DNA Polymerase
Structural Biology
Year 2015
Rebekah Sibbald
Guillem Pina
Júlia Melià
Contents
1.
2.
3.
4.
5.
6.
7.
8.
Introduction to Polymerases
Function
Context
Families
SCOP
Architecture
Focus on Family A
1.
Sequence and Structural Alignment
2.
Secondary Structure
General Mechanism
1.
Thumb
2.
Finger
3.
Hydrophobic Pocket
4.
Hydrogen Bonds
5.
Metal Ions
Introduction to Polymerases
What exactly is a polymerase?
T7 DNA Polymerase
T7 RNA Polymerase
Function
(Public Domain)
Lagging Strand Formation
Source: Harvard Biotext and Animations
http://sites.fas.harvard.edu/~biotext/animations/replication1.html
Larger Context
Even Larger Context
Multiple Families
• Although there are multiple types of polymerase, there are
also multiple families
• Families are based on sequence homology and crystal
structure analysis
– These families do not correspond to SCOP classifications
• Based on comparison of amino acid sequences, DNA
polymerase families all seem to be unrelated
– However, based on some common biochemical and
structural features, it is possible that families A, B and C
are related
– Specifically, possible that exonuclease domains are
homologs
Meet the Families
Filee et al., 2001.
Family Portraits
• As would be expected, polymerases in different families look
quite different
SCOP Classification
Family A in SCOP
Implications
• It does not make sense to do sequence or structure
comparison between DNA polymerase families
• If they are homologous, the common ancestor is very distant
• Believe us, we tried...
Conserved Architecture: The Right Hand
• Although there is little conserved
sequence or structure between families,
there is a conserved architecture:
– Finger Domain
• Template and dNTP interactions
– Palm Domain
• Phosphoryl transfer reaction
– Thumb Domain
• Processivity and translocation
Photo: Royal Collection Trust / © Her
Majesty Queen Elizabeth II 2013
Conserved Architecture: The Right Hand
Yellow = Thumb
Blue = Finger
Pink = Palm
Family A
Meet Family A
• Replicative and repair enzymes
• Includes polymerases in the bacteriophage, as well as eukaryotic
and prokaryotic domains
• We focus on Polymerase I
Anyone remember it’s function?
Answer: Processing Okazaki
fragments
Bonus: Nucleotide excision
and repair
Which means…. it has exonuclease and
polymerase functions!
Palm Domain
Finger Domain
Thumb Domain
Conserved residues
Asp 610
Arg 659
Arg 573
His 639
Ile 614
Glu 615
Phe 667
Lys 663
Tyr 671
Asp 785
Family A structure: 4 Members of
Family A
RMSD = 2.34
Yellow = Taq polymerase
Blue = T7 DNA Polymerase
Pink = E. Coli DNA polymerase I
Green = B. Stearothermophilus DNA polymerase I
Family A structure: 3 Members of
Family A
RMSD = 1.72
Yellow = Taq polymerase
Pink = E. Coli DNA polymerase I
Green = B. Stearothermophilus DNA polymerase I
G
H
6
α-helices
β-strands
H
H1
H2
H2
I
7
Palm Domain
8
Finger Domain
Thumb Domain
K
8
L
9
N
O
P
O2
Q
10
O1
O2
Q
11
12
14
M
13
R
General Mechanism
● To start, primer is already bound to template
● Step 1: Primer and template bind to enzyme, which involves
a conformational change
● Step 2: dNTP binds to primer/template-enzyme complex
● Step 3: Conformational change in fingers domain (open to
closed)
● Step 4: Nucleophilic attack forming a phosphodiester bond
● Step 5: Release of inorganic phosphate
● Finally, enzyme can dissociate the primer/template
(distributive synthesis) or translocate the template for a new
round of synthesis (processive synthesis)
General Mechanism
The conformational change is the rate limiting step!
.
Rothwell and Waksman, 2005
Conformational Change: Thumb Domain
1. Entire domain rotates by 17º resulting in an opening of the
DNA-binding crevice
2. Only helices H1 and H2 of the domain rotate by 12º to bring
the tip of the thumb domain closer to the DNA
There are further conformational changes (mostly in the H1H2
loop), finally resulting in a cylinder that almost completely
surrounds the DNA.
Conformational Change: Thumb Domain
Blue: unbound DNA state
Pink: bound DNA state
Conformational Change
• Conformational change activates the enzyme complex
• Involves shift from open structure to closed structure
• Open structure:
– The tip of the fingers domain is rotated outward by 46º,
so that a crevice is clearly accessible
• Closed structure:
– Reorientation of the tip of the fingers domain so that it is
oriented inwards towards the template and primer
Conformational Change: Finger Domain
Blue = open
Yellow = closed
Conformational Change: Finger Domain
Conformational Change: Finger Domain
Conformational Change: Finger Domain
1. Rigid body rotation of helices N, O, O1 and O by 6º causing a
partial closing of the crevice
2. Rotation of N and O helices by 40º
The orientation of the O helix is dramatically affected by this
transition, and many residues are directly involved with dNTP
binding and incorporation
Conformational Change: Finger Domain
Hydrophobic Pocket
Ile 614
Phe 667
Tyr 671
Hydrophobic pocket
Hydrophobic pocket
Hydrophobic pocket
Nucleotide stabilization
(H bonds)
Glu 615
Arg 659
Arg 573
His 639
Lys 663
Hydrogen Bonds
Metal Ions interaction
(active site)
Asp 610
Asp 785
Metal Ions
Metal Ions
Metal Ions
Metal Ions
Conclusions: Requirements for
Polymerization




Thumb changes conformation when primer and
template bind to enzyme
Fingers change conformation (rate limiting) when
nucleotide binds to enzyme
Interactions with incoming nucleotide through a
hydrophobic pocket and hydrogen bonds
Metal ion induced nucleophilic attack causing
phosphodiester bond formation
Arg 573
Asp 610
Ile 614
Glu 615
Phe 667
His 639
Asp 785
Arg 659 Lys 663
Tyr 671
Overall Conclusions


Common architecture between families
In Family A, conservation is due to functional role
 There
is more conservation in structure than sequence
 Sequence is conserved when residues have a specific
function
References:
Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.
Li Y, Kerolev S, Waksman G. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA
polymerase I: structural basis for nucleotide incorporation. EMBO J. 1998;17(24):7514-7525.
Nakamura T, Zhao Y, Yamagata Y, Hua Y, Yang W. Watching DNA polymerase g make a phosphodiester bond. Nature. 2012 Jul 12;487(7406):196-201.
Filee J, Forterre P, Sen-Lin T, Laurent J. Evolution of DNA polyemerase Families: Evidence for Multiple Gene Exchange Between Cellular and Viral Proteins. J
Mol Evol. 2002;54:763-773.
Patel PH, Loeb LA. Getting a grip on how DNA polymerases function. Nat Struct Biol. 2001;8(8):656-9.
Steitz TA. DNA polymerases: structural diversity and common mechanisms. J Biol Chem. 1999;274(25):17395-8.
Astatke M, Grindley ND, Joyce CM. How E. coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides. J Mol Biol.
1998;278(1):147-65.
Sheriff A, Motea E, Lee I, Berdis AJ. Mechanism and dynamics of translesion DNA synthesis catalyzed by the Escherichia coli Klenow fragment. Biochemistry.
2008;47(33):8527-37.
Ogawa T, Okazaki T. Discontinuous DNA Replication. Ann Rev Biochem. 1980;49:421-457.
Ramanathan S, Chary KV, Rao BJ. Incoming nucleotide binds to Klenow ternary complex leading to stable physical sequestration of preceding dNTP on DNA.
Nucleic Acids Res. 2001;29(10):2097-105.
Beese LS, Derbyshire V, Steitz TA. Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science. 1993;260(5106):352-5.
Kim Y, Eom SH, Wang J, Lee DS, Suh SW, Steitz TA. Crystal structure of Thermus aquaticus DNA polymerase. Nature. 1995 Aug 17;376(6541):612-6.
Eom SH, Wang J, Steitz TA. Structure of Taq polymerase with DNA at the polymerase active site. Nature. 1996 Jul 18;382(6588):278-81.
Rothwell JP, Waksman G. Structure and Mechanism of DNA Polymarases. Adv Protein Chem. 2005;71:401-440.
Beese LS, Steitz TA. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 1991
Jan;10(1):25-33.
Patel PH, Suzuki M, Adman E, Shinkai A, Loeb LA. Prokaryotic DNA polymerase I: evolution, structure, and "base flipping" mechanism for nucleotide selection.
J Mol Biol. 2001 May 18;308(5):823-37.
Questions
1. DNA Polymerases' classic and SCOP classifications are different, more specifically:
a) SCOP classification has more polymerases in each category.
b) SCOP classification is based on function while classic classification is based on sequence homology.
c) SCOP classification is based on folding of the polymerase domain while classic classification is based
on whole sequence sequence homology and crystal structure analysis.
d) The classic classification is based in polymerase activity, as well as SCOPE is, but have different polymerases
within them.
e) Non of the above are correct.
2. Regarding DNA polymerases:
a) All DNA polymerases have the same architecture (finger, palm and thumb domains).
b) Only DNA polymerases from the same SCOP family of Taq polymerase have the same architecture.
c) Taq polymerase is the only polymerase with both polymerase and exonuclease activity.
d) The thumb domain is responsible for the interactions with the template.
e) Non of the above are correct.
3. When performing a sequence alignment between taq, e. coli, T7 and b. stearothermophilus (the 4
polymerases obtained using the HMM of family A), the resulting alignment taking into account the
structural features explained in the presentation:
a) shows a lot of sequence homology throughout the whole sequence alignment but the finger, thumb and palm
domains are not conserved.
b) shows a lot of sequence homology throughout the whole sequence alignment and the finger, thumb and palm
domains are conserved.
c) doesn't show a lot of sequence homology throughout the whole sequence and neither does the secondary
structure, but some key residues are conserved.
d) doesn't show a lot of sequence homology throughout the whole sequence but secondary structure is
highly conserved as well as the key residues.
e) T7 is completely different from the other family A polymerases and this can be explained because is a
polymerase from a bacteriophage while the others are bacteria polymerases.
4. How is the DNA polymerase activated?
a) By a conformational change in the thumb domain after binding of the primer and template.
b) By a conformational change from open to closed in fingers domain after binding of a dNTP.
c) It is always active.
d) The binding of several inductors activates it.
e) All of the above are incorrect.
5. What is the role of the hydrophobic pocket that is formed in the fingers domain?
a) Interacts with the sugar and the base of the incoming nucleotide.
b) Interacts with the phosphate group of the incoming nucleotide.
c) A and B are correct.
d) Interacts with the last base pair of the primer/template.
e) All of the above are incorrect.
6. When does the thumb change its conformation?
a) After binding of the primer and template, forming a cylinder that surrounds them.
b) After binding of a dNTP to the template/primer-enzyme complex.
c) When the template is translocated for the addition of a new nucleotide.
d) Once the phosphodiester bond between the primer and the dNTP has been formed.
e) All of the above are incorrect.
7. Which residue forms stacking interactions with the DNA template in the open conformation of family A
polymerases?
a) A tyrosine of the O helix in the fingers domain.
b) An aspartate of the active site in the palm domain.
c) A phenylalanine of the H1 helix in the thumb domain.
d) An arginine of the N helix in the fingers domain.
e) Stacking interactions are only formed in the closed conformation.
8. How are metal ions involved in DNA polymerase activity?
a)
b)
c)
d)
e)
By causing the enzyme to dissociate.
By stabilizing the transition state.
By slowing the reaction progress.
By initiation DNA transposition.
Trick question, they are not involved.
9. Which of the following is true?
a) Different organisms contain different types of polymerases with the same function.
b) Each organism contains only one type of polymerase.
c) All polymerases are clearly homologous.
d) Unlike RNA polymerase, DNA polymerase involves only one subunit.
e) Different organisms contain different types of polymerases for specialized functions.
10. Which of the following is false concerning DNA polymerase I?
a) Residues with specific functions are more likely to be conserved.
b) DNA polymerase I is specialized in lagging strand formation.
c) Polymerization involves a metal ion mechanism.
d) Aside from common architecture, there are no similarities between polymerases.
e) DNA polymerases share a common architecture.