Download Document

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)
DNA Replication is semi-conservative
Meselson, Stahl 1958
• Both strands serve as templates
for DNA synthesis
• Each DNA molecule contains one
strand from original DNA and one
new strand
DNA Polymerization Reaction
Requirements for DNA polymerization
•
Template DNA (single stranded or double-stranded with a “nick”)
•
A primer strand with a free 3'- hydroxyl group (usually RNA)
•
Deoxynucleoside 5'-triphosphates (dATP, dGTP, dTTP and dCTP)
•
Mg2+ to activate the dNTPs
•
Polymerase and other accessory enzymes
General reaction:
(DNA)n bases + dNTP
(DNA)n+1 + PPi
2Pi
H2O
O
O
HO P O P OH
OO-
O
HO P O
O-
O
HO P O
O-
DNA Polymerization Reaction
DNA Synthesis: addition of new dNTPs
follows Watson-Crick rules
N
O
N
H 2N
NH
N
N
N
NH 2
O
N
N
N
NH 2
N
N
O
O
A•T
G•C
Template base
G
C
T
A
HN
Incoming base
C
G
A
T
3’ 5’ Exonuclease
5’  3’ -exonuclease
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)