DNA Polymerases
Klentaq
(Rothwell & Waksman)
DNA Polymerases
I) Historical perspective.
II) Review of DNA structure.
III) Delarue (1990) pile-up of amino acid sequences for classic
enzymes. Highlight key residues discussed today.
IV) Overview of catalytic cycle
V) Key DNA polymerase functions in the catalytic pathway, and
amino acids involved.
• E/DNA binary complex
• Binding dNTPs & excluding rNTPs
• Base specificity is substantially driven by steric constraints
• Phosphodiester bond formation
• Translocation
Nanopore Group, U.C. Santa Cruz
Oswald Avery (1877-1955)
NIH
3
Avery-MacLeod-McCarty experiment
Non-virulent
strain
Virulent
strain
Heat-killed
virulent
strain
Heat-killed
virulent
strain
Extracted
virulent
proteins
Extracted
virulent
DNA
+
+
+
Non-virulent
strain
Non-virulent
strain
Non-virulent
strain
4
DNA Fibers
Photo 51,
Franklin
Watson & Crick
Nature, 1953
DNA Crystals
Dickerson & Colleagues
PNAS, 1981
Calladine group
Table 1. Terminal tetranucleotides whose UL current signatures will be measured in Aim
2. These sequences were chosen because they are representative of rigid ‘A tract’ DNA,
‘G tract’ DNA, or because they are known to be highly flexible. The values in column
three are the predicted flexibility (slide) of the central dinucleotide step for each
sequence in the context of the tetranucleotide [20]. Only the sense strand is shown.
Tetranucleotide
Structural
Flexibility of Central Dinucleotide
Classification
Step
kJ mol-1 Å2
AAAG
A tract
24.5
AAAA
A tract
23.8
AAAC
A tract
27.2
TACA
Canonical B form
1.9
CATA
Canonical B form
3.2
TATA
Canonical B form
3.6
GGGG
G tract
16.4
CCGG
G tract
16.6
CGGC
G tract
14.5
7
8
DNA End Processing by HIV Integrase
CA dinucleotide
Diagram F.D. Bushman, Salk Institute
9
Ribose Sugar Pucker
What double helix structures arise from these sugar puckers?
A Form (strongly favored by dsRNA)
B Form (favored by dsDNA
but interconverts)
11
100 trillion cells
1 cell
Zygotee
100 trillion cells X 12 billion DNA bases copied = 1X 1025 bases
new cell
Error rate overall ~ 1 in 109
; Error rate of polymerase ~ 1 in 105
+
Labeled dATP
Labeled dCTP
Labeled dTTP
Labeled dGTP
Sinsheimer & coworkers, 1960s
GGGG
5′
5′
A
6 Replicates
Bases per DNA Molecule
DNA polymerase I
4
1
A
0
0
C
T
G
Addition of a dideoxynucleotide
prevents addition of the next.
-
H
+
Sanger & Coworkers
Megabases Sequenced
Human Mitochondrial DNA Sequenced
circa 16,000 bases
3000
1965
1970
1975
1980
1985
1990
1995
2000
Megabases Sequenced
Automated
Capillary Array
Sequencer
ABI 3700
3000
1965
1970
1975
1980
1985
1990
1995
2000
Cost per finished bp in
US Dollars
High Speed DNA Sequencing
100.00
10.00
1.00
0.10
$20 million
1E-3
1E-5
$1000 per
mammalian
genome
X
1980 1990 2000 2010 2020 2030 2040 2050
Year
Cost per finished bp in
US Dollars
High Speed DNA Sequencing
100.00
10.00
1.00
0.10
$20 million
1E-3
$100,000
genome X
1E-5
$1000
genome
X
1980 1990 2000 2010 2020 2030 2040 2050
Year
Cost per finished bp in
US Dollars
High Speed DNA Sequencing
100.00
40 million clusters/flow cell
10.00
1.00
0.10
1E-3
1E-5
Watson
20 microns
454 Inc.
$2 million
Illumina Corp.
$48,000
$5,000 ?
Complete Genomics
1980 1990 2000 2010 2020 2030 2040 2050
Year
Sequence alignment of four closely related A family DNA polymerases & more distantly
related Bacteriophage SPO2 DNA polymerase
D
Motif A
R
K
F
YG
Motif B
D
Motif C
21
Delarue 1990
Motif B
(R,K,F,Y,G)
Motif A
(D,E)
Catalysis
O helix
Motif C
(D)
Klentaq
(Rothwell & Waksman)
22
General Steps in the DNA Polymerase Catalytic Cycle
Rothwell & Waksman, 2005
E = DNA polymerase enzyme
p/t = primer template duplex
dNTP = deoxynucleotide triphosphate
PPi = pyrophosphate
E′ = activated DNA polymerase enzyme
23
Typical DNA polymerase
catalytic cycle at medium precision
KF
dNTP
dN
TP
~
PPi
1
2
3
4
covalent
bond
5
Is finger closing the rate-limiting step?
Rothwell & Waksman, 2005
E = DNA polymerase enzyme
p/t = primer template duplex
Phosphodiester bond
is formed (fast)
dNTP = deoxynucleotide triphosphate
PPi = pyrophosphate
E′ = activated DNA polymerase enzyme
25
It was widely assumed that this significant motion of
the fingers domain was the rate limiting step in catalysis
that precedes phosphodiester bond formation -- but there
were no data to support this.
“The rate-limiting step for replication is thought to
involve a conformational change between an ‘open
fingers’ state in which the active site samples
nucleotides, and a ‘closed’ state in which nucleotide
incorporation occurs.”. Nature 2000.
Biophysical Journal, 2004
26
Forster Resonance Energy Transfer
No FRET
FRET
Fluorescence
Emission
Fluorescence
Emission
Ro
Wavelength (nm)
Wavelength (nm)
27
FRET experimental evidence suggests that finger closing is
faster than phosphodiester bond formation & therefore
cannot be the rate limiting pre-chemistry step.
Dabcyl T(-8)
Distance from 744
744
AEDANS
Fingers
Thumb
T(-8)
dabcyl
labeled
DNA
Joyce, 2008
Open
Closed
50.4Å
43.8Å
Finger closing
Rate limiting step
(pulse chase)
29
FRET experimental evidence suggests that finger closing is
not the rate limiting pre-chemistry step.
E + DNA
Step 1
Step 2
Step 2.1
fast
fast
Eo•DNA*•dNTP-Mg2+
Eo•DNA•dNTP-Mg2+
Eo•DNA
dNTP-Mg2+
fast
Step 2.2 (finger-closing)
Ec•DNA*•dNTP-Mg2+
Step 3 (catalytic site
rearrangement?)
KF Catalytic
Cycle
Ec•DNA*•dNTP-(Mg2+)2
Bond
Formation
PPi, 2Mg2+
Eo•DNA+1•PPi-(Mg2+)2
Eo•DNA+1
Step 6
Step 4 (phosphodiester
bond formation)
Ec•DNA+1•PPi-(Mg2+)2
Step 5
Building a catalytic complex where molecular recognition is critical at
each step
3.4Å
DNA
KF
dNTP
dN
TP
1
2
~
3
4
covalent
bond
DNA+1
PPi
5
6
1) Binary complex: DNA binding to A family polymerases
3.4Å
DNA
KF
dNTP
dN
TP
1
2
~
3
4
covalent
bond
DNA+1
PPi
5
6
Apo Enzyme (E)
Binary complex (E/p/t)
4)Y on O helix occupies
n=0 position against terminal
base
1) Helix-turn-helix on
thumb loops around DNA
Kd ≃ 5nM
2) Residues in beta sheets
(palm) bind DNA minor
groove
3) ssDNA template (~5 nt
drape over finger domain)
Klentaq (Rothwell & Waksman)
Tyrosine ‘Pawl’ that registers ssDNA/dsDNA junction at the catalytic site
Motif A
YG
Motif B
34
Delarue 1990
Klentaq
(Rothwell & Waksman)
O helix
Y671
Bst DNA polymerase
(Beese & coworkers)
base-pair at
n=-1
Primer
n=-1
Y714
n=0
Template
n=0 base
O Helix
36
2) dNTP binding to the O helix
3.4Å
DNA
KF
dNTP
dN
TP
1
2
~
3
4
covalent
bond
DNA+1
PPi
5
6
dNTPs bind initially to conserved R & K residues on O helix
(fingers open conformation)
Klentaq
(Rothwell & Waksman)
Bst DNA polymerase
(Beese & coworkers)
O helix
Y 714
K
R
O helix
38
How do DNA polymerases discriminate between dNTP and rNTP?
39
Classes of polymerases compared by Catherine Joyce in her 1997 review:
Choosing the right sugar (PNAS 94:1619-1622)
A family
DNA polymerase
Reverse
transcriptase
40
A steric gate five positions C terminal of conserved aspartate (D) in A motif excludes rNTPs in
DNA polymerases. This gate is glutamate (E710) in A family DNA polymerases Steric
and F or Y in reverse transcriptases.
D Gate
R
K
YG
Motif A F,Y in
RT
Motif B
D
Motif C
41
Delarue 1990
A family
DNA polymerase
Source
Mutation that alters
sugar specificity
Outcome
KF
Astatke & Joyce
PNAS 95:3402–3407
E710
A710
Steric gate removed
(dNTP preference reduced)
Reverse
transcriptase
Gao et al.
PNAS 94: 407-411
MoMLV
F155
V155
HIVRT
Cases-Gonzalez et al.
V115
JBC 275:19759 –19767 Y115
Steric gate removed
(dNTP preference reduced)
42
Extension of primer PG5–25 using dNTPs or rNTPs as nucleotide substrates, by WT RT , and
mutants Y115V and Y115G
Cases-González C E et al. J. Biol. Chem. 2000;275:19759-19767
©2000 by American Society for Biochemistry and Molecular Biology
Position of the steric gate that inhibits rNTP binding in A family DNA
polymerases
E710
~
44
A phenyalanine in motif B strongly influences selectivity for dNTPs (bearing a 3 prime OH on
Steric
the sugar ) vs. ddNTPs which lack a 3 prime OH on the sugar.
D
Gate
Motif A
R
K
F
YG
Motif B
D
Motif C
45
Delarue 1990
Addition of a dideoxynucleotide
prevents addition of the next.
H
What accounts for base specificity in DNA polymerases?
47
Structures of thymidine mimics having gradually increasing size
Kim T W et al. PNAS 2005;102:15803-15808
©2005 by National Academy of Sciences
Histogram of nucleotide insertion efficiencies vs. varied base pair size
Kim T W et al. PNAS 2005;102:15803-15808
©2005 by National Academy of Sciences
3) Closing of the fingers domain and movement of the O helix
3.4Å
DNA
KF
dNTP
dN
TP
1
2
~
3
4
covalent
bond
DNA+1
PPi
5
6
3)Closing of the fingers domain brings the O helix proximal
to the catalytic site and the correct dNTP into the catalytic site
O helix
O helix
~
Klentaq
(Rothwell & Waksman)
Details of open to closed transition (Bst DNA polymerase)
& Central role of O helix
A family DNA polymerase from Bacillus stearothermophilus (Johnson & Beese, 2003)
Adapted by Temiakov 2004
52
4) Phosphodiester bond formation
3.4Å
DNA
KF
dNTP
dN
TP
1
2
~
3
4
covalent
bond
DNA+1
PPi
5
6
Key aspartate residues involved in catalysis
D
Motif A
Motif B
D
Motif C
54
Delarue 1990
Details of the catalytic site
n = -1
n=+1
n=0
5′
n=0
n=-1
Template
Thumb
Fingers
1) Conserved aspartate residues in
motif A (D705) & motif C (D882)......
2) coordinate essential Mg2+ ions
‘A’ and ‘B’.
3) Mg2+ (A) promotes de-protonation
of 3′ hydroxyl at end of primer strand,
which allows nucleophilic attack on the
α phosphate of the incoming dNTP.
H+
5′
Mg2+
A
D882
4) During nucleophilic attack, excess (-) charge
is transfered to the triphosphate forming a
high energy transition state. This state is
D705
stabilized by coordination of 5 O atoms by
T7 DNA Polymerase
2+
2+
Mg (A) and Mg (B).
Mg2+
B
Steitz 1998
55
Eyring/Arrhenius Theory
Mg2+
Mg2+
Potential Energy
Transition
state
E*•DNA•dNTP
DNA + dNTP
Reactants
DNA+1 + PPi
Products
Reaction Coordinate
56
5)Pyrophosphate release
3.4Å
DNA
KF
dNTP
dN
TP
1
2
~
3
4
covalent
bond
DNA+1
PPi
5
6
6)Translocation
3.4Å
DNA
KF
dNTP
dN
TP
1
2
~
3
4
covalent
bond
DNA+1
PPi
5
6
DNA must advance 1bp for O helix tyrosine to re-stack
and fingers to re-open for next dNTP binding
Primer
Template
Duplex
DNA bound
region
x
Primer
Template
Duplex
DNA bound
region
(1)
O helix
Fingers closed
Bst polymerase(Johnson & Beese, 2003)
O helix
(2)
Fingers open
59
60
61