Helicases (double-hexamers)
????Nelson, D. L. and Cox, M. M. (2005)
Lehninger Principles of Biochemistry. 4th Ed.,
Worth Publishers. Fig. 25-18a
55
Origin
Cells divide
Replication begins
Chromosome
Terminator
Replisomes
Origins separate
Chromosomes separate
Cell elongates as replication continues
????Nelson, D. L. and Cox, M. M. (2005)
Lehninger Principles of Biochemistry. 4th Ed.,
Worth Publishers. Fig. 25-18a