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Chapter 2: DNA Synthesis (Replication)
Required reading: Stryer’s Biochemistry 5th edition p. 127-128, 750-754,
759-766, 768-773
(or Stryer’s Biochemistry 4th edition p. 88-93, 799-809, 982-986, 809-814)
Normal recitation time: Thursdays 11-12, B-185 PWB
Exam: Wed., December 1
Proposed recitation time for this exam: Tue, Nov. 30, 11-12
DNA Polymerization Reaction
E. coli DNA Polymerases
Characteristic
Mol. Weight (Da)
Number of
polypeptides
Polymerase 5' 3'
rate (nucleotides/sec)
3' 5' exonuclease
5' 3' exonuclease
# molecules/cell
function
Pol I
103,000
1
Pol II
88,000
4
Pol III
900,000
10
yes
16-20
yes
yes
400
Primer removal,
gap filling
yes
7
yes
no
100
unknown
yes
250-1000
yes
no
10
Major replicative
polymerase
E. coli DNA Polymerase I
Klenow Fragment
N
5' 3' Nucl. 3' 5‘ Nucl. Polymerase
36 kDa
•
•
•
•
C
67 kDa
a large cleft for binding duplex DNA
flexible "finger" and "thumb" regions for positioning DNA duplex
and the incoming dNTP
polymerase site located in the "palm" region
3' 5' and 5'- nuclease catalytic sites
Typical Polymerase Structure: E. Coli Pol I
fingers
thumb
palm
polymerase
exonuclease
Polymerase with bound DNA
Mechanism of phosphoryl transfer
Polymerase fidelity mechanisms
1. Watson-Crick base pairing between the incoming dNTP
and the corresponding base in the template strand.
2. H-bond formation between the minor groove of the new base pair
and the amino acids in the polymerase active site.
3. Proofreading mechanism via 3' exonuclease that excises
incorrectly added nucleotides.
1. Correct Watson-Crick base pairing between the incoming
dNTP and the corresponding base in the template strand
induces conformational change required for polymerization
reaction:
Thumb
Fingers
2. H-bond formation between the minor groove of the new base pair
and amino acids in the polymerase active site:
All Watson-Crick base pairs contain two Hbond acceptors at the same sites of the
minor groove
N
O
N
H 2N
NH
N
N
N
NH 2
O
N
N
N
NH 2
HN
N
N
O
O
A•T
G•C
C:G
T:A
3. 3’-Exonuclease Proofreading function of DNA polymerases
excises incorrectly added nucleotides.
Fidelity of DNA Polymerization: Absolutely Essential!!
Error Probability = Polymerization error (10-4)
X
3' 5' Nuclease error (10-3)
= 10-7 (1 in 10,000,000 nt)
DNA Polymerization Has Three
Stages
1) Initiation
2) Priming
3) Processive Synthesis
Problems to overcome: DNA Polymerization
1. The two strands must be separated, and local DNA over-winding
must be relaxed. The single stranded DNA must be prevented from
re-annealing and protected from degradation by cellular nucleases.
2. Both antiparallel strands must be synthesized simultaneously in
the 5’ 3’ direction.
3’
3. A primer strand is required.
5’
DNA Polymerization: Initiation
•
DNA replication begins at a specific site.
•
Example: oriC site from E. coli.
• 245 bp out of 4,000,000 bp
• contains a tandem array of three 13-mers;
GATCTNTTNTTTT
•
Synthesis takes place in both directions from the origin (two
replication forks)
E. coli replication origin
•GATC common motif in oriC
•AT bp are common to facilitate duplex unwinding
DNA Polymerization: Initiation
•
DNA replication begins at a specific site.
•
Example: oriC site from E. coli.
• 245 bp out of 4,000,000 bp
• contains atandem array of three 13-mers;
GATCTNTTNTTTT
•GATC common motif in oriC
•AT bp are common to facilitate duplex unwinding
•
Synthesis takes place in both directions from the origin (two
replication forks)
Enzymes involved in the initiation of DNA Polymerization
Enzyme
Function
dnaA
recognize replication origin and
melts DNA duplex at several sites
Helicase (dnaB)
unwinding of ds DNA
DNA gyrase
generates (-) supercoiling
SSB
stabilize unwound ssDNA
Primase (dnaG)
an RNA polymerase, generates
primers for DNA Pol
Crystal structure of bacterial DNA helicase
Stryer Fig. 27.16
DNA helicase: proposed mechanism
B1
A1
Stryer Fig. 27.17
Problems to overcome: DNA Polymerization
1. The two strands must be separated, and local DNA over-winding
must be relaxed. The single stranded DNA must be prevented from
re-annealing and protected from degradation by cellular nucleases.
2. Both antiparallel strands must be synthesized simultaneously in
the 5’ 3’ direction.
3’
3. A primer strand is required.
5’
(overall
direction)
Lagging strand is synthesized in short fragments
(1000-2000 nucleotides long) using
multiple primers
3’
5’
Problems to overcome: DNA Polymerization
1. The two strands must be separated, and local DNA over-winding
must be relaxed. The single stranded DNA must be prevented from
re-annealing and protected from degradation by cellular nucleases.
2. Both antiparallel strands must be synthesized simultaneously in
the 5’ 3’ direction.
3’
3. A primer strand is required.
5’
A short stretch of RNA is used as a primer for DNA
synthesis
(dnaG)
What is the function of RNA
priming?
• DNA polymerase tests the correctness of the preceding base pair
before forming a new phosphodiester bond
•de novo synthesis does not allow proofreading of the first
nucleotide
•Low fidelity RNA primer is later replaced with DNA
Lagging strand synthesis in E. coli
Okazaki fragment
3'
5'
5'
3'
Primase
3'
5'
3'
5'
3'
5'
DNA Pol III
RNA primer
3'
5'
3'
5'
3'
5'
DNA Pol I
Template DNA
3'
5'
3'
5'
New DNA
3'
5'
5'
3'
DNA Ligase
3'
5'
5'
3'
DNA Synthesis
5'
3'
Helicase
Gyrase
Primase
SSB
Leading Strand
Lagging Strand
3'
5'
DNA
Pol
III
DNA
Pol
I
3'
5'
DNA Ligase
3'
5'
E. coli DNA Polymerase III
Processive DNA Synthesis
The bulk of DNA synthesis in E. coli is carried out by the
DNA polymerase III holoenzyme.
• Extremely high processivity: once it combines with the
DNA and starts polymerization, it does not come off until
finished.
• Tremendous catalytic potential: up to 2000
nucleotides/sec.
• Low error rate (high fidelity) 1 error per 10,000,000
nucleotides
• Complex composition (10 types of subunits) and large
size (900 kd)
E. coli Pol III: an asymmetrical dimer
Sliding clamp
clamp loader
Polymerase
3'-5' exonuclease
Polymerase
Stryer Fig. 27.30
2 sliding clamp is important for processivity of Pol III
Lagging strand loops to enable the simultaneous replication
of both DNA strands by dimeric DNA Pol III
Stryer Fig. 27.33
DNA Ligase seals the nicks
AMP + PPi
O
O
OH
-O
P
O-
O
O
DNA Ligase +
(ATP or NAD+)
P
O
O-
• Forms phosphodiester bonds between 3’ OH and 5’ phosphate
• Requires double-stranded DNA
• Activates 5’phosphate to nucleophilic attack by trans-esterification
with activated AMP
DNA Ligase -mechanism
ENZYME
1.
E + ATP  E-AMP + PPi
(+)H2N
O
P
Ade
O(-)
O
O
2. E-AMP + P-5’-DNA  AMP-O
P
O
O
5'-DNA
OH
OH
OO
3. DNA-3'
OH
+ AMP-O
P
O
O
5'-DNA
DNA-3'
O
O-
+
P
O
OAMP-OH
5'-DNA
DNA Synthesis in bacteria: Take Home Message
1) DNA synthesis is carried out by DNA polymerases
with high fidelity.
2) DNA synthesis is characterized by initiation, priming,
and processive synthesis steps and proceeds in the 5’
3’ direction.
3) Both strands are synthesized simultaneously by the
multisubunit polymerase enzyme (Pol III). One strand is
made continuously (leading strand), while the other one
is made in fragments (lagging strand).
4) Pol I removes the RNA primers and fills the resulting
gaps, and the nicks are sealed by DNA ligase