DNA polymerases

Nucleic Acid Chemistry

Third term 2022-2023

Randy Bryant

Department of Biochemistry and Molecular Biology

Johns Hopkins Bloomberg School of Public Health

DNA polymerases Catalyze the synthesis of DNA chains

3´ 3´
3´ 3´

5´
5´ 5´

dNTP
dNTP 3´
3´

Biochemistry, 6 th ed. Freeman 2007

Use deoxyribonucleoside triphosphates (dNTPs) as precursors

Extend DNA chains by adding one nucleotide unit at a time to

the 3´-end of a primer strand
 Template-directed DNA polymerization

primer

template Biochemistry, 6th ed. Freeman 2007

DNA polymerase uses single-stranded DNA as a template

Selects and adds the nucleotide that is complementary to
the corresponding nucleotide in the template strand
 Key characteristics of DNA polymerase activity

Processivity: How many nucleotides are incorporated

before DNA polymerase dissociates from
the primer-template

Range: one to thousands of nucleotides

Fidelity: How often a DNA polymerase incorporates

an incorrect nucleotide
Range: 1/100 to 1/1,000,000 nucleotides
 General kinetic mechanism for DNA polymerases

E + p/t ⇌ E-p/t + dNTP ⇌ E-p/t-dNTP fidelity

dissociation
distributive

⇌ kpol
E-p+1/t + PPi ⇌ E-p+1/t-PPi ⇌ E*-p/t-dNTP

processivity

E = DNA polymerase
p/t = primer/template
 DNA polymerase: 3´- 5´ exonuclease activity

3´ 3´
OH
OH

OH
OH
H20 5´

5´
3´

3´
dNMP

Hydrolysis reaction removes nucleotides from 3´-end of primer strand

DNA polymerase: 3´- 5´ exonuclease activity

Serves as a proofreading
mechanism

Removes mis-incorporated
nucleotides from the primer
terminus
Increases the fidelity of
DNA synthesis
 DNA polymerase: 5´- 3´ exonuclease activity
OH
OH
dNMP
OH

5´

5´
3´
H
OH
3´
OH
OH
OH 3´

OH
H20 5´ 5´

dNMP

Hydrolysis reaction removes nucleotides from 5´-end of primer strand

 DNA polymerase: 5´- 3´ exonuclease activity

Degrades Okazaki fragments

during DNA replication

Also degrades strands during

DNA repair mechanisms

Results in nick translation

Principles of Biochemistry, 5th ed. Freeman, 2008

 E. coli DNA polymerase I (Pol I)

Discovered by Arthur Kornberg and colleagues (1956)

Monomeric enzyme: 928 amino acids (109 kDa)

Has: DNA polymerase

3´- 5´ exonuclease

5´- 3´ exonuclease

Processivity: ~10 - 50 nucleotides

Error rate: ~1/100,000 nucleotides

Discovery of DNA polymerase
Arthur Kornberg and Bob Lehman
 Domain organization of E. coli DNA polymerase I

protease-sensitive
site

Structure of full-length protein still has not been determined

E. coli DNA polymerase I: Klenow fragment

Limited proteolysis cleaves Pol I into two distinct

fragments:

Small fragment (41 kDa): 5´- 3´ exonuclease

Large fragment (68 kDa): DNA polymerase

3´- 5´ exonuclease

Large fragment is referred to as Klenow fragment

Error rate: ~1/50,000 nucleotides

Proteolytic cleavage of DNA polymerase I
 Proteolytic cleavage of DNA polymerase I

Brutlag et. al., “An active fragment of DNA polymerase produced by proteolytic cleavage”, Biochem. Biophys. Res. Comm. 37, 982 (1969)
First DNA polymerase structure

DNA polymerase I (Klenow)

Steitz and colleagues

Nature 313, 762 (1985)
 DNA polymerase I (Klenow) structure

thumb

palm

3´- 5´ exonuclease

fingers

Structure resembles a right hand

Ollis et. al., “Structure of a large fragment of Escherichia coli DNA polymerase I complexed with dTMP”, Nature 313, 762 (1985)
 Original model for the Klenow-DNA complex

Proposed that DNA could fit into a large crevice in the protein

Ollis et. al., “Structure of a large fragment of Escherichia coli DNA polymerase I complexed with dTMP”, Nature 313, 762 (1985)
Two-metal ion mechanism of DNA polymerase I

Metal A: interacts with the 3´-OH

of primer

Metal B: interacts with the β and γ

phosphates of dNTP
3´

Both metals may stabilize the transition

state of the reaction

Metal A and B = Mg2+

Active site metal ions are bound by: Asp705 and Asp882

Steitz , ”DNA polymerases: Structural diversity and common mechanisms”, J. Biol. Chem. 274, 17395 (1999)
Structure of a Klenow-DNA complex

A structure of a Klenow-DNA complex

was reported by Steitz and colleagues
in 1988

Was a structure of an editing complex

rather than a polymerization complex
 Klenow polymerase: Editing complex

template

primer

Polymerase and exonuclease active sites are 30 Å apart

Freemont et. al., “Cocrystal structure of an editing complex of Klenow fragment with DNA”, PNAS 89, 8934 (1988)
 Shuttle mechanism of editing in DNA polymerase I

primer

primer

Primer strand dissociates from template and binds to exonuclease site

Mismatched nucleotide is removed

Steitz , “DNA polymerases: Structural diversity and common mechanisms”, J. Biol. Chem. 274, 17395 (1999)
Shuttle mechanism of editing in DNA polymerase I

fingers thumb

polymerase
mode

3´-5´ exonuclease
mode

Steitz , “DNA polymerases: Structural diversity and common mechanisms”, J. Biol. Chem. 274, 17395 (1999)
 3´- 5´ exonuclease active site

dCMP

Active site metal ions bound by: Asp355, Asp424, Asp501, Glu357

Derbyshire et. al. “Structure of DNA polymerase I Klenow fragment bound to duplex DNA”, Science 260, 352 (1993)
 3´- 5´exonuclease reaction mechanism

Metal A: interacts with the

primer water molecule

Metal B: interacts with the

phosphodiester
H2O oxygens

Metal A and B = Mg2+

Analogous to the DNA polymerase active site mechanism

Freemont et. al., “Cocrystal structure of an editing complex of Klenow fragment with DNA”, PNAS 89, 8934 (1988)
 Klenow polymerase (exo-) mutant

Change Asp355 and Glu357 to

alanines

Eliminates the ability to bind

the catalytic metal ions at the
3´- 5´ exonuclease site
And eliminates the 3´- 5´ exonuclease
activity (exo- mutant)

Error rate ~1/10,000 nucleotides

Elimination of 3´- 5´ exonuclease activity increases the polymerization

error rate by only ~ 5-10 fold
 Taq I DNA polymerase

Thermus aquaticus – found in hot springs and thermal vents

Monomeric enzyme: 832 amino acids (94 kDa)

optimal temperature: 70-80 ºC
half life: > 2 hr at 92 ºC

Homologous to E. coli Pol I (32% amino acid identity)

But has no 3´- 5´ exonuclease activity

Error rate: ~1/9,000 nucleotides

Low fidelity polymerase: similar to exo- version of Klenow

 Taq I polymerase: full length protein

thumb

palm
fingers 5´- 3´ exonuclease

protease-sensitive
“3´- 5´ exonuclease” site

Kim et. al. “Crystal structure of Thermus aquaticus DNA polymerase” Nature 376, 612 (1995)
Taq I polymerase: vestigial 3´- 5´ exonuclease domain

Active site carboxylates have been

replaced:

Pol I: D424, D501, D355, E357

Taq I: L356, R405, G308, V310

So does not bind metal ions at this site

And has no 3´- 5´ exonuclease activity

Kim et. al. “Crystal structure of Thermus aquaticus DNA polymerase” Nature 376, 612 (1995)
 Klentaq I DNA polymerase

N-terminal 5´- 3´ exonuclease domain of Taq I

DNA polymerase has been deleted

Analogous to Klenow fragment of E. coli Pol I

Structure and mechanism have been examined

in detail
 Klentaq I polymerase

fingers thumb

palm

3 ´- 5´exonuclease
(vestigial)

Structure is similar to Klenow polymerase

Superimposition of Klenow and Klentaq I structures

Korolev et. al. “Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5 Å resolution: Structural basis for thermostability” PNAS 92, 9264 (1995)
 Basis for the thermostability of Taq I polymerase

Three additional ion pairs in Klentaq I that are not

present in Klenow

Global rearrangement of charged amino acids reduces

the number of unfavorable electrostatic interactions

Increased number of hydrophobic amino acids in the

core

More prolines: 13 in Taq I; 6 in Klenow

Korolev et. al. “Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5 Å resolution: Structural basis for thermostability” PNAS 92, 9264 (1995)
 Klentaq I polymerase

fingers thumb

palm

3 ´-5´ exonuclease
(vestigial)

Rothwell and Waksman, “Structure and mechanism of DNA polymerases” Adv. Protein Sci. 71, 401 (2005)
 Klentaq I-primer/template complex

fingers thumb

primer/template
palm

3´- 5´ exonuclease
(vestigial)

Rothwell and Waksman, “Structure and mechanism of DNA polymerases” Adv. Protein Sci. 71, 401 (2005)
 Conformational change: thumb subdomain

thumb thumb thumb

Klentaq I Klentaq I-p/t superimposition

Conformational change in the thumb subdomain when p/t binds

Rothwell and Waksman, “Structure and mechanism of DNA polymerases” Adv. Protein Sci. 71, 401 (2005)
 Klentaq I-primer/template-ddCTP complex

fingers thumb

ddCTP
primer/template
palm

3 ´- 5´ exonuclease
(vestigial)

Rothwell and Waksman, “Structure and mechanism of DNA polymerases” Adv. Protein Sci. 71, 401 (2005)
 Conformational change: O-helix

O-helix O-helix

Klentaq I-p/t Klentaq I-p/t-ddCTP superimposition

Change in conformation of O-helix in the finger subdomain when dNTP binds

Open to closed transition
Rothwell and Waksman, “Structure and mechanism of DNA polymerases” Adv. Protein Sci. 71, 401 (2005)
Klentaq I DNA polymerase reaction sequence

Primer/template binds to thumb

and palm subdomains

Loop in thumb subdomain closes

over the primer-template

dNTP binds in finger subdomain

Open to closed transition brings

dNTP to the primer terminus

Nucleotide addition occurs

Rothwell and Waksman, “Structure and mechanism of DNA polymerases” Adv. Protein Sci. 71, 401 (2005)
Klentaq I DNA polymerase reaction sequence

Waksman lab website

 General kinetic mechanism for DNA polymerases

E + p/t ⇌ E-p/t + dNTP ⇌ E-p/t-dNTP fidelity

dissociation
distributive

⇌ kpol
E-p+1/t + PPi ⇌ E-p+1/t-PPi ⇌ E*-p/t-dNTP

processivity

E = DNA polymerase
p/t = primer/template
 T7 DNA polymerase

Gene 5 protein from bacteriophage T7 (E. coli)

Two subunits: T7 DNA polymerase (80 kDa)

E. coli thioredoxin (12 kDa)
Bind in a 1:1 ratio (Kd ~ 5 nM)

Thioredoxin is a processivity factor:

T7 DNA polymerase: 1-15 nts
T7 DNA polymerase/thioredoxin: > 2000 nts

Has 3´- 5´ exonuclease, but no 5´- 3´ exonuclease

Error rate: ~1/66,000 nts

 T7 DNA polymerase

thioredoxin
thumb
closed conformation
palm thioredoxin binding
domain
fingers
template:primer

no 5´- 3´ exonuclease domain

3´- 5´ exonuclease

Richardson lab web site

 Thioredoxin acts as a processivity factor

Thioredoxin binds to the thioredoxin binding domain

and covers primer/template site

Inhibits dissociation of T7 DNA polymerase from the

primer/template

Increases affinity of T7 DNA polymerase for primer/template

by 80-fold

And increases processivity from < 20 nucleotides to

thousands of nucleotides per binding event

Thioredoxin binding domain is not present in Pol I or Taq I

Chimeric Klenow-TBD polymerase

A chimeric Klenow-TBD protein was

created by Charles Richardson and
colleagues (1997)

Inserted a thioredoxin binding domain

into E. coli Klenow polymerase
 Chimeric Klenow-TBD polymerase

Klenow

Added a T7 thioredoxin binding domain to E. coli Klenow DNA polymerase

Bedford et. al., “The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I” PNAS 94, 479 (1997)
 Processivity of Klenow-TBD chimeric protein

T7 GP5: increased processivity when

longer
thioredoxin is added

Klenow: not affected by addition of

thioredoxin

Klenow-TBD: increased processivity when

thioredoxin is added

Were able to re-engineer the processivity

shorter of Klenow DNA polymerase

Bedford et. al., “The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I” PNAS 94, 479 (1997)
Designer DNA polymerases
 Polymerase chain reaction (PCR)

A cyclical procedure that allows a specific DNA sequence

to be amplified exponentially

Invented by Kary Mullis

in 1983

The idea for PCR came to Mullis in 1983 while he was driving his Honda Civic on a moonlit mountain road
in Northern California. That night Mullis knew he was onto something significant. "It was difficult for me
to sleep with deoxyribonuclear bombs exploding in my brain," he later wrote.
 PCR primers

Start with two oligonucleotide primers that are complementary to the

flanking sequences at either end of the target DNA sequence

Biochemistry, 6th Ed Freeman 2007

Primers are positioned to prime DNA synthesis - by a DNA polymerase - across

the target sequence using the two strands of the target DNA as templates
 PCR cycle

* Taq I

95 oC

54 oC

72 oC

Taq I polymerase is stable

to the high temperatures
that are used to separate
the DNA strands after each
reaction cycle.
 PCR amplification

Thermal cycler changes the temperature of the reaction tube:

95 ºC 54 ºC 72 ºC repeat
denature anneal DNA
target primers synthesis

Ideally: The desired target sequence will be amplified by 2n fold,

where n = number of cycles

After 20 cycles 220 ~1 million fold

After 30 cycles 230 ~1 billion fold

Allows specific DNA sequences to be amplified from extremely

small amounts of DNA