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Lecture #20 :
DNA Replication- I
Recommended reading:
Chapter 11 – DNA Replication
1
Today’s lecture content:
① Universal principles of DNA replication

DNA replication is semiconservative

DNA synthesis needs priming

DNA replication is polar (5’ to 3’): leading and lagging
strands
① DNA replication in E. coli

DNA polymerase activity: error rate and processivity

Other activities required at the replication fork
2
① Universal Principles of DNA Replication
3
① Universal Principles of DNA Replication
 Replication is semiconservative
• The complementary base pairing of the DNA helix suggested that
each strand could act as a template for the synthesis of its
complementary strand
4
① Universal Principles of DNA Replication
• What is the mechanism of DNA replication:
•
•
•

Conservative?
Semi-Conservative?
Dispersive?
Meselson and Stahl experiments (1958)
5
① Universal Principles of DNA Replication
• Meselson and Stahl grew E. coli in growth medium containing
ammonium 15NH4 (heavy Nitrogen Isotope) as a source of Nitrogen
• They then switched to 14NH4 (light Nitrogen Isotope)
• DNA with heavy or light Nitrogen sediment differently in a Cesium
Chloride density gradient
6
① Universal Principles of DNA Replication
• Meselson and Stahl grew E. coli in growth medium containing
ammonium 15NH4 (heavy Nitrogen Isotope) as a source of Nitrogen
• They then switched to 14NH4 (light Nitrogen Isotope)
• DNA with heavy or light Nitrogen sediment differently in a Cesium
Chloride density gradient
• In addition, they used either:
•
Native conditions where DNA was double stranded
•
or Alkaline, or denatured conditions, where DNA strands were separated
7
① Universal Principles of DNA Replication
8
9
① Universal Principles of DNA Replication
10
① Universal Principles of DNA Replication
• DNA replication is catalyzed by DNA polymerases, which have two
properties with major implications for DNA replication:
• They cannot initiate DNA synthesis de novo and require a
“primer” with a free 3′-OH group at the 3’ end
• They can extend DNA only in the 5′→3′ direction
11
① Universal Principles of DNA Replication
 DNA Synthesis needs priming
• Problem 1:
They cannot initiate DNA synthesis de novo and require a “primer” with
a free 3′-OH group at the end
 Solution:
DNA synthesis is primed by an RNA!
12
① Universal Principles of DNA Replication
 DNA replication is polar (5’ to 3’)
• Problem 2:
DNA polymerases can extend DNA only in the 5’ to 3’ direction but the
two parental strands are anti-parallel
 Solution:
Replication is semidiscontinuous:
• The 3’-5’ strand (Crick strand) serves as template for the
leading strand synthesis
• The 5’-3’ strand (Watson strand) serves as template for the
lagging strand synthesis
13
The leading strand can be extended
continuously, with the polymerase moving
from the 5’ terminus to the 3’ terminus in
the same direction as fork movement.
The lagging strand synthesis is
discontinuous with short fragments (called
Okazaki fragments) synthesized by the
polymerase moving opposite to the direction
of fork movement.
The Okazaki fragments are then stitched
together by the enzyme DNA ligase.
Reiji & Tuneko Okazaki
14
Today’s lecture content:
 Universal principles of DNA replication

DNA replication is semiconservative

DNA synthesis needs priming

DNA replication is polar (5’ to 3’): leading and lagging
strands
② DNA replication in E. coli

DNA polymerase activity: error rate and processivity

Other activities required at the replication fork
15
② DNA replication in E. coli
• E. coli has been a crucial model system for dissecting DNA
replication
• It is a simpler model with a well defined origin and termination of
replication sites, as well as replication forks moving in opposite
direction
16
17
② DNA replication in E. coli
 DNA polymerase activity
• The E. coli DNA pol I is the most well studied DNA polymerase:
18
② DNA replication in E. coli
 DNA polymerase activity
• DNA pol I extends the DNA in the 5′→3′ direction incorporating a
dNMP complementary to the template strand (A-T, G-C):
19
② DNA replication in E. coli
 DNA polymerase activity
• In addition to its DNA polymerase activity, the E. coli DNA pol I also
has two exonuclease activities:
• 3' →5' exonuclease activity that digests the DNA strand from
the 3' terminus
• 5' →3' exonuclease activity that digests the DNA strand from
the 5' terminus
20
② DNA replication in E. coli
 DNA polymerase activity
• The 3' →5' exonuclease activity of DNA pol I is responsible for
removing mis-incorporated dNMPs
• This activity is called proofreading
• DNA pol I has an error rate of 10-4 or 10-5, but this error rate is
improved 102- to 103-fold by 3' →5' exonuclease proofreading
activity.
21
22
② DNA replication in E. coli
 DNA polymerase activity
• The 5' →3' exonuclease activity of DNA pol I is responsible for
repairing nicks and gaps between Okazaki fragments
• This activity is called nick translation
23
At the gap between lagging strand
fragments, DNA Pol I degrades the RNA
primer in the 5'→3' direction, releasing
NMPs, and simultaneously extends the
3' terminus with dNTPs in the same
direction.
The net result is movement of the nick in
the 5'→3' direction along the DNA until
all RNA is removed. DNA ligase can then
seal the fragments (not shown here).
24
② DNA replication in E. coli
 DNA polymerase activity
• DNA Pol I is actually not the DNA polymerase responsible for the E.
coli chromosome replication
25
② DNA replication in E. coli
 DNA polymerase activity
• DNA Pol III is the DNA polymerase responsible for the E. coli
chromosome replication
• The Pol III core is a heterotrimer that contains one each of:
• α subunit, containing the DNA polymerase activity
• ε subunit, containing the proofreading 3' →5' exonuclease
activity
• θ subunit, of unknown function
26
② DNA replication in E. coli
 DNA polymerase activity
• The Pol III core has a processivity of 1 to 10 nucleotides and
synthesizes ~ 10 nucleotides per second
• However, the replication of the E. coli chromosome occurs at a rate
of 1000 bp per second!
• During replication, Pol III associates with the β sliding clamp which
dramatically increases its processivity and rate of DNA
polymerization
27
② DNA replication in E. coli
 DNA polymerase activity
• The β sliding clamp forms a “doughnut” that completely surrounds
the double-stranded DNA and thus keep the DNA Pol III associated
with its DNA template
28
② DNA replication in E. coli
 DNA polymerase activity
• The β sliding clamp is loaded onto the primed DNA template by the
γ complex clamp loader
29
② DNA replication in E. coli
 DNA polymerase activity
• The β sliding clamp increases the speed and processivity of DNA
Pol III
30
② DNA replication in E. coli
 DNA polymerase activity
• For example, with a preloaded Pol III core and β sliding clamp, the
phage ΦX174 DNA is synthesized in 11 sec!
31
② DNA replication in E. coli
 Other activities required at the replication
fork
• Apart from the DNA polymerase activity, many other activities are
required at the replication fork:
• β sliding clamp and γ complex clamp loader for processivity
32
② DNA replication in E. coli
 Other activities required at the replication
fork
• Apart from the DNA polymerase activity, many other activities are
required at the replication fork:
• DNA B 5’- 3’ helicase required for unwinding the DNA
33
② DNA replication in E. coli
 Other activities required at the replication
fork
• Apart from the DNA polymerase activity, many other activities are
required at the replication fork:
• DNA topoisomerases required for relieving the positive
supercoiling of the DNA ahead of the helicase
34
② DNA replication in E. coli
 Other activities required at the replication
fork
• Apart from the DNA polymerase activity, many other activities are
required at the replication fork:
• Okazaki fragment priming and sealing
35
② DNA replication in E. coli
 Other activities required at the replication
fork
• Apart from the DNA polymerase activity, many other activities are
required at the replication fork:
• Okazaki fragment priming and sealing
36
② DNA replication in E. coli
 Other activities required at the replication
fork
• Apart from the DNA polymerase activity, many other activities are
required at the replication fork:
• Single strand protection
37
38
Next Lecture:
#21 – DNA Replication - II
On Monday, March 11th
Recommended reading:
Chapter 11: DNA replication
39
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